In situ bioassessment of dredging and disposal activities in a contaminated ecosystem: Toronto Harbour

The contamination of Toronto Harbour is a very serious problem. The major sources of pollution are the Don River and sewer outflows, as well as industrial, and municipal effluents. The problem is further compounded by perturbations of the toxic sediment caused by dredging, dredge-disposal, navigation, and recreational activities. The impact of contamination and nutrient enrichment was reflected in the size-fractionated primary productivity experiments. Generally, microplankton/netplankton (> 20 µm) productivity was enhanced whereas ultraplankton (< 20 µm) productivity was inhibited. These observations are attributable to interactions between ameliorating nutrients and toxic contaminants as well as to the differential sensitivity of natural phytoplankton size assemblages to the bioavailable chemical regime. In situ environmental techniques applied in Toronto Harbour were effective, sensitive, and rapid, and provided a better understanding of the impact of dredging/disposal activities under natural conditions. These techniques have great potential in the assessment of the ecotoxicology of harbours and other stressed environments.     

Myogenic stem cell commitment probability remains constant as a function of organismal and mitotic age

Chicken myogenic stem cells can undergo symmetric and asymmetric cell divisions. Symmetric divisions produce two stem cells or two cells committed to terminal muscle differentiation. Asymmetric divisions produce one stem cell and one committed cell. Committed cells undergo four divisions, and their progeny differentiate into postmitotic, biochemically distinct muscle cells, which can be identified immunocytochemically. The control of stem cell commitment was investigated in vitro by means of cell cloning and subcloning experiments, and computer modeling. We found that stem cell commitment is a process which can be modeled as a stochastic event, with a central tendency or probability of 0.2 +/- 0.1. This value is independent of organismal or mitotic age of the stem cells, cell density, or growth in a mitogen-poor environment. Myogenic stem cells stop dividing after approximately 30 divisions in vitro. Since the probability of commitment to terminal differentiation remains below 0.5, clonal senescence and terminal differentiation are separate processes in this system.     

Measures of association between disease and genotype.

This paper is concerned with the statistical aspects of the phenomenon of disease occurring more frequently in individuals with some genotypes than in individuals with others. A correlation coefficient is defined to quantify association between disease and genotype. A distinction is made between the concepts of independence of allele and disease and independence of genotype and disease. This distinction is used to define two components of association which describe separate aspects of association of disease with genotype. One component is a measure of the association of disease with allele; the other a measure of the effect of allele interaction on association of disease and genotype. One aspect of the usefulness of the partition into components which is discussed is in expressing the recurrence risk of disease for a relative of an affected individual. A chi-squared analysis is provided to test hypotheses about the components of association and other hypotheses of genetic interest. This analysis is illustrated using a study done to determine the effect of the sex-linked dwarfing gene in male chickens on resistance to E. coli infection. This analysis shows a significant allele interaction effect on resistance to disease but no association of disease with alleles. In conclusion, some extensions and limitations of the proposed concepts and procedures are discussed.     

Variegated translocation mosaicism in human skin fibroblast cultures.

The term "variegated translocation mosaicism" is used to describe the repeated occurrence, within cultures of human skin fibroblasts, of a multiplicity of chromosomal rearrangements. With respect to the frequencies of such cytogenetically aberrant clones we found that they (1) were not detectable in routine diagnostic skin fibroblast cultures from 29 subjects with a wide variety of indications for biopsy; (2) were not detectable during in vitro aging of diploid strains with four normal individuals; (3) could be detected after rescue from bacterial contamination of a culture from an otherwise normal diploid male; (4) occurred with high frequencies in independent cultures from another apparently normal subject; (5) occurred with high frequencies in multiple biopsies obtained at autopsy from a patient with Werner's syndrome who died of sepsis; (6) were of pseudodiploid nature; and (7) involved a different spectrum of chromosomes in different individuals. A consistent association with mycoplasma contamination could not be made.     

A novel human gene encoding HECT domain and RCC1-like repeats interacts with cyclins and is potentially regulated by the tumor suppressor proteins

Cyclin E-Cdk2 is an evolutionary conserved cyclin-dependent kinase (CDK) complex that drives the G1 to S phase transition of the cell cycle. A novel cDNA encoding a HECT family protein also containing RCC1-like repeats was isolated by a yeast two-hybrid screening using both cyclin E and its inhibitor p21. The protein product of this cDNA, Ceb1, interacts with various cyclin subunits of CDKs in mammalian cells. Expression of Ceb1 is specifically detected in testis and ovary and is highly elevated when the functions of the tumor suppressor proteins, p53 and RB, are compromised by mutations or viral oncoproteins. The present results suggest that Ceb1 may play a critical role when its expression and the CDK activity are upregulated by inactivation of p53 and RB.     

An atomic model for actin binding by the CH domains and spectrin-repeat modules of utrophin and dystrophin

Utrophin and dystrophin link cytoskeletal F-actin filaments to the plasmalemma. Genetic strategies to replace defective dystrophin with utrophin in individuals with muscular dystrophy requires full characterization of these proteins. Both contain homologous N-terminal actin-binding motifs composed of a pair of calponin-homology (CH) domains (CH1 and CH2) that are connected by spectrin-repeat modules to C-terminal membrane-binding sequences. Here, electron microscopy and 3D reconstruction of F-actin decorated with utrophin and dystrophin actin-binding constructs were performed using Utr261 (utrophin's CH domain pair), Utr416 (utrophin's CH domains and first spectrin-repeat) and Dys246 (dystrophin's CH domain pair). The lozenge-like utrophin CH domain densities localized to the upper surface of actin subdomain 1 and extended azimuthally over subdomain 2 toward subdomains 3 and 4. The cylinder-shaped spectrin-repeat was located at the end of the CH domain pair and was aligned longitudinally along the cleft between inner and outer actin domains, where tropomyosin is present when on thin filaments. The connection between the spectrin-repeat module and the CH domains defined the orientation of CH1 and CH2 on actin. Resolution of utrophin's CH domains and spectrin-repeats permitted docking of crystal structures into respective EM densities, leading to an atomic model where both CH and spectrin-domains bind actin. The CH domain-actin interaction for dystrophin was found to be more complex than for utrophin. Binding assays showed that Utr261 and Utr416 interacted with F-actin as monomers, whereas Dys246 appeared to associate as a dimer, consistent with a bilobed Dys246 structure observed on F-actin in electron microscope reconstructions. One of the lobes was similar in shape, position and orientation to the monomeric CH domains of Utr261, while the other lobe apparently represented a second set of CH domains in the dimeric Dys246. The extensive contact made by dystrophin on actin may be used in vivo to help muscles dissipate mechanical stress from the contractile apparatus to the extracellular matrix.     

Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains approximately 10% by weight of soluble transition metals, was added (0-200 microg/ml) to BALF or sELF and exposed to 20% (18)O(2) (24 degrees C, 4 h). Oxygen incorporation was quantified as excess (18)O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.     

Store-operated calcium entry and increased endothelial cell permeability

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.     

Structure and Membrane Binding Properties of the Endosomal Tetratricopeptide Repeat (TPR) Domain-containing Sorting Nexins SNX20 and SNX21

Sorting nexins (SNX) orchestrate membrane trafficking and signaling events required for the proper distribution of proteins within the endosomal network. Their phox homology (PX) domain acts as a phosphoinositide (PI) recognition module that targets them to specific endocytic membrane domains. The modularity of SNX proteins confers a wide variety of functions from signaling to membrane deformation and cargo binding, and many SNXs are crucial modulators of endosome dynamics and are involved in a myriad of physiological and pathological processes such as neurodegenerative diseases, cancer, and inflammation. Here, we have studied the poorly characterized SNX20 and its paralogue SNX21, which contain an N-terminal PX domain and a C-terminal PX-associated B (PXB) domain of unknown function. The two proteins share similar PI-binding properties and are recruited to early endosomal compartments by their PX domain. The crystal structure of the SNX21 PXB domain reveals a tetratricopeptide repeat (TPR)-fold, a module that typically binds short peptide motifs, with three TPR α-helical repeats. However, the C-terminal capping helix adopts a highly unusual and potentially self-inhibitory topology. SAXS solution structures of SNX20 and SNX21 show that these proteins adopt a compact globular architecture, and membrane interaction analyses indicate the presence of overlapping PI-binding sites that may regulate their intracellular localization. This study provides the first structural analysis of this poorly characterized subfamily of SNX proteins, highlighting a likely role as endosome-associated scaffolds.