Population Genetic Structure of the Yangtze Finless Porpoise (Neophocaena phocaenoides asiaeorientalis): Implications for Management and Conservation

Understanding the population genetic structure is a prerequisite for conservation of a species. The degree of genetic variability characteristic of the mitochondrial DNA control region has been widely exploited in studies of population genetic structure and can be useful in identifying meaningful population subdivisions. To estimate the genetic profile of the Yangtze finless porpoise (Neophocaena phocaenoides asiaeorientalis), an endangered freshwater population endemic to China, the complete mtDNA control region was examined in 39 individuals belonging to seven different stocks inhabiting the middle and lower reaches of the Yangtze River. Very low genetic diversity was found (nucleotide diversity 0.0011± 0.0002 and haplotypic diversity 0.65± 0.05). The mtDNA genetic pattern of the Yangtze population appears to indicate a founder event in its evolutionary history and to support the marine origin for this population. Analyses by F_st and Φ_st yielded statistically significant population genetic structure (F_st = 0.44, P < 0.05; Φ_st = 0.36, P < 0.05). These results may have significant implications for the management and conservation of the Yangtze finless porpoise in the future.     

The eag potassium channel binds and locally activates calcium/calmodulin-dependent protein kinase II

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of neuronal excitability in many systems. Recent studies suggest that local regulation of membrane potential can have important computational consequences for neuronal function. In Drosophila, CaMKII regulates the eag potassium channel, but if and how this regulation was spatially restricted was unknown. Using coimmunoprecipitation from head extracts and in vitro binding assays, we show that CaMKII and Eag form a stable complex and that association with Eag activates CaMKII independently of CaM and autophosphorylation. Ca(2+)/CaM is necessary to initiate binding of CaMKII to Eag but not to sustain association because binding persists when CaM is removed. The Eag CaMKII-binding domain has homology to the CaMKII autoregulatory region, and the constitutively active CaMKII mutant, T287D, binds Eag Ca(2+)-independently in vitro and in vivo. These results favor a model in which the CaMKII-binding domain of Eag displaces the CaMKII autoinhibitory region. Displacement results in autophosphorylation-independent activation of CaMKII which persists even when Ca(2+) levels have gone down. Activity-dependent binding to this potassium channel substrate allows CaMKII to remain locally active even when Ca(2+) levels have dropped, providing a novel mechanism by which CaMKII can regulate excitability locally over long time scales.     

Lipoxygenase-catalyzed polymerization of phenolic lipids suggests a new mechanism for allergic contact dermatitis induced by urushiol and its analogs

Lipoxygenase was found to catalyze the oxidative polymerization of phenolic lipids containing a (Z,Z)-pentadiene in the side chain, the model compounds of urushiol and its analog, yielding methanol-soluble and insoluble polymers. The structural analysis of the resulted polymers suggested that the polymerization occurred at both the phenol and the unsaturated side chain. The key step of the polymerization was the generation of the hydroperoxide at the unsaturated side chain by lipoxygenase. The decomposition of hydroperoxide and concomitant dehydrogenation of phenol ring catalyzed by lipoxygenase might produce radicals that could be coupled to form cross-linked polymers. This lipoxygenase-mediated reaction implies a new mechanism for contact allergy of urushiol and its analogs.     

A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C-->T transition at -1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 -1306 C-->T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055-3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT+TT genotypes. Our data suggest that MMP-2 -1306 C-->T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.     

Structure--activity relationships of hainantoxin-IV and structure determination of active and inactive sodium channel blockers

Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The sequence of native HNTX-IV is ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCKYEI-NH(2). In the present study, to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude. (1)H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore, in the determined structures of K27A and R29A, the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV. These results suggested that residues Ser(12), Arg(26), Lys(27), and Arg(29) were not responsible for stabilizing the distinct conformation of HNTX-IV, but Lys(27) and Arg(29) were critical for the bioactivities. The potency reductions produced by Ala substitutions were primarily due to the direct interaction of the essential residues Lys(27) and Arg(29) with sodium channels rather than to a conformational change. After comparison of these structures and activities with correlated toxins, we hypothesized that residues Lys(27), Arg(29), His(28), Lys(32), Phe(5), and Trp(30) clustered on one face of HNTX-IV were responsible for ligand binding.     

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.     

TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells

TL1A, a recently described TNF-like cytokine that interacts with DR3, costimulates T cells and augments anti-CD3 plus anti-CD28 IFN-gamma production. In the current study we show that TL1A or an agonistic anti-DR3 mAb synergize with IL-12/IL-18 to augment IFN-gamma production in human peripheral blood T cells and NK cells. TL1A also enhanced IFN-gamma production by IL-12/IL-18 stimulated CD56(+) T cells. When expressed as fold change, the synergistic effect of TL1A on cytokine-induced IFN-gamma production was more pronounced on CD4(+) and CD8(+) T cells than on CD56(+) T cells or NK cells. Intracellular cytokine staining showed that TL1A significantly enhanced both the percentage and the mean fluorescence intensity of IFN-gamma-producing T cells in response to IL-12/IL-18. The combination of IL-12 and IL-18 markedly up-regulated DR3 expression in NK cells, whereas it had minimal effect in T cells. Our data suggest that TL1A/DR3 pathway plays an important role in the augmentation of cytokine-induced IFN-gamma production in T cells and that DR3 expression is differentially regulated by IL-12/IL-18 in T cells and NK cells.