Characterization of the interaction between the N-terminal extension of human cardiac troponin I and troponin C

The N-terminal extension of cardiac troponin I (TnI) is bisphosphorylated by protein kinase A in response to beta-adrenergic stimulation. How this signal is transmitted between TnI and troponin C (TnC), resulting in accelerated Ca(2+) release, remains unclear. We recently proposed that the unphosphorylated extension interacts with the N-terminal domain of TnC stabilizing Ca(2+) binding and that phosphorylation prevents this interaction. We now use (1)H NMR to study the interactions between several N-terminal fragments of TnI, residues 1-18 (I1-18), residues 1-29 (I1-29), and residues 1-64 (I1-64), and TnC. The shorter fragments provide unambiguous information on the N-terminal regions of TnI that interact with TnC: I1-18 does not bind to TnC whereas the C-terminal region of unphosphorylated I1-29 does bind. Bisphosphorylation greatly weakens this interaction. I1-64 contains the phosphorylatable N-terminal extension and a region that anchors I1-64 to the C-terminal domain of TnC. I1-64 binding to TnC influences NMR signals arising from both domains of TnC, providing evidence that the N-terminal extension of TnI interacts with the N-terminal domain of TnC. TnC binding to I1-64 broadens NMR signals from the side chains of residues immediately C-terminal to the phosphorylation sites. Binding of TnC to bisphosphorylated I1-64 does not broaden these NMR signals to the same extent. Circular dichroism spectra of I1-64 indicate that bisphosphorylation does not produce major secondary structure changes in I1-64. We conclude that bisphosphorylation of cardiac TnI elicits its effects by weakening the interaction between the region of TnI immediately C-terminal to the phosphorylation sites and TnC either directly, due to electrostatic repulsion, or via localized conformational changes.     

Evidence that Early Carboniferous ostracods colonised coastal flood plain brackish water environments

A study of the stable isotope composition (δ¹⁸O, δ¹³C) of biogenic (ostracod, mollusc) and authigenic carbonates in the Ballagan Formation, Lower Carboniferous of Scotland, coupled with evidence from sedimentology and associated fossil fauna and flora, supports the argument that this formation was deposited in a coastal flood plain setting, in brackish (0.5 < 30‰ NaCl) and hypersaline (> 40‰ NaCl) waters, but in the absence of persistent normal marine conditions. The oxygen isotope data from the Ballagan Formation divide into three clusters: a diagenetic field defined by low δ¹⁸O (< − 11‰ VPDB); an intermediary field (δ¹⁸O − 11‰ to − 9‰) composed of a mixture of known primary and secondary (diagenetic) carbonates; and samples within the range of − 9‰ to − 4‰ which, as far as we can ascertain, are largely unaltered. No samples give typical Early Carboniferous δ¹⁸O marine values. Average marine carbonates from Europe have δ¹⁸O between − 4‰ to − 3‰. The Ballagan Formation carbonates were probably deposited in evaporated freshwater and/or brackish water. This conclusion is supported by the presence of evaporites (gypsum, anhydrite, halite pseudomorphs) and common desiccation-cracked mudstone surfaces throughout the Ballagan Formation, suggesting conditions of fluctuating salinity in ephemeral bodies of water. The stable isotope data support the notion that the ostracod assemblages of the Ballagan Formation were colonising brackish water and hypersaline ecologies on a coastal flood plain during the Early Carboniferous, a stage of development that may have encouraged their colonisation of fully non-marine (limnetic) environments during the later Carboniferous. The ostracods include cytherellacean and kloedenellacean species known from marginal marine sites elsewhere, but probably tolerant of brackish water, podocopid species such as ‘Bythocypris’ aequalis that may have been adapted for brackish water settings on coastal flood plains (ephemeral lakes and lagoons), and paraparchitacean-dominated assemblages that may signal harsh (hypersaline or desiccating) environments.     

The 2 micron plasmid purloins the yeast cohesin complex: a mechanism for coupling plasmid partitioning and chromosome segregation?

The yeast 2 micron plasmid achieves high fidelity segregation by coupling its partitioning pathway to that of the chromosomes. Mutations affecting distinct steps of chromosome segregation cause the plasmid to missegregate in tandem with the chromosomes. In the absence of the plasmid stability system, consisting of the Rep1 and Rep2 proteins and the STB DNA, plasmid and chromosome segregations are uncoupled. The Rep proteins, acting in concert, recruit the yeast cohesin complex to the STB locus. The periodicity of cohesin association and dissociation is nearly identical for the plasmid and the chromosomes. The timely disassembly of cohesin is a prerequisite for plasmid segregation. Cohesin-mediated pairing and unpairing likely provides a counting mechanism for evenly partitioning plasmids either in association with or independently of the chromosomes.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

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.     

The development of early host response to Pseudomonas aeruginosa lung infection is critically dependent on myeloid differentiation factor 88 in mice

Toll-like receptors (TLR) induce distinct patterns of host responses through myeloid differentiation factor 88 (MyD88)-dependent and/or -independent pathways, depending on the nature of the pathogen. Pseudomonas aeruginosa is a cause of serious lung infection in immunocompromised individuals and cystic fibrosis patients. The role of the TLR-MyD88 pathway in P. aeruginosa-induced lung infection in vivo was examined in this study. MyD88-/- mice demonstrated an impaired clearance of P. aeruginosa from the lung. Little or no neutrophil recruitment was observed in the airways of MyD88-/- mice following P. aeruginosa lung infection. This observation was associated with a reduced production of inflammatory mediators that affect neutrophil recruitment, including macrophage-inflammatory protein-2, tumor necrosis factor, and interleukin-1beta in the airways of MyD88-/- mice. Similarly, MyD88-/- mice showed inhibited NF-kappaB activation in the lung following P. aeruginosa infection. Interestingly, P. aeruginosa infection induced a 7.5-fold increase of TLR2 mRNA expression in the lungs of MyD88+/+ mice. Furthermore, host responses to P. aeruginosa lung infection in TLR2-/- and TLR4 mutant mice were partially inhibited compared with the responses of respective control mice. Taken together, our results indicate that the MyD88-dependent pathway is essential for the development of early host responses to P. aeruginosa infection, leading to the clearance of this bacterium, and that TLR2 and TLR4 are involved in this process.     

Both estrogen receptor subtypes, ERα and ERβ, prevent aldosterone-induced oxidative stress in VSMC via increased NADPH bioavailability

Activation of vascular mineralocorticoid (MR) or estrogen receptors (ER) exerts opposing effects on vascular remodeling. As we have previously shown, activation of either estrogen receptor subtype, ERα or ERβ, is fully sufficient to attenuate vascular remodeling in aldosterone salt-treated rats. To further elucidate the underlying mechanism(s) we tested the hypothesis that ER and MR activation might differentially modulate vascular reactive oxygen species (ROS) generation. In support of this concept, aldosterone increased ROS generation in vascular smooth muscle cells as determined by quantitative dihydroethidium fluorescence microscopy. Co-treatment with the selective ERα agonist 16α-LE2, the selective ERβ agonist 8β-VE2 or the non-selective ER agonist 17β-estradiol (E2) significantly reduced aldosterone-induced ROS generation. The pure ER antagonist ICI 182,780 completely blocked these salutary effects of E2, 16α-LE2 and 8β-VE2. Activation of ERα or ERβ fully blocked the reduction of intracellular nicotinamide adenine dinucleotide phosphate (NADPH) levels observed in aldosterone treated vascular smooth muscle cells. Intracellular NADPH levels were closely associated with expression and activity of the NADPH generating enzyme glucose-6-phosphate dehydrogenase. In conclusion, estrogens attenuate the detrimental vascular effects of excessive MR activation at least in part by preventing the depletion of intracellular NADPH levels.     

Growth and Maximum Size of Tiger Sharks (Galeocerdo cuvier) in Hawaii

Tiger sharks (Galecerdo cuvier) are apex predators characterized by their broad diet, large size and rapid growth. Tiger shark maximum size is typically between 380 & 450 cm Total Length (TL), with a few individuals reaching 550 cm TL, but the maximum size of tiger sharks in Hawaii waters remains uncertain. A previous study suggested tiger sharks grow rather slowly in Hawaii compared to other regions, but this may have been an artifact of the method used to estimate growth (unvalidated vertebral ring counts) compounded by small sample size and narrow size range. Since 1993, the University of Hawaii has conducted a research program aimed at elucidating tiger shark biology, and to date 420 tiger sharks have been tagged and 50 recaptured. All recaptures were from Hawaii except a single shark recaptured off Isla Jacques Cousteau (24°13′17″N 109°52′14″W), in the southern Gulf of California (minimum distance between tag and recapture sites  =  approximately 5,000 km), after 366 days at liberty (DAL). We used these empirical mark-recapture data to estimate growth rates and maximum size for tiger sharks in Hawaii. We found that tiger sharks in Hawaii grow twice as fast as previously thought, on average reaching 340 cm TL by age 5, and attaining a maximum size of 403 cm TL. Our model indicates the fastest growing individuals attain 400 cm TL by age 5, and the largest reach a maximum size of 444 cm TL. The largest shark captured during our study was 464 cm TL but individuals >450 cm TL were extremely rare (0.005% of sharks captured). We conclude that tiger shark growth rates and maximum sizes in Hawaii are generally consistent with those in other regions, and hypothesize that a broad diet may help them to achieve this rapid growth by maximizing prey consumption rates.