Oocyte-specific differences in cell-cycle control create an innate susceptibility to meiotic errors

Segregation of homologs at the first meiotic division (MI) is facilitated by crossovers and by a physical constraint imposed on sister kinetochores that facilitates monopolar attachment to the MI spindle. Recombination failure or premature separation of homologs results in univalent chromosomes at MI, and univalents constrained to form monopolar attachments should be inherently unstable and trigger the spindle assembly checkpoint (SAC). Although univalents trigger cell-cycle arrest in the male, this is not the case in mammalian oocytes. Because the spindle assembly portion of the SAC appears to function normally, two hypotheses have been proposed to explain the lack of response to univalents: (1) reduced stringency of the oocyte SAC to aberrant chromosome behavior, and (2) the ability of univalents to satisfy the SAC by forming bipolar attachments. The present study of Mlh1 mutant mice demonstrates that metaphase alignment is not a prerequisite for anaphase onset and provides strong evidence that MI spindle stabilization and anaphase onset require stable bipolar attachment of a critical mass--but not all--of chromosomes. We postulate that subtle differences in SAC-mediated control make the human oocyte inherently error prone and contribute to the age-related increase in aneuploidy.     

The assay of Met-enkephalin aminopeptidase with [125I]Met-enkephalin

Presented here are procedural modifications whic permit the utilization of ¹²⁵I-labeled Met-enkephalin as substrate in the assay of rat brain enkephalin amipeptidase. The hydrolysis of enkephalin is monitored by the release of [¹²⁵I]tyrosine separated on Porapak Q. The release of tyrosine is proportionate with both increasing time and tissue concentration. The estimated K_m is near 10^?4 M and the enzyme activity can be inhibited more than 95% with puromycin. The majority of the enzyme activity remains in the 100 000 × g supernatant following differential centrifugation.     

Expression analysis of Arabidopsis thaliana NAD-dependent isocitrate dehydrogenase genes shows the presence of a functional subunit that is mainly expressed in the pollen and absent from vegetative organs

NAD-dependent isocitrate dehydrogenase (IDH) is a Krebs cycle enzyme situated in mitochondria. In Arabidopsis thaliana, five genes encode functional IDH subunits that can be classed into two groups based on gene structure and subunit amino acid sequence. Arabidopsis contains two 'catalytic' and three 'regulatory' subunits according to their homology with yeast IDH. To date, an active IDH is believed to be heteromeric, containing at least one of each subunit type. This was verified in Arabidopsis by the complementation of yeast IDH mutants with the different Arabidopsis IDH-encoding cDNAs. Indeed, a single 'catalytic' and 'regulatory' subunit was sufficient to restore acetate growth of the yeast IDH double mutant. To gain information on possible IDH subunit interactions in planta, Arabidopsis IDH gene expression was analysed by Northern blot, PCR on cDNA libraries, in silico and in 'promoter'-reporter gene transgenic plants. Four of the IDH genes were expressed in all plant organs tested, while one gene (At4g35650) was not expressed in vegetative organs but was mainly expressed in the pollen. In leaves, the IDH genes were highly expressed in the veins, and to a lesser extent in mesophyll cells. The data are discussed with respect to IDH in other plant species.     

Modelling the Canada lynx and snowshoe hare population cycle: the role of specialist predators

Mathematical models of the snowshoe hare (Lepus americanus) and Canada lynx (Lynx canadensis) population cycles in the boreal forest have largely focused on the interaction between a single specialist predator and its prey. Here, we consider the role that other hare predators play in shaping the cycles, using a predator–prey model for up to three separate specialist predators. We consider the Canada lynx, coyote (Canis latrans) and great horned owl (Bubo virginianus). Our model improves on past modelling efforts in two ways: (1) our model solutions more closely represent the boreal hare and predator cycles with respect to the cycle period, maximum and minimum hare densities and maximum and minimum predator densities for each predator, and (2) our model sheds light on the role each specialist plays in regulation of the hare cycle, in particular, the dynamics of the raptor appear to be crucial for characterising the low hare densities correctly.     

Solution structure of the C-terminal antiparallel coiled-coil domain from Escherichia coli osmosensor ProP

Bacteria respond to increasing medium osmolality by accumulating organic solutes that are compatible with cellular functions. Transporter ProP of Escherichia coli, a proton symporter and a member of the major facilitator superfamily, senses osmotic shifts and responds by importing osmolytes such as glycine betaine. ProP contains a cytoplasmic, C-terminal extension that is essential for its activity. A peptide corresponding to the C-terminal extension of ProP forms a homodimeric alpha-helical coiled-coil even though some of its heptad a positions are not occupied by hydrophobic amino acid residues. Unexpectedly, amino acid replacement R488I, occurring at a heptad a position, destabilized the coiled-coil formed by the ProP peptide and attenuated the response of the intact transporter to osmotic upshifts in vivo. Thus, ProP was proposed to dimerize via an antiparallel coiled-coil. We used nuclear magnetic resonance (NMR) spectroscopy to determine the structure of the synthetic peptide corresponding to residues 468-497 of ProP. This region did form an antiparallel coil-coil in which critical residue R488 specifies the antiparallel coiled-coil orientation by forming stabilizing salt-bridges. Charged residues (both acidic and basic) are clustered on the c/g surface of the coiled-coil whereas polar residues are distributed on the b/e surface. This causes the structure to be bent, in contrast to other known antiparallel coiled-coils (those from the hepatitis delta antigen (PDB ID code 1A92) and the bovine F(1) ATPase inhibitor protein (PDB ID code 1HF9)). The coiled-coil and its possible importance for osmosensing are discussed.     

Neutrophil-Mediated Phagocytic Host Defense Defect in Myeloid Cftr-Inactivated Mice

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.