Dimerization of endogenous MT1-MMP is a regulatory step in the activation of the 72-kDa gelatinase MMP-2 on fibroblasts and fibrosarcoma cells

The secreted gelatinase matrix metalloprotease-2 (MMP-2) and the membrane-anchored matrix metalloprotease MT1-MMP (MMP-14), are central players in pericellular proteolysis in extracellular matrix degradation. In addition to possessing a direct collagenolytic and gelatinolytic activity, these enzymes take part in a cascade pathway in which MT1-MMP activates the MMP-2 proenzyme. This reaction occurs in an interplay with the matrix metalloprotease inhibitor, TIMP-2, and the proposed mechanism involves two molecules of MT1-MMP in complex with one TIMP-2 molecule. We provide positive evidence that proMMP-2 activation is governed by dimerization of MT1-MMP on the surface of fibroblasts and fibrosarcoma cells. Even in the absence of transfection and overexpression, dimerization of MT1-MMP markedly stimulated the formation of active MMP-2 products. The effect demonstrated here was brought about by a monoclonal antibody that binds specifically to MT1-MMP as shown by immunofluorescence experiments. The antibody has no effect on the catalytic activity. The effect on proMMP-2 activation involves MT1-MMP dimerization because it requires the divalent monoclonal antibody, with no effect obtained with monovalent Fab fragments. Since only a negligible level of proMMP-2 activation was obtained with MT1-MMP-expressing cells in the absence of dimerization, our results identify the dimerization event as a critical level of proteolytic cascade regulation.     

Oxygen isotope fractionation between human phosphate and water revisited

The oxygen isotope composition of human phosphatic tissues (δ¹⁸O_P) has great potential for reconstructing climate and population migration, but this technique has not been applied to early human evolution. To facilitate this application we analyzed δ¹⁸O_P values of modern human teeth collected at 12 sites located at latitudes ranging from 4°N to 70°N together with the corresponding oxygen composition of tap waters (δ¹⁸O_W) from these areas. In addition, the δ¹⁸O of some raw and boiled foods were determined and simple mass balance calculations were performed to investigate the impact of solid food consumption on the oxygen isotope composition of the total ingested water (drinking water + solid food water). The results, along with those from three, smaller published data sets, can be considered as random estimates of a unique δ¹⁸O_W/δ¹⁸O_P linear relationship: δ¹⁸O_W = 1.54(±0.09) × δ¹⁸O_P−33.72(±1.51) (R² = 0.87: p [H₀:R² = 0] = 2 × 10⁻¹⁹). The δ¹⁸O of cooked food is higher than that of the drinking water. As a consequence, in a modern diet the δ¹⁸O of ingested water is +1.05 to 1.2‰ higher than that of drinking water in the area. In meat-dominated and cereal-free diets, which may have been the diets of some of our early ancestors, the shift is a little higher and the application of the regression equation would slightly overestimate δ¹⁸O_W in these cases.     

The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells

Pluripotent stem cell lines can be derived from blastocyst embryos, which yield embryonic stem cell lines (ES cells), as well as the postimplantation epiblast, which gives rise to epiblast stem cell lines (EpiSCs). Remarkably, ES cells and EpiSCs display profound differences in the combination of growth factors that maintain their pluripotent state. Molecular and functional differences between these two stem cell types demonstrate that the tissue of origin and/or the growth factor milieu may be important determinants of the stem cell identity. We explored how developmental stage of the tissue of origin and culture growth factor conditions affect the stem cell pluripotent state. Our findings indicate that novel stem cell lines, with unique functional and molecular properties, can be generated from murine blastocyst embryos. We demonstrate that the culture growth factor environment and cell-cell interaction play a critical role in defining several unique and stable stem cell ground states.     

A protein domain-based interactome network for C. elegans early embryogenesis

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.     

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities?

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sinapis phylogeny and evolution of glucosinolates and specific nitrile degrading enzymes

Levels of sinalbin (4-hydroxybenzylglucosinolate) and 28 other glucosinolates were determined in leaves and roots of 20 species that were either phylogenetically close to Sinapis alba, Sinapis arvensis, or Sinapis pubescens (tribe Brassiceae, Brassicaceae), or were expected to contain arylalkyl nitrilase activity. Comparison with a molecular phylogenetic tree based on ITS DNA sequences identified two separate occurrences of sinalbin. The first in a group of species related to S. alba (including members of the genera Coincya and Kremeriella); and the second in S. arvensis, nested among sinalbin deficient species. Significant 4-hydroxyphenylacetonitrile degrading enzyme activity was found in both S. alba and S. arvensis, but in S. alba the major product was the corresponding carboxylic acid, while in S. arvensis the major product was the amide. Both investigated enzyme activities, nitrilase and nitrile hydratase, were specific, accepting only certain arylacetonitriles such as 4-hydroxy and 4-methoxyphenylacetonitrile. Only the S. alba enzyme required an oxygen in para position of the substrate, as found in sinalbin. Indole-3-acetonitrile, arylcyanides, and arylpropionitriles were poor substrates. The nitrilase activity of S. alba was quantitatively comparable to that reported in the monocot Sorghum bicolor (believed to be involved in cyanogenic glycoside metabolism). Glucosinolates derived from methionine were found in all Sinapis clades. Glucosinolate patterns suggested a complex evolution of glucosinolates in the investigated species, with several apparent examples of abrupt changes in glucosinolate profiles including chain length variation and appearance of glucosinolates derived from branched-chain amino acids. NMR data for desulfated homosinalbin, 9-methylsulphonylnonylglucosinolate, 3-methylpentylglucosinolate and related glucosinolates are reported, and a facultative connection between sinalbin and specific nitrilases is suggested.     

Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years

Background

Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles.

Methodology/Principal Findings

We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a “clean- laboratory” dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the “unhandled” teeth and there was no indication of contamination, while the latter was the case with half of the “handled” teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (<2% in modern Scandinavians) supports our previous findings of a pronounced frequency of this haplogroup in Viking and Iron Age Danes.

Conclusion

The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate precautions are taken and well-considered sampling is applied.     

Radiocarbon dating of the human eye lens crystallines reveal proteins without carbon turnover throughout life

Background

Lens crystallines are special proteins in the eye lens. Because the epithelial basement membrane (lens capsule) completely encloses the lens, desquamation of aging cells is impossible, and due to the complete absence of blood vessels or transport of metabolites in this area, there is no subsequent remodelling of these fibers, nor removal of degraded lens fibers. Human tissue ultimately derives its ¹⁴C content from the atmospheric carbon dioxide. The ¹⁴C content of the lens proteins thus reflects the atmospheric content of ¹⁴C when the lens crystallines were formed. Precise radiocarbon dating is made possible by comparing the ¹⁴C content of the lens crystallines to the so-called bomb pulse, i.e. a plot of the atmospheric ¹⁴C content since the Second World War, when there was a significant increase due to nuclear-bomb testing. Since the change in concentration is significant even on a yearly basis this allows very accurate dating.

Methodology/Principal Findings

Our results allow us to conclude that the crystalline formation in the lens nucleus almost entirely takes place around the time of birth, with a very small, and decreasing, continuous formation throughout life. The close relationship may be further expressed as a mathematical model, which takes into account the timing of the crystalline formation.

Conclusions/Significance

Such a life-long permanence of human tissue has hitherto only been described for dental enamel. In confront to dental enamel it must be held in mind that the eye lens is a soft structure, subjected to almost continuous deformation, due to lens accommodation, yet its most important constituent, the lens crystalline, is never subject to turnover or remodelling once formed. The determination of the ¹⁴C content of various tissues may be used to assess turnover rates and degree of substitution (for example for brain cell DNA). Potential targets may be nervous tissues in terms of senile or pre-senile degradation, as well as other highly specialised structures of the eyes. The precision with which the year of birth may be calculated points to forensic uses of this technique.     

Greater temperature sensitivity of plant phenology at colder sites: implications for convergence across northern latitudes

Warmer temperatures are accelerating the phenology of organisms around the world. Temperature sensitivity of phenology might be greater in colder, higher latitude sites than in warmer regions, in part because small changes in temperature constitute greater relative changes in thermal balance at colder sites. To test this hypothesis, we examined up to 20 years of phenology data for 47 tundra plant species at 18 high‐latitude sites along a climatic gradient. Across all species, the timing of leaf emergence and flowering was more sensitive to a given increase in summer temperature at colder than warmer high‐latitude locations. A similar pattern was seen over time for the flowering phenology of a widespread species, Cassiope tetragona. These are among the first results highlighting differential phenological responses of plants across a climatic gradient and suggest the possibility of convergence in flowering times and therefore an increase in gene flow across latitudes as the climate warms.