Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.     

Monitoring proton dissociation and solution conformation of chiral ytterbium complexes with near-IR CD

The ytterbium complex [Yb((S)-THP)](3+) ((S)-THP = (1S,4S,7S,10S-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane) is investigated in solution through NMR, near-IR absorption, and CD spectroscopy. Quantitative analysis of the paramagnetic pseudocontact NMR shift shows Lambda helicity of the ligand cage around the metal. The NIR CD spectrum recorded at acidic pH is found to be very similar to that of [Yb((R)-DOTMA)](-) ((R)-DOTMA = (1R,4R,7R,10R)-alpha,alpha',alpha',alpha'-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), which in solution assumes a twisted square antiprism (TSA) conformation. The similarity of the NIR CD spectra is discussed, and it is the first proof of the Lambda(lambda,lambda,lambda,lambda) conformation of [Yb((S)-THP)](3+). NIR CD spectra recorded in the pH range of 2-9 allow one to easily follow proton dissociation and to calculate the pK of this equilibrium in water (pK(A) = 6.4 +/- 0.1). This value agrees well with that determined for [Lu((S)-THP)](3+) using potentiometric methods. This demonstrates once again that NIR CD spectroscopy is a powerful technique for investigating the solution structure and dynamics of these complexes.     

Hypoglycosylation with increased fucosylation and branching of serum transferrin N-glycans in untreated galactosemia

Untreated classic galactosemia (galactose-1-phosphate uridyltransferase [GALT] deficiency) is known as a secondary congenital disorders of glycosylation (CDG) characterized by galactose deficiency of glycoproteins and glycolipids (processing defect or CDG-II). The mechanism of this undergalactosylation has not been established. Here we show that in untreated galactosemia, there is also a partial deficiency of whole glycans of serum transferrin associated with increased fucosylation and branching as seen in genetic glycosylation assembly defects (CDG-I). Thus galactosemia seems to be a secondary "dual" CDG causing a processing as well as an assembly N-glycosylation defect. We also demonstrated that in galactosemia patients, transferrin N-glycan biosynthesis is restored upon dietary treatment.     

Biological quality of running waters in protected areas: the influence of size and land use

There are still substantial questions about whether protected areas affect the quality and biodiversity of surface waters within their borders. In this study, the size and land use of 19 protected areas of Latium Region (central Italy) were related to the biological quality of 32 streams running inside them. Additionally, the biological quality of 18 out of the 32 streams was compared with the quality recorded on the same streams outside the boundaries of the protected areas. The biological quality was assessed using the Extended Biotic Index, which indicates the macroinvertebrate community health. The quality of 32 study streams running through the protected areas was not related to the size of these areas, but it did reflect land use. On average, the 18 study sites inside protected areas had biological quality similar to external control sites. In the protected areas, the biological quality of streams was higher than for the same streams in the surrounding territory provided that anthropogenic changes were fewer. These data indicate that the creation of protected areas per se does not increase freshwater biodiversity and that land use has a major impact on the biological quality of the stream in a protected area. As a consequence, a higher number of reserves or landscape designations specifically created for aquatic conservation is necessary and recovery programs aimed at restoring physical habitats and reducing sources of impact to aquatic life have to be pursued. Also, where the anthropogenic impact is high (e.g., as in the case of strongly urbanised areas), the creation of effective protected areas might improve the biological quality of water courses.     

Climate change causing phase transitions of walleye pollock (Theragra chalcogramma) recruitment dynamics

In 1976 the North Pacific climate shifted, resulting in an average increase of the water temperature. In the Gulf of Alaska the climate shift was followed (i.e. early 1980s) by a gradual but dramatic increase in the abundance of groundfish species that typically prey on pre-recruitment stages of walleye pollock. In the present study we used a previously parameterized model to investigate the effect of these climate and biological changes on the recruitment dynamics of walleye pollock in the Gulf of Alaska. Simulations covered the 1970-2000 time frame and emphasized the medium-to-long temporal scale (i.e. about 5-10 years) of environmental variability. Results showed that during periods characterized by high sea surface temperature and high predation on juvenile pollock stages, recruitment variability and magnitude were below average, and recruitment control was delayed to stages older than the 0-group. Opposite dynamics (i.e. high abundance and variability, and early recruitment control) occurred during periods characterized by low temperature and predation. These results are in general agreement with empirical observations, and allowed us to formulate causal explanations for their occurrence. We interpreted the delay of recruitment control and the reduction of variability as an effect of increased constraint on the abundance of post age-0 stages, in turn imposed by high density dependence and predation mortality. On the other hand, low density-dependence and predation favoured post age-0 survival, and allowed for an unconstrained link between larval and recruitment abundance. Our findings demonstrate that the dominant mechanisms of pollock survival change over contrasting climate regimes. Such changes may in turn cause a phase transition of recruitment dynamics with profound implications for the management of the entire stock.     

Determination of allele frequency in pooled DNA: comparison of three PCR-based methods

Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).     

Relationships between familial risks of cancer and the effects of heritable genes and their SNP variants

Familial risks for cancer can be used in many ways in guiding gene identification efforts and, more broadly, in understanding cancer etiology. Gene identification efforts may be properly designed and targeted if the familial risks are well characterized and the mode of inheritance is identified. Single nucleotide polymorphisms (SNPs) are extensively used in case-control studies of practically all cancer types. They are used for the identification of inherited cancer susceptibility genes and those that may interact with environmental factors. However, being genetic markers, they are applicable only on heritable conditions, which is often a neglected fact. Based on the data in the nationwide Swedish Family-Cancer Database, we review familial risks for all main cancers and discuss the evidence for a heritable component in cancer. The available evidence, including differences in cancer incidence between regions and temporal changes within regions, indicates that cancer is mainly an environmental disease, with a minor heritable etiology. The large environmental component will hamper the success of SNP-based genetic association studies. Empirical familial risks should be used to evaluate the feasibility of such studies. We develop figures for the assessment of genetic parameters based on familial risks. Such data are helpful in the estimation of the expected genetic effects in cancer. Overall, we consider the likelihood of a successful application of SNPs in gene-environment studies small, unless established environmental risk factors are tested on proven candidate genes.     

Molecular and functional characterization of NKG2D, NKp80, and NKG2C triggering NK cell receptors in rhesus and cynomolgus macaques: monitoring of NK cell function during simian HIV infection

An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.