Selected contribution: Bone adaptation with aging and long-term caloric restriction in Fischer 344 x Brown-Norway F1-hybrid rats.

Rodents are commonly used as models for human aging because of their relatively short life span, the ease of obtaining age-specific tissue samples, and lower cost. However, age-associated disease may confound inbred animal studies. For example, numerous physiologically significant lesions, such as chronic nephropathy, are more common in aged Fischer 344 (F344) rats than in other strains (Bronson RT, Genetic Effects of Aging, 1990). Conversely, F344 x Brown-Norway F1-hybrid (F344BN) rats, developed by the National Institute on Aging for aging research, live considerably longer and have fewer pathologies at any given age vs. inbred strains (Lipman RD, Chrisp CE, Hazzard DG, and Bronson RT, J Gerontol A Biol Sci Med Sci 51: 54-59, 1996). To our knowledge, there are no data regarding the effect of age on bone geometry and mechanics in this strain of rat. Furthermore, caloric restriction (CR) extends the mean and maximal life span of animals and significantly reduces age-associated disease but may have adverse consequences for bone growth and mechanics. Thus we investigated the effects of age and CR on bone geometry and mechanics in the axial and appendicular skeleton of F344 Brown-Norway rats. Ad libitum fed rats were assessed at 8 mo (young adult; n = 6), 28 mo (late middle age; n = 5), and 36 mo (senescence; n = 6). CR rats were assessed at 28 mo (n = 6). Tibiae and the sixth lumbar vertebrae (L6) were dissected, scanned (micro-computed tomography) to determine geometry, and tested mechanically. From 8 to 36 mo, there were no significant changes in L6 geometry, and only the cross-sectional moment of inertia changed (increased) with the tibia. CR-induced body mass reductions accounted for changes in L6 load at proportional limit, maximal load, and stiffness (structural properties), but altered tibial structural properties were independent of body mass. In tibiae, geometric changes dominated alterations in structural properties. Those data demonstrated that, whereas aging in ad libitum-fed animals induced minor changes in bone mechanics, axial and appendicular bones were adversely influenced by CR in late-middle-aged animals in different manners.     

Chorusing Behaviour,a Density-dependent Alternative Mating Strategy in Male Common Toads (Bufo bufo)

Male chorusing behaviour was studied in a population of common toads (Bufo bufo) on the island of Öland south Sweden, and the functional role of male advertisement calling in this species was experimentally examined. Calling males were larger and heavier than non-calling males (t = 2.41, p < 0.025 and t = 2.36, p < 0.025, respectively). However, small males were also found to call. This indicates that large males persisted in calling for longer and/or called more often. The proportion of calling males decreased as population size increased during the breeding season, indicating that calling is a low density strategy. Females responded more readily to calls than males. There were insufficient data to determine if the dominant frequencies of advertisement calls were inversely correlated with male body size, however, this relationship was found for the similar release calls. Females were found not to discriminate between high and low frequency calls, but when given a choice between two calls of different sound pressure levels (SPL), females were attracted to the louder calls. Thus, the function of chorusing is to advertise the position of males to mate-seeking females when the population density is low.     

Protein phosphatase 2A regulates MPF activity and sister chromatid cohesion in budding yeast

Background Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28–cyclin B complexes. Increasing levels of cyclin B abundance and the loss of inhibitory phosphates from Cdc2/28 drives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are arrested in mitosis by the spindle-assembly checkpoint, which prevents the destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint, cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis.Results The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gene, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, shows a different checkpoint defect. In the presence of a defective spindle, these cells separate their sister chromatids and leave mitosis without inducing cyclin B destruction. Despite the persistence of B-type cyclins, cdc55 mutant cells inactivate MPF. Two experiments show that this inactivation is due to inhibitory phosphorylation on Cdc28: phosphotyrosine accumulates on Cdc28 in cdc55Δ cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phosphorylation sites on Cdc28 allows spindle defects to arrest cdc55 mutants in mitosis with active MPF and unseparated sister chromatids.Conclusions We conclude that perturbations of protein phosphatase activity allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separation appears to be regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pathway plays a role in adaptation to prolonged activation of the spindle-assembly checkpoint.     

Overexpression of the yeast MCK1 protein kinase suppresses conditional mutations in centromere-binding protein genes CBF2 and CBF5

We find that overexpression in yeast of the yeast MCK1 gene, which encodes a meiosis and centromere regulatory kinase, suppresses the temperature-sensitive phenotype of certain mutations in essential centromere binding protein genes CBF2 and CBF5. Since Mck1p is a known serine/threonine protein kinase, this suppression is postulated to be due to Mck1p-catalyzed in vivo phosphorylation of centromere binding proteins. Evidence in support of this model was provided by the finding that purified Mck1p phosphorylates in vitro the 110 kDa subunit (Cbf2p) of the multimeric centromere binding factor CBF3. This phosphorylation occurs on both serine and threonine residues in Cbf2p.     

Effects of wall shear stress and fluid recirculation on the localization of circulating monocytes in a three-dimensional flow model

There is a correlation between the location of early atherosclerotic lesions and the hemodynamic characteristics at those sites. Circulating monocytes are key cells in the pathogenesis of atherosclerotic plaques and localize at sites of atherogenesis. The hypothesis that the distribution of monocyte adhesion to the vascular wall is determined in part by hemodynamic factors was addressed by studying monocyte adhesion in an in vitro flow model in the absence of any biological activity in the model wall.

Suspensions of U937 cells were perfused (Re = 200) through an axisymmetric silicone flow model with a stenosis followed by a reverse step. The model provided spatially varying wall shear stress, flow separation and reattachment, and a three-dimensional flow pattern. The cell rolling velocity and adhesion rates were determined by analysis of videomicrographs. Wall shear stress was obtained by numerical solution of the equations of fluid motion. Cell adhesion patterns were also studied in the presence of chemotactic peptide gradients.

The cell rolling velocity varied linearly with wall shear stress. The adhesion rate tended to decrease with increasing local wall shear stress, but was also affected by the radial component of velocity and the dynamics of the recirculation region and flow reattachment. Adhesion was increased in the vicinity of chemotactic peptide sources downstream of the expansion site. Results with human monocytes were qualitatively similar to the U937 experiments.

Differences in the adhesion rates of U937 cells occurring solely as a function of the fluid dynamic properties of the flow field were clearly demonstrated in the absence of any biological activity in the model wall.     

Newt Myotubes Reenter the Cell Cycle by Phosphorylation of the Retinoblastoma Protein

Withdrawal from the cell cycle is an essential aspect of vertebrate muscle differentiation and requires the retinoblastoma (Rb) protein that inhibits expression of genes needed for cell cycle entry. It was shown recently that cultured myotubes derived from the Rb^−/−mouse reenter the cell cycle after serum stimulation (Schneider, J.W., W. Gu, L. Zhu, V. Mahdavi, and B. Nadal-Ginard. 1994. Science (Wash. DC). 264:1467– 1471). In contrast with other vertebrates, adult urodele amphibians such as the newt can regenerate their limbs, a process involving cell cycle reentry and local reversal of differentiation. Here we show that myotubes formed in culture from newt limb cells are refractory to several growth factors, but they undergo S phase after serum stimulation and accumulate 4N nuclei. This response to serum is inhibited by contact with mononucleate cells. Despite the phenotypic parallel with Rb^−/− mouse myotubes, Rb is expressed in the newt myotubes, and its phosphorylation via cyclin-dependent kinase 4/6 is required for cell cycle reentry. Thus, the postmitotic arrest of urodele myotubes, although intact in certain respects, can be undermined by a pathway that is inactive in other vertebrates. This may be important for the regenerative ability of these animals.     

Analysis of physiological and molecular changes in melon (Cucumis melo L.) varieties with different rates of ripening

Seven melon varieties (Alpha, Delada, Marygold, Sirio, Topper,Tornado, and Viva) known to exhibit differences in their ripeningbehaviour were used in this study. The expression of mRNAs forACC oxidase (MEL1) and phytoene synthase (MEL5), required forsynthesis of ethylene and carotenoids, respectively, and tworipening-related cDNAs (MEL2 and MEL7), of unknown function,was examined and correlated with the development of colour andsoftening of fruits. The MEL2 and MEL7 mRNAs were present andaccumulated in all varieties, indicating their importance inmelon fruit ripening. The fruits of Delada and Marygold didnot show any change in the colour of the flesh even at 50 daa(days after anthesis). All other varieties changed colour fromgreen to orange between 25–30 daa. The phytoene synthasemRNA levels in most varieties seemed to be unrelated to changein fruit flesh colour. The firmness of all the fruits was reducedsignificantly between 25 and 40 daa. The expression of ACC oxidasemRNA showed the most variation among the different varitiesand was delayed in Sirio and undetectable in Marygold fruitseven at 40 daa. Varieties with delayed expression of ACC oxidasemRNAs after anthesis also showed delayed softening during ripening.The prospects of genetic engineering and breeding for melonfruits with improved quality characteristics and extended storagelife are discussed. Key words: Cucumis melo, colour development, melon varieties, ripening genes, softening