Crustacean plankton community (Crustacea: Copepoda and Cladocera) in gypsum karst lakes and their relation to abiotic parameters

Two gypsum karst lakes and one non-gypsum karst lake were studied for copepod and cladoceran species composition in relation to specific habitat characteristics. The investigation was conducted from March to June 2007. Four copepod and six cladoceran species were recorded. Gypsum karst lakes are characterized as sulphate lakes and they show significant differences from non-gypsum karst lakes in conductivity, TDS, alkalinity, calcium and sulphates. Data on environmental variables and zooplankton were analyzed using redundancy analysis (RDA). The model explained 65.73% of the variance of the crustacean zooplankton and environmental data by the first two axes. The analysis confirmed that the major environmental variables influencing zooplankton in gypsum karst lakes are conductivity, TDS, calcium and sulphates. In the non-gypsum karst lake, on the contrary, the major variables were oxygen concentration and alkalinity. Specific habitat characteristics of gypsum karst lakes influence the zooplankton community by reducing the number of species and leading to the dominance of one of them.     

Enantioselectivity of resolved Δ and Λ orthoruthenated 2-phenylpyridine complexes [Ru(o-C6H4-2-py)(LL)2]PF6 (LL = bpy and phen) toward glucose oxidase

Cyclometalated 2-phenylpyridine complexes [Ru^II(o-C₆H₄-2-py)(LL)₂]PF₆, LL = 2,2′-bipyridine (1) and 1,10-phenanthroline (2) were resolved into Δ and Λ enantiomers using column chromatography on SP Sephadex C-25 in the presence of (+)-2,3-dibenzoyl-D-tartrate. The absolute configuration of enantiomers was established using circular dichroism spectroscopy. The rate constants k_et for the electron transfer from reduced glucose oxidase (GO from Aspergillus niger) and PQQ-dependent glucose dehydrogenase (GDH) at the generated Ru^III species were measured by cyclic voltammetry and UV–vis spectroscopy. The electron transfer shows enantioselectivity. In the case of GO, the bell-shaped pH profile for the ratio k_Λ/k_Δ has a maximum at pH 7 (k_Λ/k_Δ equals 3.4 and 3.9 for 1 and 2, respectively), but its inversion is observed at pH around 5 and 9. The k_Λ/k_Δ ratio equals 2.0 for 2 and GDH at pH 7. The results of theoretical modeling of biological electron transfer for GO using functional docking Monte-Carlo simulations are presented and analyzed together with the experimental observations.     

Change in rigidity in the activated form of the glucose/galactose receptor from Escherichia coli: a phenomenon that will be key to the development of biosensors

Recently a periplasmic glucose/galactose binding protein, GGRQ26C, immobilized on a gold surface has been used as an active part of a glucose biosensor based on quartz microbalance technique. However the nature of the glucose detection was not clear. Here we have found that the receptor protein film immobilized on the gold surface increases its rigidity when glucose is added, which explains the unexpected detection signal. To study the rigidity change, we developed a new fast and simple method based on using atomic force microscopy (AFM) in tapping mode. The method was verified by explicit measurements of the Young's modulus of the protein film by conventional AFM methods. Since there are a host of receptors that undergo structural change when activated by ligand, AFM can play a key role in the development and/or optimization of biosensors based on rigidity changes in biomolecules.     

Amino acids defining the acyl pocket of an invertebrate cholinesterase

Amphioxus (Branchiostoma floridae) cholinesterase 2 (ChE2) hydrolyzes acetylthiocholine (AsCh) almost exclusively. We constructed a homology model of ChE2 on the basis of Torpedo californica acetylcholinesterase (AChE) and found that the acyl pocket of the enzyme resembles that of Drosophila melanogaster AChE, which is proposed to be comprised of Phe330 (Phe290 in T. californica AChE) and Phe440 (Val400), rather than Leu328 (Phe288) and Phe330 (Phe290), as in vertebrate AChE. In ChE2, the homologous amino acids are Phe312 (Phe290) and Phe422 (Val400). To determine if these amino acids define the acyl pocket of ChE2 and its substrate specificity, and to obtain information about the hydrophobic subsite, partially comprised of Tyr352 (Phe330) and Phe353 (Phe331), we performed site-directed mutagenesis and in vitro expression. The aliphatic substitution mutant F312I ChE2 hydrolyzes AsCh preferentially but also butyrylthiocholine (BsCh), and the change in substrate specificity is due primarily to an increase in k_cat for BsCh; K_m and K_ss are also altered. F422L and F422V produce enzymes that hydrolyze BsCh and AsCh equally due to an increase in k_cat for BsCh and a decrease in k_cat for AsCh. Our data suggest that Phe312 and Phe422 define the acyl pocket. We also screened mutants for changes in sensitivity to various inhibitors. Y352A increases the sensitivity of ChE2 to the bulky inhibitor ethopropazine. Y352A decreases inhibition by BW284c51, consistent with its role as part of the choline-binding site. Aliphatic replacement mutations produce enzymes that are more sensitive to inhibition by iso-OMPA, presumably by increasing access to the active site serine. Y352A, F353A and F353V make ChE2 considerably more resistant to inhibition by eserine and neostigmine, suggesting that binding of these aromatic inhibitors is mediated by π–π or cation–π interactions at the hydrophobic site. Our results also provide information about the aromatic trapping of the active site histidine and the inactivation of ChE2 by sulfhydryl reagents.     

Protein and DNA sequence determinants of thermophilic adaptation

There have been considerable attempts in the past to relate phenotypic trait—habitat temperature of organisms—to their genotypes, most importantly compositions of their genomes and proteomes. However, despite accumulation of anecdotal evidence, an exact and conclusive relationship between the former and the latter has been elusive. We present an exhaustive study of the relationship between amino acid composition of proteomes, nucleotide composition of DNA, and optimal growth temperature (OGT) of prokaryotes. Based on 204 complete proteomes of archaea and bacteria spanning the temperature range from −10 °C to 110 °C, we performed an exhaustive enumeration of all possible sets of amino acids and found a set of amino acids whose total fraction in a proteome is correlated, to a remarkable extent, with the OGT. The universal set is Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL), and the correlation coefficient is as high as 0.93. We also found that the G + C content in 204 complete genomes does not exhibit a significant correlation with OGT (R = −0.10). On the other hand, the fraction of A + G in coding DNA is correlated with temperature, to a considerable extent, due to codon patterns of IVYWREL amino acids. Further, we found strong and independent correlation between OGT and the frequency with which pairs of A and G nucleotides appear as nearest neighbors in genome sequences. This adaptation is achieved via codon bias. These findings present a direct link between principles of proteins structure and stability and evolutionary mechanisms of thermophylic adaptation. On the nucleotide level, the analysis provides an example of how nature utilizes codon bias for evolutionary adaptation to extreme conditions. Together these results provide a complete picture of how compositions of proteomes and genomes in prokaryotes adjust to the extreme conditions of the environment.     

Neurotransmitter receptor and time dependence of the synaptic plasticity disrupting actions of Alzheimer's disease Aβ in vivo

Many endogenous factors influence the time course and extent of the detrimental effects of amyloid β-protein (Aβ) on synaptic function. Here, we assessed the impact of varying endogenous glutamatergic and cholinergic transmission by pharmacological means on the disruption of plasticity at hippocampal CA3-to-CA1 synapses in the anaesthetized rat. NMDA receptors (NMDARs) are considered critical in mediating Aβ-induced inhibition of long-term potentiation (LTP). However, intracerebroventricular injection of Aβ_1–42 inhibited not only NMDAR-dependent LTP but also voltage-activated Ca²⁺-dependent LTP induced by strong conditioning stimulation during NMDAR blockade. On the other hand, another form of NMDAR-independent synaptic plasticity, endogenous acetylcholine-induced muscarinic receptor-dependent long-term enhancement, was not hindered by Aβ_1–42. Interestingly, augmenting endogenous acetylcholine activation of nicotinic receptors prior to the injection of Aβ_1–42 prevented the inhibition of NMDAR-dependent LTP, whereas the same intervention when introduced after the infusion of Aβ was ineffective. We also examined the duration of action of Aβ, including water soluble Aβ from Alzheimer''s disease (AD) brain. Remarkably, the inhibition of LTP induction caused by a single injection of sodium dodecyl sulfate-stable Aβ dimer-containing AD brain extract persisted for at least a week. These findings highlight the need to increase our understanding of non-NMDAR mechanisms and of developing novel means of overcoming, rather than just preventing, the deleterious synaptic actions of Aβ.     

Investigation and molecular mimicry of the antigen involved in the interaction between the monoclonal antibody 5D10 and the human breast cancer cell line MCF-7

Monoclonal antibody (mAb) 5D10 is directed against the human breast cancer cell line MCF-7. Biochemical characterization of the antibody epitope was attempted and revealed a complex, most likely carbohydrate-linked nature, which prevented isolation and further studies of the interaction. A major goal of this work was to generate structural mimics of the 5D10 epitope to serve as putative substitutes in such studies. A peptide library displayed on filamentous phage was used to select for mimotope peptide sequences. All positive phage clones selected from the library displayed the amino acid sequence H(2)N-QMNPMYYR-CO(2)H. This peptide sequence, as well as a branched form of the peptide, was found to bind mAb 5D10. Moreover, both peptide sequences were able to inhibit the binding of 5D10 to the MCF-7 cells in a concentration-dependent manner, with an EC(50) value in the range of 65 microM. According to these results, random phage peptide libraries can serve to identify mimotopic peptides for unknown complex cell surface epitopes.     

Preconditioning improves postischemic mitochondrial function and diminishes oxidation of mitochondrial proteins

This study examines the hypothesis that ischemic or pharmacologic preconditioning improves postischemic mitochondrial function by attenuating oxidation of mitochondrial proteins. Isolated rat hearts were perfused for 38 min preischemia, followed by 25 min global ischemia and then 60 min reperfusion. Hearts were preconditioned by two episodes of 3 min global ischemia, followed by 2 min of reflow (IP), or by perfusion with 50 micromol/l nicorandil (Nic) for 10 min, followed by 10 min washout. IP and Nic significantly (p <.05) improved postischemic function, which was abolished by bracketing the protocols with 200 micromol/l 5-hydroxydecoanate (5HD) or 300 micromol/l alpha-mercaptopropionylglycine (MPG). After isolation of cardiac mitochondria, the respiratory control index (RCI) was calculated from State 3 and State 4 respiration. Both IP and Nic significantly (p <.05) improved postischemic RCI, which was depressed 71% from preischemic values in control hearts. The protective effects of IP and Nic were partially abolished by bracketing with 5HD or MPG. Furthermore, mitochondria from ischemic hearts had significantly (p <.05) less ability to resist swelling on Ca2+ loading, which was improved by both IP and Nic. By use of an immunoblot technique, carbonyl content of multiple bands of mitochondrial proteins was observed to be elevated after 25 min ischemia, and still elevated by the end of 60 min reperfusion. Both IP and Nic attenuated the increased protein oxidation observed at the end of ischemia. The protective effect of IP was almost completely abolished by MPG and partially by 5HD, which also partially abolished the protective effect of Nic. These studies support the conclusion that one mechanism for enhanced postischemic function in the preconditioned heart is improved mitochondrial function as a result of decreased oxidation of mitochondrial proteins.     

Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed.