Contraceptive efficacy of a novel intrauterine device (IUD) in white-tailed deer

Overabundant white-tailed deer (Odocoileus virginianus) pose risks to property, health, and safety of human beings. Public concerns about lethal management can impair efforts to address these issues, particularly in urban settings. Several techniques developed for reducing reproductive output of deer have limited utility because they require repeated dosing to achieve permanent effect and face uncertain regulatory approval for use beyond experimentation. From 10 August 2006 through 30 December 2007, we evaluated the contraceptive efficacy of copper-containing intrauterine devices (IUDs) implanted trans-cervically in white-tailed deer at the E.S. George Reserve in Pinckney, Michigan. Intrauterine devices were implanted before (n = 9) and shortly after (n = 10) the breeding season. Post-breeding season IUD treatment was in conjunction with a 5 cm³ dose of 5 mg/ml prostaglandin F_2α (PGF_2α), delivered subcutaneously. Intrauterine devices reduced pregnancy rates when administered prior to breeding (P < 0.001) and prevented pregnancy for up to 2 years (the duration of the study). Two of 8 does that received IUDs prior to the breeding season and survived to the end of the study became pregnant (due to loss of the implant) during the second year while all (n = 16) does without implants conceived. Cervical changes associated with early pregnancy made trans-cervical implantation after the breeding season challenging, and resulted in improperly placed IUDs in 2 treated does. The apparent expulsion of IUDs by pregnant does that received the combined treatment after breeding suggests IUD treatment should be limited to the pre-breeding season. Intrauterine devices show potential as a tool for small-scale deer population management via non-steroidal reproductive inhibition.     

Limitations of Augmentation Index in the Assessment of Wave Reflection in Normotensive Healthy Individuals

Objectives

Augmentation index (AIx) is widely used as a measure of wave reflection. We compared the relationship between AIx and age, height and sex with ‘gold standard’ measures of wave reflection derived from measurements of pressure and flow to establish how well AIx measures wave reflection.

Materials and Methods

Measurements of carotid pressure and flow velocity were made in the carotid artery of 65 healthy normotensive individuals (age 21–78 yr; 43 male) and pulse wave analysis, wave intensity analysis and wave separation was performed; waveforms were classified into type A, B or C. AIx, the time of the first shoulder (T_s), wave reflection index (WRI) and the ratio of backward to forward pressure (P_b/P_f) were calculated.

Results

AIx did not correlate with log WRI or P_b/P_f. When AIx was restricted to positive values AIx and log WRI were positively correlated (r = 0.33; p = 0.04). In contrast log WRI and P_b/P_f were closely correlated (r = 0.66; p<0.001). There was no correlation between the T_s and the timing of P_b or the reflected wave identified by wave intensity analysis. Wave intensity analysis showed that the morphology of type C waveforms (negative AIx) was principally due to a forward travelling (re-reflected) decompression wave in mid-systole. AIx correlated positively with age, inversely with height and was higher in women. In contrast log WRI and P_b/P_f showed negative associations with age, were unrelated to height and did not differ significantly by gender.

Conclusions

AIx has serious limitations as a measure of wave reflection. Negative AIx values derived from Type C waves should not be used as estimates of wave reflection magnitude.     

Interactions of ecosystem processes with spatial heterogeneity in the puzzle of nitrogen limitation

We examined the potential effects of spatial heterogeneity and its development on the distribution, abundance, and functioning of nitrogen fixing and non-fixing components of a model ecosystem. CAECO, a spatially explicit model individual based approach, simulated the interactions between nitrogen fluxes and plant species community dynamics. Self-organized spatial patterns of nitrogen concentrations and plant occupancy were observed as the system approached an apparently meta-stable state. Nitrogen limitation was tested using chronic and gradient nitrogen amendments to the landscape. The dynamic arrangement of ecosystem components was sufficient to maintain indefinite nitrogen limitation at a local scale. However, landscape scale productivity was not similarly increased with nitrogen amendments. Landscape productivity was independent of nitrogen additions while fixers were present in the ecosystem. The probability of fixer loss from the system responded non-linearly to increasing nitrogen addition. The results of these model experiments suggest local and landscape constraints of primary productivity may be fundamentally distinct.     

An Estimate of the Average Number of Recessive Lethal Mutations Carried by Humans

The effects of inbreeding on human health depend critically on the number and severity of recessive, deleterious mutations carried by individuals. In humans, existing estimates of these quantities are based on comparisons between consanguineous and nonconsanguineous couples, an approach that confounds socioeconomic and genetic effects of inbreeding. To overcome this limitation, we focused on a founder population that practices a communal lifestyle, for which there is almost complete Mendelian disease ascertainment and a known pedigree. Focusing on recessive lethal diseases and simulating allele transmissions, we estimated that each haploid set of human autosomes carries on average 0.29 (95% credible interval [0.10, 0.84]) recessive alleles that lead to complete sterility or death by reproductive age when homozygous. Comparison to existing estimates in humans suggests that a substantial fraction of the total burden imposed by recessive deleterious variants is due to single mutations that lead to sterility or death between birth and reproductive age. In turn, comparison to estimates from other eukaryotes points to a surprising constancy of the average number of recessive lethal mutations across organisms with markedly different genome sizes.     

Interaction of calpastatin with calpain: a review

Calpastatin is a multiheaded inhibitor capable of inhibiting more than one calpain molecule. Each inhibitory domain of calpastatin has three subdomains, A, B, and C; A binds to domain IV and C binds to domain VI of the calpains. Crystallographic evidence shows that binding of C to domain VI involves hydrophobic interactions at a site near the first EF-hand in domain VI. Sequence homology suggests that binding of A to calpain domain IV also involves hydrophobic interactions near the EF1-hand of domain IV. Neither subdomain A nor C have inhibitory activity without subdomain B, but both increase the inhibitory activity of B. Subdomain B peptides have no inhibitory activity unless they contain at least 13 amino acids, and inhibitory activity increases with the number of amino acid residues, suggesting that inhibition requires interaction over a large area of the calpain molecule. Although subdomain B inhibition kinetically is competitive in nature, subdomain B does not seem to interact with the active site of the calpains directly, but may bind to domain III of the calpains and act to block access to the active site. It is possible that subdomain B binds to calpain only after it has been activated by Ca2+.     

Calpain cleaves RhoA generating a dominant-negative form that inhibits integrin-induced actin filament assembly and cell spreading

Integrin-induced cell adhesion results in transmission of signals that induce cytoskeletal reorganizations and resulting changes in cell behavior. The cytoskeletal reorganizations are regulated by transient activation and inactivation of Rho GTPases. Previously, we identified mu-calpain as an enzyme that is activated by signaling across beta1 and beta3 integrins. We showed that it mediates cytoskeletal reorganizations in bovine aortic endothelial (BAE) and Chinese hamster ovary (CHO) cells and does so by acting upstream of Rac1 activation. Here we show that mu-calpain is also involved in inactivating RhoA during integrin-induced signaling. Cleavage of RhoA was detectable in BAE cells plated on an integrin substrate; it did not occur in cells plated on poly-l-lysine. Cleavage was inhibited by calpain inhibitors. In vitro, mu-calpain cleaved RhoA generating a fragment of the same size as in intact cells. The cleavage site was identified, an HA-tagged construct expressing calpain-cleaved RhoA generated, and the construct expressed in BAE and CHO cells. Calpain-cleaved RhoA inhibited integrin-induced stress fiber assembly and decreased cell spreading. Together, our data show that calpain cleaves RhoA and generates a form that inhibits integrin-induced stress fiber assembly and cell spreading.     

Kinetics and time-temperature equivalence of polymer degradation

We studied the hydrolysis kinetics of amorphous polylactide. It was found the hydrolysis rate had a slow-to-fast transition at a certain molecular weight (Mn). This transition was not correlated with the mass loss and water uptake of samples, nor the pH values of testing media. We speculated that this transition was due to the slow diffusion of polymer chain ends. The chain ends did not significantly promote the hydrolysis of samples until their concentrations (approximately 1/Mn) reached a critical value. The degradation tests were also conducted over a temperature range from 37 to 90 degrees C. A time-temperature equivalent relationship of degradation processes was established and a master curve spanning a time range equivalent to 3-5 years at 37 degrees C was constructed. This master curve can be used to predict polymer degradation processes based on accelerated tests. The functional time and disappearance time of degradable polymers were also discussed.     

Effects of autolysis on properties of mu- and m-calpain

Although the biochemical changes that occur during autolysis of mu- and m-calpain are well characterized, there have been few studies on properties of the autolyzed calpain molecules themselves. The present study shows that both autolyzed mu- and m-calpain lose 50-55% of their proteolytic activity within 5 min during incubation at pH 7.5 in 300 mM or higher salt and at a slower rate in 100 mM salt. This loss of activity is not reversed by dialysis for 18 h against a low-ionic-strength buffer at pH 7.5. Proteolytic activity of the unautolyzed calpains is not affected by incubation for 45 min at ionic strengths up to 1000 mM. Size-exclusion chromatography shows that ionic strengths of 100 mM or above cause dissociation of the two subunits of autolyzed calpains and that the dissociated large subunits (76- or 78-kDa) aggregate to form dimers and trimers, which are proteolytically inactive. Hence, instability of autolyzed calpains is due to aggregation of dissociated heavy chains. Autolysis removes the N-terminal 19 (m-calpain) or 27 (mu-calpain) amino acids from the large subunit and approximately 90 amino acids from the N-terminus of the small subunit. These regions form contacts between the two subunits in unautolyzed calpains, and their removal leaves only contacts between domain IV in the large subunit and domain VI in the small subunit. Although many of these contacts are hydrophobic in nature, ionic-strength-induced dissociation of the two subunits in the autolyzed calpains indicates that salt bridges have an important, possibly indirect, role in the domain IV/domain VI interaction.     

Digestion of mu- and m-calpain by trypsin and chymotrypsin

Proteolytic digestion by trypsin and chymotrypsin was used to probe conformation and domain structure of the mu- and m-calpain molecules in the presence and the absence of Ca(2+). Both calpains have a compact structure in the absence of Ca(2+); incubation with either protease for 120 min results in only three or four major fragments. A 24-kDa fragment was produced by removal of the Gly-rich area in domain V of the 28-kDa subunit. The other fragments were from the 80-kDa subunit. Except for trypsin digestion of m-calpain, the region between amino acids 245 and 265 (human sequence) was very susceptible to cleavage by both proteases in the absence of Ca(2+); this region is in domain II (IIb of the crystallographic structure). Although no proteolytically active fragments could be isolated from either tryptic or chymotryptic digests, the calpain molecule can remain assembled in a proteolytically active complex even after the 80-kDa subunit has been completely degraded. The results suggest that interaction among different regions of the entire calpain molecule is required for its full proteolytic activity. In the presence of 1 mM Ca(2+), both calpains are degraded to fragments less than 40-kDa in less than 5 min. The C-terminal ends of both subunits, from amino acids 503 to 506 to the end of the 80-kDa subunit and from amino acids 85 to 88 to the end of the 28-kDa subunit, were resistant to degradation by either protease in the presence or in the absence of Ca(2+). Hence, this part of the calpain molecule is in a compact structure that does not change significantly in the presence of Ca(2+).