Factors affecting the daily rhythm of body temperature of captive mouse lemurs (<Emphasis Type="Italic">Microcebus murinus</Emphasis>)

Microcebus murinus, a small nocturnal Malagasy primate, exhibits adaptive energy-saving strategies such as daily hypothermia and gregarious patterns during diurnal rest. To determine whether ambient temperature (T_a), food restriction and nest sharing can modify the daily body temperature (T_b) rhythm, T_b was recorded by telemetry during winter in six males exposed to different ambient temperatures (T_a=25, 20, 15°C) and/or to a total food restriction for 3 days depending on social condition (isolated versus pair-grouped). At 25°C, the daily rhythm of T_b was characterized by high T_b values during the night and lower values during the day. Exposure to cold significantly decreased minimal T_b values and lengthened the daily hypothermia. Under food restriction, minimal T_b values were also markedly lowered. The combination of food restriction and cold induced further increases in duration and depth of torpor bouts, minimal T_b reaching a level just above T_a. Although it influenced daily hypothermia less than environmental factors, nest sharing modified effects of cold and food restriction previously observed by lengthening duration of torpor but without increasing its depth. In response to external conditions, mouse lemurs may thus adjust their energy expenditures through daily modifications of both the duration and the depth of torpor.     

Mutagenesis of the bovSERPINA3-3 demonstrates the requirement of aspartate-371 for intermolecular interaction and formation of dimers

The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This "A-sheet polymerization" has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new "non-A-sheet swap" model of serpin homodimer in which D371 is involved at the molecular interface.     

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M₁₄₂, N/D₁₄₆, S/D₁₄₆, R/Q₂₁₁, and Q/K₂₂₂) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I₁₄₂R₁₅₄R₂₁₁Q₂₂₂) goats and homozygous and/or heterozygous I/M₁₄₂, R/H₁₅₄, R/Q₂₁₁, and Q/K₂₂₂ goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M₁₄₂ PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H₁₅₄, Q₂₁₁, and K₂₂₂ PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H₁₅₄ and Q₂₁₁ allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K₂₂₂ and a small proportion of homozygous K₂₂₂ goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K₂₂₂ goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.     

Expression of SERPINA3s in cattle: focus on bovSERPINA3-7 reveals specific involvement in skeletal muscle

α₁-Antichymotrypsin is encoded by the unique SERPINA3 gene in humans, while it is encoded by a cluster of eight closely related genes in cattle. BovSERPINA3 proteins present a high degree of similarity and significant divergences in the reactive centre loop (RCL) domains which are responsible for the antiprotease activity. In this study, we analysed their expression patterns in a range of cattle tissues. Even if their expression is ubiquitous, we showed that the expression levels of each serpin vary in different tissues of 15-month-old Charolais bulls. Our results led us to focus on bovSERPINA3-7, one of the two most divergent members of the bovSERPINA3 family. Expression analyses showed that bovSERPINA3-7 protein presents different tissue-specific patterns with diverse degrees of N-glycosylation. Using a specific antibody raised against bovSERPINA3-7, Western blot analysis revealed a specific 96 kDa band in skeletal muscle. BovSERPINA3-7 immunoprecipitation and mass spectrometry revealed that this 96 kDa band corresponds to a complex of bovSERPINA3-7 and creatine kinase M-type. Finally, we reported that the bovSERPINA3-7 protein is present in slow-twitch skeletal myofibres. Precisely, bovSERPINA3-7 specifically colocalized with myomesin at the M-band region of sarcomeres where it could interact with other components such as creatine kinase M-type. This study opens new prospects on the bovSERPINA3-7 function in skeletal muscle and promotes opportunities for further understanding of the physiological role(s) of serpins.     

The Limits of Test-Based Scrapie Eradication Programs in Goats

Small ruminant post-mortem testing programs were initially designed for monitoring the prevalence of prion disease. They are now considered as a potential alternative to genetic selection for eradicating/controlling classical scrapie at population level. If such policy should be implemented, its success would be crucially dependent on the efficiency of the surveillance system used to identify infected flocks. In this study, we first determined the performance of post-mortem classical scrapie detection in eight naturally affected goat herds (total n = 1961 animals) according to the age at culling. These results provided us with necessary parameters to estimate, through a Monte Carlo simulation model, the performance of scrapie detection in a commercial population. According to this model, whatever the number of tests performed, post mortem surveillance will have limited success in identifying infected herds. These data support the contention that scrapie eradication programs relying solely on post mortem testing in goats will probably fail. Considering the epidemiological and pathological similarities of scrapie in sheep and goats, the efficiency of scrapie surveillance in both species is likely to be similar.     

RU 58668: Further in vitro and in vivo pharmacological data related to its antitumoral activity

Previous studies with the pure antiestrogen RU 58668 showed that this compound proved to be highly antiproliferative in vitro, and to be the only antiestrogenic compound so far known to induce long-term regression of MCF-7 tumours implanted into nude mice. In order to obtain more insight into the therapeutic potential of this molecule, we performed a new set of experiments in vitro and in vivo in comparison with tamoxifen and/or ICI 182,780. In vitro, 1 nM RU 58668 induced an accumulation of MCF-7 cells in G0/G1 phases of the cell cycle within 48 h and, in contrast to trans-4-hydroxy-tamoxifen, blocked the invasiveness of ras-transfected MCF-7 cells into the chick embryo heart during the three weeks of co-culture. An in vivo dose-effect relationship study showed that RU 58668 induced a regression of MCF-7 tumour with as low a dose as 10 mg/kg/week, and that such an effect can not be obtained either with a sublethal dose of adriamycin or with ICI 182,780, (2–250 mg/kg/week). This reduction in the tumour volumes accords with histological modifications of the tumours, which showed a decrease in the ratio of epithelial cells over the tumoral mass, and with a concomitant decrease in their regrowth potential when reimplanted into naive nude mice. Taken together, these results suggest a promising usefulness for RU 58668 in the treatment of metastatic breast cancer in women.     

Purification of the skeletal muscle protein Endopin 1B and characterization of the genes encoding Endopin 1A and 1B isoforms

In the present work, a new endopin-like serpin designed mEndopin 1B was purified from bovine muscle. Biochemical characterizations (amino acid sequencing and Maldi-Tof mass spectrometry peptide mapping) demonstrated that the purified protein is different from the previously described Endopin 1, renamed mEndopin 1A. The genes and cDNA of both endopins were characterized. The cDNA sequence of mEndopin 1B encodes a predicted protein of 411 amino-acids with a molecular mass of 43808Da. The mEndopin 1B gene comprised four coding exons and an additional 5' untranslated exon. The reactive site sequence of mEndopin 1B is somewhat different from that of mEndopin 1A. Nevertheless, both serpins have a similar peptidase inhibitory pattern against examined proteases (elastase, trypsin, plasmin and chymotrypsin). The high expression of both mEndopin 1A and 1B in bovine serum and tissues and their high efficiency to inhibit elastase (k(ass) approximately 10(6)-10(7) M(-1) s(-1)) suggested that these serpins might play a major role in inflammatory processes.     

Maximum effect of the heterogeneity of tissue mineralization on the effective cortical bone elastic properties

Biomechanics and Modeling in Mechanobiology - The mineralization level is heterogeneous in cortical bone extracellular matrix as a consequence of remodeling. Models of the effective elastic...