1.56 Å structure of mature truncated human fibroblast collagenase

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 Å resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded β-sheet and three α-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase. © 1994 Wiley-Liss, Inc.     

Histone acetylation and the deoxyribonuclease I sensitivity of the Tetrahymena ribosomal gene

Under appropriate conditions, up to 8.5% of the total acetate can be removed from the histones of isolated Tetrahymena macronuclei by an endogenous histone deacetylase activity. After in vitro deacetylation, the ribosomal genes are still preferentially digested by DNase I. These observations suggested that either the majority of histone-bound acetate is unnecessary to maintain the DNase I sensitive state or ribosomal chromatin (rChromatin) histones remain acetylated under these conditions. The characteristics of histones acetylation were studied in Tetrahymena rChromatin, which can be isolated in a relatively pure form. Histones associated with the presumably active, DNase I sensitive ribosomal genes have a high steady-state level of histone acetylation which, surprisingly, is maintained by very low acetate turnover rates.     

Roles of two large serine recombinases in mobilizing the methicillin‐resistance cassette SCCmec

Methicillin‐resistant Staphylococcus aureus (MRSA) emerged via acquisition of a mobile element, staphylococcal cassette chromosome mec (SCCmec). Integration and excision of SCCmec is mediated by an unusual site‐specific recombination system. Most variants of SCCmec encode two recombinases, CcrA and CcrB, that belong to the large serine family. Since CcrA and CcrB are always found together, we sought to address their specific roles. We show here that CcrA and CcrB can carry out both excisive and integrative recombination in Escherichia coli in the absence of any host‐specific or SCCmec‐encoded cofactors. CcrA and CcrB are promiscuous in their substrate choice: they act on many non‐canonical pairs of recombination sites in addition to the canonical ones, which may explain tandem insertions into the SCCmec attachment site. Moreover, CcrB is always required, but CcrA is only required if one of the four half‐sites is present. Recombinational activity correlates with DNA binding: CcrA recognizes only that half‐site, which overlaps a conserved coding frame on the host chromosome. Therefore, we propose that CcrA serves as a specificity factor that emerged through modular evolution to enable recognition of a bacterial recombination site that is not an inverted repeat.     

How fast is fast? Eco‐evolutionary dynamics and rates of change in populations and phenotypes

It is increasingly recognized that evolution may occur in ecological time. It is not clear, however, how fast evolution – or phenotypic change more generally – may be in comparison with the associated ecology, or whether systems with fast ecological dynamics generally have relatively fast rates of phenotypic change. We developed a new dataset on standardized rates of change in population size and phenotypic traits for a wide range of species and taxonomic groups. We show that rates of change in phenotypes are generally no more than 2/3, and on average about 1/4, the concurrent rates of change in population size. There was no relationship between rates of population change and rates of phenotypic change across systems. We also found that the variance of both phenotypic and ecological rates increased with the mean across studies following a power law with an exponent of two, while temporal variation in phenotypic rates was lower than in ecological rates. Our results are consistent with the view that ecology and evolution may occur at similar time scales, but clarify that only rarely do populations change as fast in traits as they do in abundance.     

Effect of nitric oxide on neointimal hyperplasia based on sex and hormone status

Nitric oxide (NO)-based therapies decrease neointimal hyperplasia; however, studies have been performed only in male animal models. Thus, we sought to evaluate the effect of NO on vascular smooth muscle cells (VSMC) in vitro and neointimal hyperplasia in vivo based on sex and hormone status. In hormone-replete medium, male VSMC proliferated at greater rates than female VSMC. In hormone-depleted medium, female VSMC proliferated at greater rates than male VSMC. However, in both hormone environments, NO inhibited proliferation and migration to a greater extent in male compared to female VSMC. These findings correlated with greater G₀/G₁ cell cycle arrest and changes in cell cycle protein expression in male compared to female VSMC after exposure to NO. Next, the rat carotid artery injury model was used to assess the effect of NO on neointimal hyperplasia in vivo. Consistent with the in vitro data, NO was significantly more effective at inhibiting neointimal hyperplasia in hormonally intact males compared to females using weight-based dosing. An increased weight-based dose of NO in females was able to achieve efficacy equal to that in males. Surprisingly, NO was less effective at inhibiting neointimal hyperplasia in castrated animals of both sexes. In conclusion, these data suggest that NO inhibits neointimal hyperplasia more effectively in males compared to females and in hormonally intact compared to castrated rats, indicating that the effects of NO in the vasculature may be sex- and hormone-dependent.     

Nitric Oxide Inhibits Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia by Increasing the Ubiquitination and Degradation of UbcH10

Nitric oxide (NO) limits formation of neointimal hyperplasia in animal models of arterial injury in large part by inhibiting vascular smooth muscle cell (VSMC) proliferation through cell cycle arrest. The ubiquitin-conjugating enzyme UbcH10 is responsible for ubiquitinating cell cycle proteins for proper exit from mitosis. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing levels of UbcH10. Western blotting and immunofluorescent staining showed that NO reduced UbcH10 levels in a concentration-dependent manner in VSMC harvested from the abdominal aortas of Sprague-Dawley rats. Treatment with NO or siRNA to UbcH10 decreased both UbcH10 levels and VSMC proliferation (P<0.001), while increasing UbcH10 levels by plasmid transfection or angiotensin II stimulation increased VSMC proliferation to 150% (P=0.008) and 212% (P=0.002) of control, respectively. Immunofluorescent staining of balloon-injured rat carotid arteries showed a ~4-fold increase in UbcH10 levels, which was profoundly decreased following treatment with NO. Western blotting of carotid artery lysates showed no UbcH10 in uninjured vessels, a substantial increase in the injury alone group, and a significant decrease in the injury+NO group (~3-fold reduction versus injury alone). Importantly, in vitro and in vivo, a marked increase in polyubiquitinated UbcH10 was observed in the NO-treated VSMC and carotid arteries, respectively, indicating that NO may be decreasing unmodified UbcH10 levels by increasing its ubiquitination. Central to our hypothesis, we report that NO decreases UbcH10 levels in VSMC in vitro and following arterial injury in vivo in association with increasing polyubiquitinated-UbcH10 levels. These changes in UbcH10 levels correlate with VSMC proliferation and neointimal hyperplasia, making UbcH10 a promising therapeutic target for inhibiting this proliferative disease.     

Predators modify the temperature dependence of life‐history trade‐offs

Although life histories are shaped by temperature and predation, their joint influence on the interdependence of life‐history traits is poorly understood. Shifts in one life‐history trait often necessitate shifts in another—structured in some cases by trade‐offs—leading to differing life‐history strategies among environments. The offspring size–number trade‐off connects three traits whereby a constant reproductive allocation (R) constrains how the number (O) and size (S) of offspring change. Increasing temperature and size‐independent predation decrease size at and time to reproduction which can lower R through reduced time for resource accrual or size‐constrained fecundity. We investigated how O, S, and R in a clonal population of Daphnia magna change across their first three clutches with temperature and size‐independent predation risk. Early in ontogeny, increased temperature moved O and S along a trade‐off curve (constant R) toward fewer larger offspring. Later in ontogeny, increased temperature reduced R in the no‐predator treatment through disproportionate decreases in O relative to S. In the predation treatment, R likewise decreased at warmer temperatures but to a lesser degree and more readily traded off S for O whereby the third clutch showed a constant allocation strategy of O versus S with decreasing R. Ontogenetic shifts in S and O rotated in a counterclockwise fashion as temperature increased and more drastically under risk of predation. These results show that predation risk can alter the temperature dependence of traits and their interactions through trade‐offs.