Identification of mutations that decrease the stability of a fragment of Saccharomyces cerevisiae chromosome III lacking efficient replicators

Eukaryotic chromosomes are duplicated during S phase and transmitted to progeny during mitosis with high fidelity. Chromosome duplication is controlled at the level of replication initiation, which occurs at cis-acting replicator sequences that are spaced at intervals of approximately 40 kb along the chromosomes of the budding yeast Saccharomyces cerevisiae. Surprisingly, we found that derivatives of yeast chromosome III that lack known replicators were replicated and segregated properly in at least 96% of cell divisions. To gain insight into the mechanisms that maintain these "originless" chromosome fragments, we screened for mutants defective in the maintenance of an "originless" chromosome fragment, but proficient in the maintenance of the same fragment that carries its normal complement of replicators (originless fragment maintenance mutants, or ofm). We show that three of these Ofm mutations appear to disrupt different processes involved in chromosome transmission. The OFM1-1 mutant seems to disrupt an alternative initiation mechanism, and the ofm6 mutant appears to be defective in replication fork progression. ofm14 is an allele of RAD9, which is required for the activation of the DNA damage checkpoint, suggesting that this checkpoint plays a key role in the maintenance of the "originless" fragment.     

Mitochondrial oxidative stress causes hyperphosphorylation of tau

Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and beta-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Ass load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD.     

DNA elements modulating the <Emphasis Type="Italic">KARS12</Emphasis> chromosomal replicator in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.     

Specification of neuronal identities by feedforward combinatorial coding

Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. Here, we use the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Amongst the progeny of neuroblast 5–6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. We show the HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5–6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, we find that the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues.     

The molecular anatomy of spontaneous germline mutations in human testes

The frequency of the most common sporadic Apert syndrome mutation (C755G) in the human fibroblast growth factor receptor 2 gene (FGFR2) is 100–1,000 times higher than expected from average nucleotide substitution rates based on evolutionary studies and the incidence of human genetic diseases. To determine if this increased frequency was due to the nucleotide site having the properties of a mutation hot spot, or some other explanation, we developed a new experimental approach. We examined the spatial distribution of the frequency of the C755G mutation in the germline by dividing four testes from two normal individuals each into several hundred pieces, and, using a highly sensitive PCR assay, we measured the mutation frequency of each piece. We discovered that each testis was characterized by rare foci with mutation frequencies 10³ to >10⁴ times higher than the rest of the testis regions. Using a model based on what is known about human germline development forced us to reject (p < 10⁻⁶) the idea that the C755G mutation arises more frequently because this nucleotide simply has a higher than average mutation rate (hot spot model). This is true regardless of whether mutation is dependent or independent of cell division. An alternate model was examined where positive selection acts on adult self-renewing Ap spermatogonial cells (SrAp) carrying this mutation such that, instead of only replacing themselves, they occasionally produce two SrAp cells. This model could not be rejected given our observed data. Unlike the disease site, similar analysis of C-to-G mutations at a control nucleotide site in one testis pair failed to find any foci with high mutation frequencies. The rejection of the hot spot model and lack of rejection of a selection model for the C755G mutation, along with other data, provides strong support for the proposal that positive selection in the testis can act to increase the frequency of premeiotic germ cells carrying a mutation deleterious to an offspring, thereby unfavorably altering the mutational load in humans. Studying the anatomical distribution of germline mutations can provide new insights into genetic disease and evolutionary change.     

Analysis of antigen-specific antibodies and their isotypes in experimental malaria.

BACKGROUND: Measuring antibody production in response to antigen exposure or vaccination is key to disease prevention and treatment. Our understanding of the mechanisms involved in the antibody response is limited by a lack of sensitive analysis methods. We address this limitation using multiplexed microsphere arrays for the semi -quantitative analysis of antibody production in response to malaria infection. METHODS: We used microspheres as solid supports on which to capture and analyze circulating antibodies. Antigen immobilized on beads captured antigen-specific antibodies for semi- quantitative analysis using fluorescent secondary antibodies. Anti-immunoglobulin antibodies on beads captured specific antibody isotypes for affinity estimation using fluorescent antigen. RESULTS: Antigen-mediated capture of plasma antibodies enables determination of antigen-specific antibody "titer," a semi-quantitative parameter describing a convolution of antibody abundance and avidity, as well as parameters describing numbers of antibodies bound/bead at saturation and the plasma concentration-dependent approach to saturation. Results were identical in single-plex and multiplex assays, and in qualitative agreement with similar parameters derived from ELISA-based assays. Isotype-specific antibody-mediated capture of plasma antibodies allowed the estimation of the affinity of antibody for antigen. CONCLUSION: Analysis of antibody responses using microspheres and flow cytometry offer significant advantages in speed, sample size, and quantification over standard ELISA-based titer methods.     

Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis

Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-alpha(2) activity by 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with beta-guanidinopropionic acid (beta-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.     

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances

Grazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half-saturation constant of 6.7 × 10⁵ prey ml⁻¹, a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST-4 and MAST-7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.     

Multiscale modelling of Potts shunt as a potential palliative treatment for suprasystemic idiopathic pulmonary artery hypertension: a paediatric case study

Biomechanics and Modeling in Mechanobiology - Potts shunt (PS) was suggested as palliation for patients with suprasystemic pulmonary arterial hypertension (PAH) and right ventricular (RV) failure....