Slow backbone dynamics of the C-terminal fragment of human centrin 2 in complex with a target peptide probed by cross-correlated relaxation in multiple-quantum NMR spectroscopy

The C-terminal domain of human centrin 2 (C-HsCen2) strongly binds to P1-XPC, a peptide comprising 17 amino acids with a NWKLLAKGLLIRERLKR sequence. This peptide corresponds to residues N847-R863 of XPC, a protein involved in the recognition of damaged DNA during the initial step of the nucleotide excision repair pathway. The slow internal dynamics of the protein backbone in the C-HsCen-P1-XPC complex was studied by measuring the relaxation rates of zero- and double-quantum coherences involving neighboring pairs of carbonyl 13C and amide 15N nuclei. These relaxation rates, which reflect dynamics on time scales in the range of micro- to milliseconds, vary significantly along the protein backbone. Analysis of the relaxation rates at different CaCl2 concentrations and ionic strengths shows that these slow motions are mainly affected by the binding of a Ca2+ ion to the lower-affinity EF-hand III. Moreover, we discuss the possible functional role of residues that undergo differential exchange in the formation of HsCen homodimers.     

The N-terminal domain of human centrin 2 has a closed structure, binds calcium with a very low affinity, and plays a role in the protein self-assembly

Centrins are well-conserved calcium binding proteins from the EF-hand superfamily implicated in various cellular functions, such as centrosome duplication, DNA repair, and nuclear mRNA export. The intrinsic molecular flexibility and the self-association tendency make difficult the structural characterization of the integral protein. In this paper we report the solution structure, the Ca2+ binding properties, and the intermolecular interactions of the N-terminal domain of two human centrin isoforms, HsCen1 and HsCen2. In the absence of Ca2+, the N-terminal construct of HsCen2 revealed a compact core conformation including four almost antiparallel alpha-helices and a short antiparallel beta-sheet, very similar to the apo state structure of other calcium regulatory EF-hand domains. The first 25 residues show a highly irregular and dynamic structure. The three-dimensional model for the N-terminal domain of HsCen1, based on the high sequence conservation and NMR spectroscopic data, shows very close structural properties. Ca2+ titration of the apo-N-terminal domain of HsCen1 and HsCen2, monitored by NMR spectroscopy, revealed a very weak affinity (10(2)-10(3) M(-1)), suggesting that the cellular role of this domain is not calcium dependent. Isothermal calorimetric titrations showed that an 18-residue peptide, derived from the N-terminal unstructured fragment, has a significant affinity (approximately 10(5) M(-1)) for the isolated C-terminal domain, suggesting an active role in the self-assembly of centrin molecules.     

Comparative analysis of cytotoxic, genotoxic and antioxidant effects of 2,2,4,7-tetramethyl-1,2,3,4-tetrahydroquinoline and ethoxyquin on human lymphocytes

2,2,4,7-Tetramethyl-1,2,3,4-tetrahydroquinoline (THQ) is a new synthetic compound with potential antioxidant activity. In this study, cytotoxic, genotoxic and antioxidant activities of THQ were studied on human lymphocytes with the use of the trypan blue exclusion assay, the TUNEL method, the comet assay and the micronucleus test. The activities of THQ were compared with those of a structurally similar compound-ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ), which is used in animal feeds as a preservative. Cytotoxic effects of THQ were observed after 1-h treatment at the concentration of 500 microM and after 24-h treatments at the concentrations of 250-500 microM. Although the micronucleus test did not reveal a genotoxic effect of THQ, in the comet assay the statistically significant increase in DNA damage was observed as compared with the control. On the other hand, the protection of human lymphocytes against DNA damage induced by hydrogen peroxide suggests an antioxidant activity of THQ. The comparative analysis of THQ and EQ activities performed in these studies revealed that THQ was less cytotoxic and less genotoxic than EQ. Slightly lower antioxidant activity of THQ was also shown in the comet assay when it was used at the lower studied doses (1-5 microM), but for the highest one (10 microM) its efficiency was similar to that of EQ. In the micronucleus assay THQ was more effective than EQ in protecting the cultured lymphocytes from clastogenicity of H2O2. We believe that THQ is worthy of further detailed studies on its antioxidant properties to confirm its usefulness as a preservative.     

Productive folding of tyrosinase ectodomain is controlled by the transmembrane anchor

Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway characterized by the post-translational formation of buried disulfides. Lacking calnexin assistance, the truncated mutant is arrested in an unstable conformation bearing exposed disulfides. We showed that the transmembrane anchor of a protein may crucially, albeit indirectly, control the folding pathway of the ectodomain.     

Charophyte growth in small temperate water bodies: Extreme isotopic disequilibrium and implications for the palaeoecology of shallow marl lakes

A small pond containing the charophyte Chara hispida was monitored over a one-year period for changes in growth, water chemistry, water level and stable isotopic composition. Chara growth was found to be seasonal, with maximum growth occurring from late April to July. During this period, pH rose to > 10 while the dissolved inorganic carbon (DIC) and calcium fell as a result of photosynthesis and calcification. Large gradients in pH, water temperature and irradiance were found within the Chara sward and measurements showed that most growth and photosynthesis occurred within the upper 20 cm of the water column. Chara oospore formation was also found to be seasonal but dependent upon environmental conditions.

δ¹⁸O_w rose rapidly during summer as evaporation progressed and this was correlated with the δ¹⁸O_DIC, and to some extent with δ¹⁸O_c of the Chara encrusted calcite. However, extreme isotopic disequilibrium was observed between the δ¹⁸O_c and the δ¹⁸O_w and also between the δ¹³C_c and the δ¹³C_DIC. This arose from the high pH allowing atmospheric CO₂ to enter the water and combine directly with OH⁻.

It is concluded that, within shallow eutrophic lakes containing Chara swards, inferences of climate (e.g. air temperature) cannot be made from observations of the isotopic composition of Chara carbonates. However in combination with other geochemical data, disequilibrium events may be identifiable in ancient lake basins and taken as evidence for lake shallowing and/or eutrophication.     

LH release by Cu and Ni salts and metal-GnRH complexes, in vitro

The present studies were undertaken to examine the effect of copper and nickel salts and their complexes with GnRH on LH release from the pig anterior pituitary cells in vitro. The potency of Cu-GnRH and Ni-GnRH binding to GnRH receptors with iodinated GnRH as a radioactive tracer was also verified. The incubation of pig pituitary cells with Cu and Ni acetate salts showed no effect of the studied ions on LH release at any concentration used. However, nickel salt at a lower dose (10(-10) and 10(-9) M) tended to decrease LH output. By contrast, the native GnRH as well as its metal complexes significantly stimulated LH release after three hours of treatment and Cu-GnRH was found to be the most effective. The results showed that Cu and Ni complexes with GnRH but not their acetate salts are effective in LH release from pig pituitary cells collected from adult female pigs.     

Mesozooplankton distribution in relation to hydrology of the Northeastern Aegean Sea, Eastern Mediterranean

The Northeastern Aegean Sea (NEA) is the area where Black Seawaters (BSW) outflow in the Mediterranean enhancing local productivityand inducing high hydrographic complexity. We describe the structureand distribution (both vertical and horizontal) of mesozooplanktonassemblages in the NEA during the thermal stratification periodin July and September 2003, in an effort to identify BSW effects.The overall standing stocks of mesozooplankton in the NEA weremuch higher than those typically reported for other pelagicwaters of the eastern Mediterranean. Higher abundance and biomassvalues and a distinctive copepod and cladoceran species assemblagewere recorded in the surface layer (directly influenced by theBSW). Variability in the supply and subsequent advection ofBSW in the NEA seemed to be the major factor affecting the structureand distribution of mesozooplankton assemblages. In July, lowersurface salinity reflected the increased supply of BSW (richin organic carbon) and, consequently, the abundance of filterfeeding taxa (cladocerans, appendicularians and doliolids) washigher than in September. During both surveys, the copepod/cladoceranassemblages of the surface (0–50 m) layer were sensitivetracers of horizontal hydrological and biological variability(e.g. location of fronts) associated with the advection of theBSW. This paper was presented at Plankton Symposium III, held atFiguera da Foz, Portugal between 17 and 20 March 2005, underthe auspices of the University of Coimbra and the Universityof Aveiro, and coordinated by Mário Jorge Pereira andUlisses M. Azeiteiro.