Na,K-ATPase from mice lacking the gamma subunit (FXYD2) exhibits altered Na+ affinity and decreased thermal stability

The gamma subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of gamma in a native tissue environment, mice lacking a functional gamma subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that gamma is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2-/- dams. In adult Fxyd2-/- mouse kidney, splice variants of gamma that have different nephron segment-specific expression patterns were absent. Purified gamma-deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that gamma and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the gamma subunit had increased thermal lability. Combined with other evidence that gamma participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.     

Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens

The effect of adsorption onto aluminum salt adjuvants on the structure and stability of three model protein antigens was studied using fluorescence and Fourier transform infrared spectroscopies, as well as isothermal titration and differential scanning calorimetric techniques. Lysozyme was preferentially adsorbed to aluminum phosphate (Adju-Phos), whereas ovalbumin and bovine serum albumin were better adsorbed to aluminum hydroxide (Alhydrogel). A linearized Langmuir adsorption isotherm was used to obtain information regarding the binding interactions between proteins and adjuvants. Binding energetics and stoichiometry data obtained from isothermal titration calorimetry measurements were complex. Based on the spectroscopic and differential scanning calorimetry studies, the structure of all three proteins, when adsorbed to the surface of an aluminum salt, was altered in such a way as to render the proteins less thermally stable. Besides the pharmaceutical significance of this destabilization, we consider the possibility that this phenomenon may facilitate the presentation of antigens and thus contribute to the adjuvant activity of the aluminum salts.     

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.     

Structural studies of inhibition of S-adenosylmethionine synthetase by slow, tight-binding intermediate and product analogues

S-Adenosylmethionine synthetase (ATP: L-methionine S-adenosyltransferase) catalyzes a two-step reaction in which tripolyphosphate (PPPi) is a tightly bound intermediate. Diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3); PNPNP), a non-hydrolyzable analogue of PPPi, is the most potent known inhibitor of AdoMet synthetase with a K(i) of 2 nM. The structural basis for the slow, tight-binding inhibition by PNPNP has been investigated by spectroscopic methods. UV difference spectra reveal environmental alterations of aromatic protein residues upon PNPNP binding to form the enzyme.2Mg(2+).PNPNP complex, and more extensive changes upon formation of the enzyme.2Mg(2+).PNPNP.AdoMet complex. Stopped-flow kinetic studies of complex formation revealed that two slow isomerizations follow PNPNP binding in the presence of AdoMet, in contrast to the lower affinity, rapid-equilibrium binding in the absence of AdoMet. (31)P NMR spectra of enzyme complexes with PNPNP revealed electronic perturbations of each phosphorus atom by distinct upfield chemical shifts for each of the three phosphoryl groups in the enzyme.2Mg(2+).PNPNP complex, and further upfield shifts of at least 2 resonances in the complex with AdoMet. Comparison of the chemical shifts for the enzyme-bound PNPNP with the enzyme complexes containing either the product analogue O(3)P-NH-PO(3) or O(3)P-O-PO(2)-NH-PO(3) indicates that the shifts on binding are largest at the binding sites corresponding to those for the alpha and gamma phosphoryl groups of the nucleotide (-3.1 to -4.1 ppm), while the resonance at the beta phosphoryl group position shifts by -2.1 ppm. EPR spectra of Mn(2+) complexes demonstrate spin coupling between the two Mn(2+) in both enzyme.2Mn(2+).PNPNP and enzyme.2Mn(2+).PNPNP.AdoMet, indicating that the metal ions have comparable distances in both cases. The combined results indicate that formation of the highest affinity complex is associated with protein side chain rearrangements and increased electron density at the ligand phosphorus atoms, likely due to ionization of an -NH- group of the inhibitor. The energetic feasibility of ionization of a -NH- group when two Mg(2+) ions are bound to O(3)P-NH-PO(3) is supported by density functional theoretical calculations on model chelates. This mode of interaction is uniquely available to compounds with P-NH-P linkages and may be possible with other proteins in which multiple cations coordinate a polyphosphate chain.     

Synthesis and utility of 14C-labeled nicotinamide cofactors

A new method for the synthesis of the reduced form of beta-nicotinamide [U-14C]adenine dinucleotide 2(')-phosphate([Ad-14C]NADPH) is presented. The present synthesis results in a radioactive material with a specific activity that is greater than 220 mCi/mmol. This method could easily be adapted for syntheses of 14C-labeled NADH, NADP(+), or any nicotinamide cofactors with radiolabels in other positions. Since these cofactors are so ubiquitous, the use and applications of such labeled material has broad implications. The utility of the labeled cofactor for determination of substrates for nicotinamide-dependent enzymes in the nano- to femtomole scale, in alternative enzymatic assays, and in kinetic isotope effect studies is discussed.     

Concordant phylogeography and cryptic speciation in two Western Palaearctic oak gall parasitoid species complexes

Little is known about the evolutionary history of most complex multi‐trophic insect communities. Widespread species from different trophic levels might evolve in parallel, showing similar spatial patterns and either congruent temporal patterns (Contemporary Host‐tracking) or later divergence in higher trophic levels (Delayed Host‐tracking). Alternatively, host shifts by natural enemies among communities centred on different host resources could disrupt any common community phylogeographic pattern. We examined these alternative models using two Megastigmus parasitoid morphospecies associated with oak cynipid galls sampled throughout their Western Palaearctic distributions. Based on existing host cynipid data, a parallel evolution model predicts that eastern regions of the Western Palaearctic should contain ancestral populations with range expansions across Europe about 1.6 million years ago and deeper species‐level divergence at both 8–9 and 4–5 million years ago. Sequence data from mitochondrial cytochrome b and multiple nuclear genes showed similar phylogenetic patterns and revealed cryptic genetic species within both morphospecies, indicating greater diversity in these communities than previously thought. Phylogeographic divergence was apparent in most cryptic species between relatively stable, diverse, putatively ancestral populations in Asia Minor and the Middle East, and genetically depauperate, rapidly expanding populations in Europe, paralleling patterns in host gallwasp species. Mitochondrial and nuclear data also suggested that Europe may have been colonized multiple times from eastern source populations since the late Miocene. Temporal patterns of lineage divergence were congruent within and across trophic levels, supporting the Contemporary Host‐tracking Hypothesis for community evolution.     

Lactobacilli-expressed single-chain variable fragment (scFv) specific for intercellular adhesion molecule 1 (ICAM-1) blocks cell-associated HIV-1 transmission across a cervical epithelial monolayer

The vaginal and cervical epithelia provide an initial barrier to sexually acquired HIV-1 infection in women. To study the interactions between HIV-1-infected cells or cell-free HIV-1 and the reproductive epithelium, the transmission of HIV-1 by infected cells or cell-free virus across human cervical epithelial cells was examined using a Transwell culture system. Cell-associated HIV-1 was transmitted more efficiently than cell-free virus, and monocyte-associated virus was transmitted most efficiently. Abs to ICAM-1 added to the apical side of the epithelium blocked cell-mediated transepithelial HIV-1 transmission in vitro. When used in a previously described model of vaginal HIV-1 transmission in human PBL-SCID mice, anti-murine ICAM-1 Abs (0.4 microg/10 microl) also blocked vaginal transmission of cell-associated HIV-1 in vivo. To evaluate a candidate delivery system for the use of this Ab as an anti-HIV-1 microbicide, anti-ICAM single-chain variable fragment Abs secreted by transformed lactobacilli were evaluated for their protective efficacy in the Transwell model. Like the intact Ab and Fab derived from it, the single-chain variable fragment at a concentration of 6.7 microg/100 microl was able to reduce HIV-1 transmission by 70 +/- 5%. These data support the potential efficacy of an anti-ICAM Ab delivered by lactobacilli for use as an anti-HIV-1 microbicide.     

Induction time for resistance to microsporidial gill disease caused by Loma salmonae following vaccination of rainbow trout (Oncorhynchus mykiss) with a spore-based vaccine

Resistance to re-infection of rainbow trout to Loma salmonae, a microsporidian gill parasite has been previously documented and this study examined how rapidly this resistance develops. Naive rainbow trout were inoculated intraperitoneally (IP) with an inactivated spore-based vaccine and were then given an oral challenge with a high dose of L. salmonae spores at various weeks after being vaccinated. Non-vaccinated naive fish (exposed group) were challenged alongside of each group of vaccinated fish to ensure that the challenges were relatively standardised. In each group of fish, four weeks after the challenge, numbers of xenomas were counted on a gill arch for all fish. Vaccinated trout were completely resistant to a L. salmonae challenge six weeks after vaccination, although the onset of resistance began at approximately week 3, as observed with a reduction in the percent infected and xenoma intensity. The maximum percent infected for the vaccinated fish was 83% following a challenge two weeks following vaccination, whereas for the exposed group the maximum prevalence of 100% was reached several times. With continued research, a spore-based vaccine for L. salmonae has the potential to become the first commercially available parasite vaccine for fish.     

Species loss of stoneflies (Plecoptera) in the Czech Republic during the 20th century

1. Rapid expansion and intensification of anthropogenic activities in the 20th century has caused profound changes in freshwater assemblages. Unfortunately, knowledge of the extent and causes of species loss (SL) is limited due to the lack of reliable historical data. An unusual data set allows us to compare changes in the most sensitive of aquatic insect orders, the Plecoptera, at some 170 locations in the Czech Republic between two time periods, 1955–1960 and 2006–2010. Historical data (1890–1911) on assemblages of six lowland rivers allow us to infer even earlier changes. 2. Regional stonefly diversity decreased in the first half of the 20th century. Streams at lower altitudes lost a substantial number of species, which were never recovered. In the second half of the century, large‐scale anthropogenic pressure caused SL in all habitats, leading to a dissimilarity of contemporary and previous assemblages. The greatest changes were found at sites affected by organic pollution and a mixture of organic pollution and channelisation or impoundment. Colonisation of new habitats was observed in only three of the 80 species evaluated. 3. Species of moderate habitat specialisation and tolerance to organic pollution were most likely to be lost. Those with narrow specialisations in protected habitats were present in both historical and contemporary collections. 4. Contemporary assemblages are the consequence of more than a 100 years of anthropogenic impacts. In particular, streams at lower altitude and draining intensively exploited landscapes host a mere fragment of the original species complement. Most stonefly species are less frequently present than before, although their assemblages remain almost intact in near‐natural mountain streams. Our analyses demonstrate dramatic restriction of species ranges and, in some cases, apparent changes in altitudinal preference throughout the area.