Physical mapping identifies DXS265 as a useful genetic marker for carrier detection and prenatal diagnosis of X-linked agammaglobulinemia

The gene responsible for X-linked agammaglobulinemia (XLA) has not been identified; however, in the course of genetic linkage studies designed to map the locus more precisely, a number of closely linked polymorphic loci have been identified. These have proved to be useful in identifying carriers and in pre-natal diagnosis of this disease. The DXS178 locus was found to be closest to the XLA locus and has been the most usefully employed probe to date. Using physical mapping techniques, we have identified a previously cloned genetic marker, DXS265, as being situated within 5kb of DXS178. So far, we have found one family that is not informative for DXS178 but that is informative for DXS265; females in this family can now be offered the possibility of carrier determination and pre-natal diagnosis for this life-threatening disease.     

The Role of Dbf4-Dependent Protein Kinase in DNA Polymerase ζ-Dependent Mutagenesis in Saccharomyces cerevisiae

The yeast Dbf4-dependent kinase (DDK) (composed of Dbf4 and Cdc7 subunits) is an essential, conserved Ser/Thr protein kinase that regulates multiple processes in the cell, including DNA replication, recombination and induced mutagenesis. Only DDK substrates important for replication and recombination have been identified. Consequently, the mechanism by which DDK regulates mutagenesis is unknown. The yeast mcm5-bob1 mutation that bypasses DDK’s essential role in DNA replication was used here to examine whether loss of DDK affects spontaneous as well as induced mutagenesis. Using the sensitive lys2ΔA746 frameshift reversion assay, we show DDK is required to generate “complex” spontaneous mutations, which are a hallmark of the Polζ translesion synthesis DNA polymerase. DDK co-immunoprecipitated with the Rev7 regulatory, but not with the Rev3 polymerase subunit of Polζ. Conversely, Rev7 bound mainly to the Cdc7 kinase subunit and not to Dbf4. The Rev7 subunit of Polζ may be regulated by DDK phosphorylation as immunoprecipitates of yeast Cdc7 and also recombinant Xenopus DDK phosphorylated GST-Rev7 in vitro. In addition to promoting Polζ-dependent mutagenesis, DDK was also important for generating Polζ-independent large deletions that revert the lys2ΔA746 allele. The decrease in large deletions observed in the absence of DDK likely results from an increase in the rate of replication fork restart after an encounter with spontaneous DNA damage. Finally, nonepistatic, additive/synergistic UV sensitivity was observed in cdc7Δ pol32Δ and cdc7Δ pol30-K127R,K164R double mutants, suggesting that DDK may regulate Rev7 protein during postreplication “gap filling” rather than during “polymerase switching” by ubiquitinated and sumoylated modified Pol30 (PCNA) and Pol32.     

Mammalian COPII Coat Component SEC24C Is Required for Embryonic Development in Mice

COPII-coated vesicles mediate the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi. SEC24 is the COPII component primarily responsible for recruitment of protein cargoes into nascent vesicles. There are four Sec24 paralogs in mammals, with mice deficient in SEC24A, -B, and -D exhibiting a wide range of phenotypes. We now report the characterization of mice with deficiency in the fourth Sec24 paralog, SEC24C. Although mice haploinsufficient for Sec24c exhibit no apparent abnormalities, homozygous deficiency results in embryonic lethality at approximately embryonic day 7. Tissue-specific deletion of Sec24c in hepatocytes, pancreatic cells, smooth muscle cells, and intestinal epithelial cells results in phenotypically normal mice. Thus, SEC24C is required in early mammalian development but is dispensable in a number of tissues, likely as a result of compensation by other Sec24 paralogs. The embryonic lethality resulting from loss of SEC24C occurs considerably later than the lethality previously observed in SEC24D deficiency; it is clearly distinct from the restricted neural tube phenotype of Sec24b null embryos and the mild hypocholesterolemic phenotype of adult Sec24a null mice. Taken together, these results demonstrate that the four Sec24 paralogs have developed unique functions over the course of vertebrate evolution.     

The Structure of Human 15-Lipoxygenase-2 with a Substrate Mimic

Atherosclerosis is associated with chronic inflammation occurring over decades. The enzyme 15-lipoxygenase-2 (15-LOX-2) is highly expressed in large atherosclerotic plaques, and its activity has been linked to the progression of macrophages to the lipid-laden foam cells present in atherosclerotic plaques. We report here the crystal structure of human 15-LOX-2 in complex with an inhibitor that appears to bind as a substrate mimic. 15-LOX-2 contains a long loop, composed of hydrophobic amino acids, which projects from the amino-terminal membrane-binding domain. The loop is flanked by two Ca²⁺-binding sites that confer Ca²⁺-dependent membrane binding. A comparison of the human 15-LOX-2 and 5-LOX structures reveals similarities at the active sites, as well striking differences that can be exploited for design of isoform-selective inhibitors.     

Biochemical and Structural Studies of 6-Carboxy-5,6,7,8-tetrahydropterin Synthase Reveal the Molecular Basis of Catalytic Promiscuity within the Tunnel-fold Superfamily

6-Pyruvoyltetrahydropterin synthase (PTPS) homologs in both mammals and bacteria catalyze distinct reactions using the same 7,8-dihydroneopterin triphosphate substrate. The mammalian enzyme converts 7,8-dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin, whereas the bacterial enzyme catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin. To understand the basis for the differential activities we determined the crystal structure of a bacterial PTPS homolog in the presence and absence of various ligands. Comparison to mammalian structures revealed that although the active sites are nearly structurally identical, the bacterial enzyme houses a His/Asp dyad that is absent from the mammalian protein. Steady state and time-resolved kinetic analysis of the reaction catalyzed by the bacterial homolog revealed that these residues are responsible for the catalytic divergence. This study demonstrates how small variations in the active site can lead to the emergence of new functions in existing protein folds.     

Place and Cause of Death in Centenarians: A Population-Based Observational Study in England, 2001 to 2010

Background

Centenarians are a rapidly growing demographic group worldwide, yet their health and social care needs are seldom considered. This study aims to examine trends in place of death and associations for centenarians in England over 10 years to consider policy implications of extreme longevity.

Methods and Findings

This is a population-based observational study using death registration data linked with area-level indices of multiple deprivations for people aged ≥100 years who died 2001 to 2010 in England, compared with those dying at ages 80-99. We used linear regression to examine the time trends in number of deaths and place of death, and Poisson regression to evaluate factors associated with centenarians’ place of death. The cohort totalled 35,867 people with a median age at death of 101 years (range: 100–115 years). Centenarian deaths increased 56% (95% CI 53.8%–57.4%) in 10 years. Most died in a care home with (26.7%, 95% CI 26.3%–27.2%) or without nursing (34.5%, 95% CI 34.0%–35.0%) or in hospital (27.2%, 95% CI 26.7%–27.6%). The proportion of deaths in nursing homes decreased over 10 years (−0.36% annually, 95% CI −0.63% to −0.09%, p = 0.014), while hospital deaths changed little (0.25% annually, 95% CI −0.06% to 0.57%, p = 0.09). Dying with frailty was common with “old age” stated in 75.6% of death certifications. Centenarians were more likely to die of pneumonia (e.g., 17.7% [95% CI 17.3%–18.1%] versus 6.0% [5.9%–6.0%] for those aged 80–84 years) and old age/frailty (28.1% [27.6%–28.5%] versus 0.9% [0.9%–0.9%] for those aged 80–84 years) and less likely to die of cancer (4.4% [4.2%–4.6%] versus 24.5% [24.6%–25.4%] for those aged 80–84 years) and ischemic heart disease (8.6% [8.3%–8.9%] versus 19.0% [18.9%–19.0%] for those aged 80–84 years) than were younger elderly patients. More care home beds available per 1,000 population were associated with fewer deaths in hospital (PR 0.98, 95% CI 0.98–0.99, p<0.001).

Conclusions

Centenarians are more likely to have causes of death certified as pneumonia and frailty and less likely to have causes of death of cancer or ischemic heart disease, compared with younger elderly patients. To reduce reliance on hospital care at the end of life requires recognition of centenarians’ increased likelihood to “acute” decline, notably from pneumonia, and wider provision of anticipatory care to enable people to remain in their usual residence, and increasing care home bed capacity. Please see later in the article for the Editors'' Summary