Limb development, evolution, and regeneration and repair:Part two

International audienc

The chick limb: embryology, genetics and teratology

MD is supported by the BBSRC through a BBSRC Institute Strategic Grant, MT is a Wellcome Trust Senior Fellow and NV’s studies on thalidomide were supported by The Wellcome Trust. We all thank lab members past and present for their help and support. We apologize to those whose work we have not referenced due to lack of space.Peer reviewedPublisher PD

The effects of oligomerization on Saccharomyces cerevisiae Mcm4/6/7 function

<p>Abstract</p> <p>Background</p> <p>Minichromosome maintenance proteins (Mcm) 2, 3, 4, 5, 6 and 7 are related by sequence and form a variety of complexes that unwind DNA, including Mcm4/6/7. A Mcm4/6/7 trimer forms one half of the Mcm2-7 hexameric ring and can be thought of as the catalytic core of Mcm2-7, the replicative helicase in eukaryotic cells. Oligomeric analysis of Mcm4/6/7 suggests that it forms a hexamer containing two Mcm4/6/7 trimers, however, under certain conditions trimeric Mcm4/6/7 has also been observed. The functional significance of the different Mcm4/6/7 oligomeric states has not been assessed. The results of such an assessment would have implications for studies of both Mcm4/6/7 and Mcm2-7.</p> <p>Results</p> <p>Here, we show that <it>Saccharomyces cerevisiae </it>Mcm4/6/7 reconstituted from individual subunits exists in an equilibrium of oligomeric forms in which smaller oligomers predominate in the absence of ATP. In addition, we found that ATP, which is required for Mcm4/6/7 activity, shifts the equilibrium towards larger oligomers, likely hexamers of Mcm4/6/7. ATPγS and to a lesser extent ADP also shift the equilibrium towards hexamers. Study of Mcm4/6/7 complexes containing mutations that interfere with the formation of inter-subunit ATP sites (arginine finger mutants) indicates that full activity of Mcm4/6/7 requires all of its ATP sites, which are formed in a hexamer and not a trimer. In keeping with this observation, Mcm4/6/7 binds DNA as a hexamer.</p> <p>Conclusions</p> <p>The minimal functional unit of Mcm4/6/7 is a hexamer. One of the roles of ATP binding by Mcm4/6/7 may be to stabilize formation of hexamers.</p

Cognitive Performance in Centenarians and the Oldest Old: Norms from the Georgia Centenarian Study

We present normative data from a large population-based sample of centenarians for several brief, global neurocognitive tasks amenable for frail elders. Comparative data from octogenarians are included. A total of 244 centenarians and 80 octogenarians from Phase III of the Georgia Centenarian Study were administered the Mini-Mental Status Examination, Severe Impairment Battery, and Behavioral Dyscontrol Scale. Centenarians (age 98–107) were stratified into three age cohorts (98–99, 100–101, 102–107), octogenarians into two 5- year cohorts (80–84, 85–89). Highly significant differences were observed between groups on all measures, with greater variation and dispersion in performance among centenarians, as well as stronger associations between age and performance. Descriptive statistics and normative ranges (unweighted and population-weighted) are provided by age cohort. Additional statistics are provided by education level. While most previous centenarian studies have used convenience samples, ours is population-based and likely more valid for comparison in applied settings. Results suggest centenarians look different than do even the oldest age range of most normative aging datasets (e.g., 85–90). Results support using global measures of neurocognition to describe cognitive status in the oldest old, and we provide normative comparisons to do so

The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization

PHOSPHO1 is a phosphoethanolamine/phosphocholine phosphatase that has previously been implicated in generating inorganic phosphate (Pi) for matrix mineralization. In this study, we have investigated PHOSPHO1 mRNA expression during embryonic development in the chick. Whole-mount in situ hybridization indicated that PHOSPHO1 expression occurred prior to E6.5 and was initially restricted to the bone collar within the mid-shaft of the diaphysis of long bones but by E11.5 expression was observed over the entire length of the diaphysis. Alcian blue/alizarin red staining revealed that PHOSPHO1 expression seen in the primary regions of ossification preceded the deposition of mineral, suggesting that it is involved in the initial events of mineral formation. We isolated MVs from growth plate chondrocytes and confirmed the presence of high levels of PHOSPHO1 by immunoblotting. Expression of PHOSPHO1, like TNAP activity, was found to be up-regulated in MVs isolated from chondrocytes induced to differentiate by the addition of ascorbic acid. This suggests that both enzymes may be regulated by similar mechanisms. These studies provide for the first time direct evidence that PHOSPHO1 is present in MVs, and its developmental expression pattern is consistent with a role in the early stages of matrix mineralization