New insights into the size and stoichiometry of the plasminogen activator inhibitor type-1.vitronectin complex

Plasminogen activator inhibitor-type 1 (PAI-1) is the primary inhibitor of endogenous plasminogen activators that generate plasmin in the vicinity of a thrombus to initiate thrombolysis, or in the pericellular region of cells to facilitate migration and/or tissue remodeling. It has been shown that the physiologically relevant form of PAI-1 is in a complex with the abundant plasma glycoprotein, vitronectin. The interaction between vitronectin and PAI-1 is important for stabilizing the inhibitor in a reactive conformation. Although the complex is clearly significant, information is vague regarding the composition of the complex and consequences of its formation on the distribution and activity of vitronectin in vivo. Most studies have assumed a 1:1 interaction between the two proteins, but this has not been demonstrated experimentally and is a matter of some controversy since more than one PAI-1-binding site has been proposed within the sequence of vitronectin. To address this issue, competition studies using monoclonal antibodies specific for separate epitopes confirmed that the two distinct PAI-1-binding sites present on vitronectin can be occupied simultaneously. Analytical ultracentrifugation was used also for a rigorous analysis of the composition and sizes of complexes formed from purified vitronectin and PAI-1. The predominant associating species observed was high in molecular weight (M(r) approximately 320,000), demonstrating that self-association of vitronectin occurs upon interaction with PAI-1. Moreover, the size of this higher order complex indicates that two molecules of PAI-1 bind per vitronectin molecule. Binding of PAI-1 to vitronectin and association into higher order complexes is proposed to facilitate interaction with macromolecules on surfaces.     

Domains I and III of the human copper chaperone for superoxide dismutase interact via a cysteine-bridged Dicopper(I) cluster

Copper binding to the human copper chaperone for superoxide dismutase (hCCS) has been investigated by X-ray absorption spectroscopy. Stoichiometry measurements on the dialyzed, as-isolated protein indicated that up to 3.5 Cu ions bound per hCCS molecule. Reduction with either sodium dithionite or dithiothreitol decreased the copper binding ratio to 2 coppers per hCCS monomer. Analysis of the as-isolated EXAFS data indicated coordination of Cu by a mixture of S and N backscatterers, suggestive of heterogeneous binding of copper between Cu-cysteine binding sites of domain I or III and copper-histidine SOD1-like metal binding sites of domain II. The best fit was obtained with 1.6 Cu-S (cysteine) at 2.24 A (2sigma(2) = 0.011 A(2)) and 1.1 N (histidine) at 1.98 A (2sigma(2) = 0.005 A(2)). A peak of variable intensity in the Fourier transform (FT) of the as-isolated protein at 2.7 A was suggestive of the presence of a heavy atom scatterer such as Cu. Analysis of the dithionite- and DTT-reduced derivatives indicated that copper was lost from the histidine coordinating sites, resulting in a S-only environment with copper coordinated to three S backscatterers at 2. 26 A. The heavy atom scatterer peak was now prominent in the FT and could be well fit by a Cu-Cu interaction at 2.72 A. The data were best interpreted by a dinuclear mu(2)()-bridged cluster with doubly bridging cysteine ligands similar to the cluster proposed to exist in the cytochrome c oxidase chaperone COX17. Analysis of primary sequence and X-ray structural information on yeast CCS strongly suggests that this cluster bridges between domains I and III in hCCS. A mechanism for copper translocation is briefly discussed.     

Bioremediation Scaleup Effectiveness: A Review

Bioremediation has been applied in laboratory-scale testing to simulate field-scale bioremediation applications. The simulations have focused on the development of concentration-time profile data in the laboratory to be modeled and used for predicting field-scale performance, particularly the time of treatment and the treatment concentration endpoints. This review reports on more than a dozen examples of bioremediation where both the laboratory-scale and field-scale data are provided. These data have been analyzed to examine how well the laboratory-scale kinetics predict the kinetics at field scale. In most cases, the laboratory-scale kinetics exceed field-scale kinetics and underpredict the time of treatment to a given endpoint in excess of 100% and by as much as 11,900%. In some cases, the laboratory-scale kinetic rate constants fall between ±100% of the field-scale kinetics, leading to predictions within 50 to 200% of the time of treatment to a given concentration level. Not enough examples nor details within the references exist to permit a detailed explanation of why these differences exist. However, several hypotheses are given in this review as to why scaleup is often problematic, and comments from other investigators on this topic are presented.     

The anatomy of the interspecific abundance–range size relationship for the British avifauna: I. Spatial patterns

Data from the British Trust for Ornithology Common Birds Census and two atlases of breeding birds were used to examine the form of the interspecific abundance–range size relationship for the British avifauna. The relationship is positive for both farmland and woodland habitats and over two different periods, with some evidence of curvilinearity, using either proportion of occupied sites or numbers of occupied 10 × 10 km squares as measures of range size, and mean density at occupied sites as a measure of abundance. A log-linear plot gives the highest correlation. The relationship is stronger if based on maximum local densities than if based on average densities, but there is no relationship using minimum local densities. Relationships based on abundances at individual sites are uniformly positive for all sites, although the relationships for many sites also show evidence of curvilinearity, especially when range size is measured as the proportion of occupied sites. Species show significant concordance in their rank abundances across sites. We discuss some implications of these results.     

Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

Quantitative Profiling of Colorectal Cancer-Associated Bacteria Reveals Associations between Fusobacterium spp., Enterotoxigenic Bacteroides fragilis (ETBF) and Clinicopathological Features of Colorectal Cancer

Various studies have presented clinical or in vitro evidence linking bacteria to colorectal cancer, but these bacteria have not previously been concurrently quantified by qPCR in a single cohort. We quantify these bacteria (Fusobacterium spp., Streptococcus gallolyticus, Enterococcus faecalis, Enterotoxigenic Bacteroides fragilis (ETBF), Enteropathogenic Escherichia coli (EPEC), and afaC- or pks-positive E. coli) in paired tumour and normal tissue samples from 55 colorectal cancer patients. We further investigate the relationship between a) the presence and b) the level of colonisation of each bacterial species with site and stage of disease, age, gender, ethnicity and MSI-status. With the exception of S. gallolyticus, we detected all bacteria profiled here in both tumour and normal samples at varying frequencies. ETBF (FDR = 0.001 and 0.002 for normal and tumour samples) and afaC-positive E. coli (FDR = 0.03, normal samples) were significantly enriched in the colon compared to the rectum. ETBF (FDR = 0.04 and 0.002 for normal and tumour samples, respectively) and Fusobacterium spp. (FDR = 0.03 tumour samples) levels were significantly higher in late stage (III/IV) colorectal cancers. Fusobacterium was by far the most common bacteria detected, occurring in 82% and 81% of paired tumour and normal samples. Fusobacterium was also the only bacterium that was significantly higher in tumour compared to normal samples (p = 6e-5). We also identified significant associations between high-level colonisation by Fusobacterium and MSI-H (FDR = 0.05), age (FDR = 0.03) or pks-positive E. coli (FDR = 0.01). Furthermore, we exclusively identified atypical EPEC in our cohort, which has not been previously reported in association with colorectal cancer. By quantifying colorectal cancer-associated bacteria across a single cohort, we uncovered inter- and intra-individual patterns of colonization not previously recognized, as well as important associations with clinicopathological features, especially in the case of Fusobacterium and ETBF.     

Genetics,Morphology, Advertisement Calls,and Historical Records Distinguish Six New Polyploid Species of African Clawed Frog (Xenopus,Pipidae) from West and Central Africa

African clawed frogs, genus Xenopus, are extraordinary among vertebrates in the diversity of their polyploid species and the high number of independent polyploidization events that occurred during their diversification. Here we update current understanding of the evolutionary history of this group and describe six new species from west and central sub-Saharan Africa, including four tetraploids and two dodecaploids. We provide information on molecular variation, morphology, karyotypes, vocalizations, and estimated geographic ranges, which support the distinctiveness of these new species. We resurrect Xenopus calcaratus from synonymy of Xenopus tropicalis and refer populations from Bioko Island and coastal Cameroon (near Mt. Cameroon) to this species. To facilitate comparisons to the new species, we also provide comments on the type specimens, morphology, and distributions of X. epitropicalis, X. tropicalis, and X. fraseri. This includes significantly restricted application of the names X. fraseri and X. epitropicalis, the first of which we argue is known definitively only from type specimens and possibly one other specimen. Inferring the evolutionary histories of these new species allows refinement of species groups within Xenopus and leads to our recognition of two subgenera (Xenopus and Silurana) and three species groups within the subgenus Xenopus (amieti, laevis, and muelleri species groups).     

Thymus-associated parathyroid hormone has two cellular origins with distinct endocrine and immunological functions

In mammals, parathyroid hormone (PTH) is a key regulator of extracellular calcium and inorganic phosphorus homeostasis. Although the parathyroid glands were thought to be the only source of PTH, extra-parathyroid PTH production in the thymus, which shares a common origin with parathyroids during organogenesis, has been proposed to provide an auxiliary source of PTH, resulting in a higher than expected survival rate for aparathyroid Gcm2 ^−/− mutants. However, the developmental ontogeny and cellular identity of these “thymic” PTH–expressing cells is unknown. We found that the lethality of aparathyroid Gcm2 ^−/− mutants was affected by genetic background without relation to serum PTH levels, suggesting a need to reconsider the physiological function of thymic PTH. We identified two sources of extra-parathyroid PTH in wild-type mice. Incomplete separation of the parathyroid and thymus organs during organogenesis resulted in misplaced, isolated parathyroid cells that were often attached to the thymus; this was the major source of thymic PTH in normal mice. Analysis of thymus and parathyroid organogenesis in human embryos showed a broadly similar result, indicating that these results may provide insight into human parathyroid development. In addition, medullary thymic epithelial cells (mTECs) express PTH in a Gcm2-independent manner that requires TEC differentiation and is consistent with expression as a self-antigen for negative selection. Genetic or surgical removal of the thymus indicated that thymus-derived PTH in Gcm2 ^−/− mutants did not provide auxiliary endocrine function. Our data show conclusively that the thymus does not serve as an auxiliary source of either serum PTH or parathyroid function. We further show that the normal process of parathyroid organogenesis in both mice and humans leads to the generation of multiple small parathyroid clusters in addition to the main parathyroid glands, that are the likely source of physiologically relevant “thymic PTH.”