Unisexual Reproduction Reverses Muller’s Ratchet

Cryptococcus neoformans is a pathogenic basidiomycetous fungus that engages in outcrossing, inbreeding, and selfing forms of unisexual reproduction as well as canonical sexual reproduction between opposite mating types. Long thought to be clonal, >99% of sampled environmental and clinical isolates of C. neoformans are MATα, limiting the frequency of opposite mating-type sexual reproduction. Sexual reproduction allows eukaryotic organisms to exchange genetic information and shuffle their genomes to avoid the irreversible accumulation of deleterious changes that occur in asexual populations, known as Muller’s ratchet. We tested whether unisexual reproduction, which dispenses with the requirement for an opposite mating-type partner, is able to purge the genome of deleterious mutations. We report that the unisexual cycle can restore mutant strains of C. neoformans to wild-type genotype and phenotype, including prototrophy and growth rate. Furthermore, the unisexual cycle allows attenuated strains to purge deleterious mutations and produce progeny that are returned to wild-type virulence. Our results show that unisexual populations of C. neoformans are able to avoid Muller’s ratchet and loss of fitness through a unisexual reproduction cycle involving α-α cell fusion, nuclear fusion, and meiosis. Similar types of unisexual reproduction may operate in other pathogenic and saprobic eukaryotic taxa.     

The epigenetic effect of glucosamine and a nuclear factor-kappa B (NF-kB) inhibitor on primary human chondrocytes--implications for osteoarthritis

Objective: Idiopathic osteoarthritis is the most common form of osteoarthritis (OA) world-wide and remains the leading cause of disability and the associated socio-economic burden in an increasing aging population. Traditionally, OA has been viewed as a degenerative joint disease characterized by progressive destruction of the articular cartilage and changes in the subchondral bone culminating in joint failure. However, the etiology of OA is multifactorial involving genetic, mechanical and environmental factors. Treatment modalities include analgesia, joint injection with steroids or hyaluronic acid, oral supplements including glucosamine and chondroitin sulfate, as well as physiotherapy. Thus, there is significant interest in the discovery of disease modifying agents. One such agent, glucosamine (GlcN) is commonly prescribed even though the therapeutic efficacy and mechanism of action remain controversial. Inflammatory cytokines, including IL-1β, and proteinases such as MMP-13 have been implicated in the pathogenesis and progression of OA together with an associated CpG demethylation in their promoters. We have investigated the potential of GlcN to modulate NF-kB activity and cytokine-induced abnormal gene expression in articular chondrocytes and, critically, whether this is associated with an epigenetic process. Method: Human chondrocytes were isolated from the articular cartilage of femoral heads, obtained with ethical permission, following fractured neck of femur surgery. Chondrocytes were cultured for 5 weeks in six separate groups; (i) control culture, (ii) cultured with a mixture of 2.5 ng/ml IL-1β and 2.5 ng/ml oncostatin M (OSM), (iii) cultured with 2 mM N-acetyl GlcN (Sigma–Aldrich), (iv) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 2 mM GlcN, (v) cultured with 1.0 μM BAY 11-7082 (BAY; NF-kB inhibitor: Calbiochem, Darmstadt, Germany) and, (vi) cultured with a mixture of 2.5 ng/ml IL-1β, 2.5 ng/ml OSM and 1.0 μM BAY. The levels of IL1B and MMP13 mRNA were examined using qRT-PCR. The percentage DNA methylation in the CpG sites of the IL1β and MMP13 proximal promoter were quantified by pyrosequencing. Result:IL1β expression was enhanced over 580-fold in articular chondrocytes treated with IL-1β and OSM. GlcN dramatically ameliorated the cytokine-induced expression by 4-fold. BAY alone increased IL1β expression by 3-fold. In the presence of BAY, IL-1β induced IL1B mRNA levels were decreased by 6-fold. The observed average percentage methylation of the -256 CpG site in the IL1β promoter was 65% in control cultures and decreased to 36% in the presence of IL-1β/OSM. GlcN and BAY alone had a negligible effect on the methylation status of the IL1B promoter. The cytokine-induced loss of methylation status in the IL1B promoter was ameliorated by both GlcN and BAY to 44% and 53%, respectively. IL-1β/OSM treatment increased MMP13 mRNA levels independently of either GlcN or BAY and no change in the methylation status of the MMP13 promoter was observed. Conclusion: We demonstrate for the first time that GlcN and BAY can prevent cytokine-induced demethylation of a specific CpG site in the IL1β promoter and this was associated with decreased expression of IL1β. These studies provide a potential mechanism of action for OA disease modifying agents via NF-kB and, critically, demonstrate the need for further studies to elucidate the role that NF-kB may play in DNA demethylation in human chondrocytes.     

铅锌镉联合染毒及营养干预对大鼠血液系统的影响研究

目的:了解铅锌镉联合染毒对大鼠血液系统的影响及营养干预对其损伤的修复作用。方法:选择SPF级初断乳Wistar大鼠72只,随机分为对照组、染毒组和干预组,分别采用生理盐水、铅锌镉联合染毒液及染毒后以营养干预液灌胃28天和56天之后,检测其血液系统中五元素和血细胞的指标。结果:染毒组较对照组大鼠血铜、血锌含量高,血钙含量低于对照组,差异均有统计学意义(P<0.05);染毒组血铜含量高于干预组,血钙含量低于干预组,差异均有统计学意义(P<0.05);干预组红细胞(RBC)计数、血红蛋白(Hb)、血细胞比容(HCT)均高于染毒组,差异均有统计学意义(P<0.05);对照组白细胞(WBC)计数高于染毒组、干预组,差异均有统计学意义(P<0.05)。结论:铅镉对大鼠血铜、血钙、血锌水平有影响;综合营养干预对重金属元素造成的血液系统损伤有明显的拮抗作用,对血液系统有一定的保护及修复作用。     

Dual fluorochrome analysis of human B lymphocytes: phenotypic examination of resting, anti-immunoglobulin stimulated, and in vivo activated B cells

B cell-enriched preparations were prepared from human peripheral blood and lymphoid tissues by the depletion of T cells and monocytes. Only B cells by virtue of their staining with anti-B1 conjugated to fluorescein were additionally examined. Dual fluorescence staining and flow cytometric analysis demonstrated that the majority of "resting" human peripheral blood and splenic B cells co-express the B cell-restricted B1 and B2 antigens and lack B5, a B cell-restricted activation antigen, and interleukin 2 receptor (IL 2R). In contrast, nearly 2/3 and 1/3 of B1+ cells isolated from lymph node expressed IL 2R and B5 antigens, respectively. When B1+ B cells from peripheral blood and spleen were "activated" by anti-Ig, they lost the B2 antigen and acquired the B5 and/or IL 2R antigens. 2/3 of B1+ cells strongly expressed IL 2R, and up to 1/2 of B1+ cells co-expressed B5. Delineation of increased numbers of B1+ cells that co-express B5 and/or IL 2R within lymphoid tissues obtained from patients with diseases characterized by "activated" B cells provides in vivo confirmation that these phenotypic changes correlate with B cell activation. We believe that the identification and isolation of these and similar subsets of cells defined by differing cell surface phenotypes should further our understanding both of normal B cell activation and the pathophysiology of B cell disease states.     

The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression

The amyloid-beta (Abeta) peptide, which likely plays a key role in Alzheimer disease, is derived from the amyloid-beta precursor protein (APP) through consecutive proteolytic cleavages by beta-site APP-cleaving enzyme and gamma-secretase. Unexpectedly gamma-secretase inhibitors can increase the secretion of Abeta peptides under some circumstances. This "Abeta rise" phenomenon, the same inhibitor causing an increase in Abeta at low concentrations but inhibition at higher concentrations, has been widely observed. Here we show that the Abeta rise depends on the beta-secretase-derived C-terminal fragment of APP (betaCTF) or C99 levels with low levels causing rises. In contrast, the N-terminally truncated form of Abeta, known as "p3," formed by alpha-secretase cleavage, did not exhibit a rise. In addition to the Abeta rise, low betaCTF or C99 expression decreased gamma-secretase inhibitor potency. This "potency shift" may be explained by the relatively high enzyme to substrate ratio under conditions of low substrate because increased concentrations of inhibitor would be necessary to affect substrate turnover. Consistent with this hypothesis, gamma-secretase inhibitor radioligand occupancy studies showed that a high level of occupancy was correlated with inhibition of Abeta under conditions of low substrate expression. The Abeta rise was also observed in rat brain after dosing with the gamma-secretase inhibitor BMS-299897. The Abeta rise and potency shift are therefore relevant factors in the development of gamma-secretase inhibitors and can be evaluated using appropriate choices of animal and cell culture models. Hypothetical mechanisms for the Abeta rise, including the "incomplete processing" and endocytic models, are discussed.     

Overexpression of glycogen synthase in mouse muscle results in less branched glycogen

Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.     

Host Exopolysaccharide Quantity and Composition Impact Erwinia amylovora Bacteriophage Pathogenesis

Erwinia amylovora bacteriophages (phages) belonging to the Myoviridae and Podoviridae families demonstrated a preference for either high-exopolysaccharide-producing (HEP) or low-exopolysaccharide-producing (LEP) bacterial hosts when grown on artificial medium without or with sugar supplementation. Myoviridae phages produced clear plaques on LEP hosts and turbid plaques on HEP hosts. The reverse preference was demonstrated by most Podoviridae phages, where clear plaques were seen on HEP hosts. Efficiency of plating (EOP) was determined by comparing phage growth on the original isolation host to the that on the LEP or HEP host. Nine of 10 Myoviridae phages showed highest EOPs on LEP hosts, and 8 of 11 Podoviridae phages had highest EOPs on HEP hosts. Increasing the production of EPS on sugar-supplemented medium or decreasing production by knocking out the synthesis of amylovoran or levan, the two EPSs produced by E. amylovora, indicated that these components play crucial roles in phage infection. Amylovoran was virtually essential for proliferation of most Podoviridae phages when phage population growth was compared to the wild type. Decreased levan production resulted in a significant reduction of progeny from phages in the Myoviridae family. Thus, Podoviridae phages are adapted to hosts that produce high levels of exopolysaccharides and are dependent on host-produced amylovoran for pathogenesis. Myoviridae phages are adapted to hosts that produce lower levels of exopolysaccharides and host-produced levan.     

Blood flow downstream of a two-dimensional bifurcation

Many cardiovascular lesions such as aneurysms, intimal cushions, and atherosclerotic plaques tend to occur near bifurcations. This suggests that hemodynamic factors may be involved. Since measuring devices (such as anemometers) are still too large to allow local measurements of flow disturbances, we have attempted to predict the nature of these factors mathematically. Biological variables include pulsatile flow of a nonNewtonian fluid in distensible branching vessels with different angles and flow rates. Our initial analysis considers the flow in a two-dimensional bifurcation with a symmetrical flow divider perfused with steady flow at variable Reynolds numbers. At all flows, high shear forces develop on either side of the flow divider (i.e. at the apex of the bifurcation). With high flows, regions of sluggish or reverse flow develop near the outer walls of the bifurcation. The analysis confirms that the flow at the apex is quite different from that at the outer angles and that the latter varies more with flow rate than the former.