Reversibility and "pH-T phase diagrams" of Rapana venosa hemocyanin and its structural subunits

We have studied the stability and reassociation behaviour of native molecules of Rapana venosa hemocyanin and its two subunits, termed RvH1 and RvH2. In the presence of different concentrations of Ca(2+) and Mg(2+) ions and pH values, the subunits differ not only in their reassociation behaviour, but also in their formation of helical tubules and multidecamers. RvH1 revealed a greater stability at higher pH values compared to RvH2. Overall, the stability of reassociated RvH and its structural subunits was found to be pH-dependent. The increasing stability of native Hc and its subunits, shown by pH-induced CD transitions (acid and alkaline denaturation), can be explained with the formation of quaternary structure. The absence of a Cotton effect at temperatures 20-40 degrees C in the pH-transition curves of RvH2 indicates that this subunit is stabilized by additional "factors", e.g.: non-ionic/hydrophobic stabilization and interactions of carbohydrate moieties. A similar behaviour was observed for the T-transition curves in a wide pH interval for RvH and its structural subunits. At higher temperatures, many of the secondary structural elements are preserved especially at neutral pH, even at extreme high temperatures above 90 degrees C the protein structures resemble a "globule state".     

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration.METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel.RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes.CONCLUSION: These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.     

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange. Here, the BTR complex, consisting of the Bloom helicase (HIM-6 in worms), topoisomerase 3 (TOP-3), and the RMI1 (RMH-1 and RMH-2) and RMI2 scaffolding proteins, is essential for dismantling joint DNA molecules to form non-crossovers (NCOs) via decatenation. In contrast, in meiosis COs are essential for accurate chromosome segregation and the BTR complex plays distinct roles in CO and NCO generation at different steps in meiotic recombination. RMI2 stabilizes the RMI1 scaffolding protein, and lack of RMI2 in mitosis leads to elevated sister chromatid exchange, as observed upon RMI1 knockdown. However, much less is known about the involvement of RMI2 in meiotic recombination. So far, RMI2 homologs have been found in vertebrates and plants, but not in lower organisms such as Drosophila, yeast, or worms. We report the identification of the Caenorhabditis elegans functional homolog of RMI2, which we named RMIF-2. The protein shows a dynamic localization pattern to recombination foci during meiotic prophase I and concentration into recombination foci is mutually dependent on other BTR complex proteins. Comparative analysis of the rmif-2 and rmh-1 phenotypes revealed numerous commonalities, including in regulating CO formation and directing COs toward chromosome arms. Surprisingly, the prevalence of heterologous recombination was several fold lower in the rmif-2 mutant, suggesting that RMIF-2 may be dispensable or less strictly required for some BTR complex-mediated activities during meiosis.     

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex_0-9 HexNAc_2-4 Hex_0-3 Pent_0-3 Fuc_0-3. A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.     

Variomics Screen Identifies the Re-entrant Loop of the Calcium-activated Chloride Channel ANO1 That Facilitates Channel Activation

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized ∼6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.     

Increased Metallothionein I/II Expression in Patients with Temporal Lobe Epilepsy

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and patients with TLE associated with tumor or dysplasia (TLE-TD) were evaluated for expression of MT-I/II, for the vesicular zinc levels, and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis, microgliosis and reduced neuronal population. In TLE-TD, the same changes were observed, except that were mainly confined to fascia dentata. Increased vesicular zinc was observed only in the inner molecular layer of MTLE patients, when compared to control cases. Correlation and linear regression analyses indicated an association between increased MT-I/II and increased astrogliosis in TLE. MT-I/II levels did not correlate with any clinical variables, but MTLE patients with secondary generalized seizures (SGS) had less MT-I/II than MTLE patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from TLE patients and our data suggest that it is associated with astrogliosis and may be associated with different seizure spread patterns.     

Bioleaching of Metals from Mineral Waste Product

The possibility of bioleaching Al, K, Na, Ca and Mg using microorganisms of the Thiobacillus thiooxidans group from industrial waste product (IWP) of copper ore flotation from the company MEDET was studied. The aim of the investigations was to establish the possible application of a combined method for processing IWP. The preliminary mechanical activation in combination with bioleaching resulted in a high extent of extraction of useful components. It was established that the removal of useful components from mechanically activated IWP is improved compared to non‐activated IWP. The effect of the concentration of Al‐containing waste product, of incubation time and time of preliminary mechanical activation on the extraction degree (α) [% w/w] of useful elements was investigated. The maximum degree of extraction of Al was achieved on Day 28 and its value reached 71% for industrial waste product mechanically activated for 4 hours. The maximum degrees of extraction of K and Na in the case of industrial waste product mechanically activated for 4 hours were achieved on Day 7 of the incubation period and their values were 78% and 91%, respectively. Under the conditions of bioleaching only Si had a low degree of extraction, accounting for 2.5%. The ability of microorganisms to leach aluminium could be used for the extraction of metals from nonbauxite raw materials and Al‐containing waste product not treatable by means of the BAYER method.     

Electrochemical sensor based on Arthrobacter globiformis for cholinesterase activity determination

The sensors applied recently for determination of cholinesterase activity are mostly enzymatic amperometric sensors, in spite of their disadvantages: short life-time at ambient temperature, instability of the response, interferences, as well as passivation of the electrode surface. In the present paper a new approach for determination of cholinesterase activity was proposed, overcoming the main drawbacks of the analysis performed with amperometric enzymatic sensors. Instead of the immobilization of enzymes on a conducting electrode surface, whole cells of Arthrobacter globiformis, containing choline oxidase were fixed on a Clark type oxygen probe. Current proportional to bacteria respiration is registered as a sensor response. The application of whole cells of bacteria as a sensing element permits to achieve high stability of the response and long life-time of the sensor at ambient temperature, due to the conservation of the enzyme in its natural micro-environment inside the immobilized cells. The proposed sensor keeps its functionality more than 7 weeks stored in deionized water at ambient temperature. For the first 2 weeks the amplitude of the response decreases with only 10% and at the end of the studied 7 weeks period the response was 50% of the initial. The other advantages of the proposed sensor are: the dissolved oxygen is used as a mediator which concentration can be reliably and interferences free measured by the aim of a Clark type oxygen probe applied as a transducer; reproducible bacterial membranes can be elaborated by filtration of resuspended bacterial culture after preliminary determination of its activity; application of membranes containing lyophilized bacteria capable to be conserved infinitely long time and activated just before their application; negligible cost compared with the sensors based on immobilized enzymes. The steady-state response of the proposed bacterial sensor to choline obtained in 200 s is linear in the investigated concentration range up to 2 x 10(-4) moldm(-3), with detection limit of 8 x 10(-8) moldm(-3) and sensitivity of 4 x 10(-1) microAcm(3)mol(-1), at pH 6, temperature of 25 degrees C and stirring rate of 300 rpm. Choline is formed as a result of the catalytic hydrolysis (depending on the cholinesterase activity) of the substrate acetylcholine. Linear calibration graph for cholinesterase activity determination was obtained in the range up to 11 mUcm(-3), with a slope of 1.97 x 10(-2) microAcm(3)mU(-1), at pH 6, temperature of 25 degrees C and stirring rate of 300 rpm. The tests with reconstituted lyophilized serum with known activity used as a control sample confirm the accuracy of the proposed method. The relative error of the determination was only 2.82%.     

Structural analysis and molecular modeling of the RvH2-e functional unit of Rapana venosa hemocyanin

Rapana venosa hemocyanin (RvH), a circulating glycoprotein of the marine snail, has a complex structure. To provide details on the stability of the protein, one functional unit, RvH2-e, was compared with the native molecule and the structural subunits, RvH1 and RvH2, via pH–T diagrams, typical phase portraits for stability and denaturation reversibility. By analyzing the T transition curves of RvH2-e at different pH values, several parameters of the thermodynamic functions were obtained. Increasing the temperature from 25 °C to 55 °C, the reversibility of the molecule of protein also increases, opening a reversibility window within the range of pH 4.0–8.0. On analyzing the pH transition curves, the start of the acid denaturation (below pH 6) and alkaline denaturation (above pH 9) was determined to be between 20 °C and 35 °C. For this range, the thermodynamic functions ΔH° and ΔG° for a standard temperature of 25 °C were calculated.