Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2

Many protein kinases are activated by a conserved regulatory step involving T-loop phosphorylation. Although there is considerable focus on kinase activator proteins, the importance of specific T-loop phosphatases reversing kinase activation has been underappreciated. We find that the protein phosphatase 6 (PP6) holoenzyme is the major T-loop phosphatase for Aurora A, an essential mitotic kinase. Loss of PP6 function by depletion of catalytic or regulatory subunits interferes with spindle formation and chromosome alignment because of increased Aurora A activity. Aurora A T-loop phosphorylation and the stability of the Aurora A-TPX2 complex are increased in cells depleted of PP6 but not other phosphatases. Furthermore, purified PP6 acts as a T-loop phosphatase for Aurora A-TPX2 complexes in vitro, whereas catalytically inactive mutants cannot dephosphorylate Aurora A or rescue the PPP6C depletion phenotype. These results demonstrate a hitherto unappreciated role for PP6 as the T-loop phosphatase regulating Aurora A activity during spindle formation and suggest the general importance of this form of regulation.     

Ants accelerate succession from mountain grassland towards spruce forest

Question: What is the role of mound‐building ants (Lasius flavus) in successional changes of a grassland ecosystem towards a spruce forest? Location: Slovenské Rudohorie Mountains, Slovakia; ca. 950 m a.s.l. near the Obrubovanec point (1020 m a.s.l.; 48°41′N, 19°39′E). Methods: Both chronosequence data along a successional gradient and temporal data from long‐term permanent plots were collected on ants, spruce establishment, and vegetation structure, together with additional data on spruce growth. Results: There are more spruce seedlings on ant mounds (4.72 m^?2) than in the surrounding vegetation (0.81 m^?2). Spruce seedlings grow faster on these mounds compared to surrounding areas. The first colonization wave of seedlings was rapid and probably occurred when grazing prevailed over mowing. Ant colony presence, mound volume, and plant species composition change along the successional gradient. Mounds become bigger when partly shaded but shrink in closed forest, when ant colonies disappear. Shade‐tolerant acidophylic species replace grassland plants both on the mounds and in surrounding areas. Conclusions: The massive occurrence of Lasius flavus anthills contributes to a runaway feedback process that accelerates succession towards forest. The effect of ants as ecosystem engineers is scale‐dependent: although they stabilize the system at the scale of an individual mound, they may destabilize the whole grassland system over a longer time scale if combined with changes in mowing regime.     

Effects of seed burial on germination,protein mobilisation and seedling survival in Dodonaea viscosa

Ecological restoration of disturbed areas requires substantial knowledge of the germination of native plants and the creation of novel methods to increase seedling establishment in the field. We studied the effects of soil matrix priming on the germination of Dodonaea viscosa seeds, which exhibit physical dormancy. To this end, we buried both pre‐scarified (in H₂SO₄, 3 min) and non‐pre‐scarified seeds in the Parque Ecológico de la Ciudad de México. After seeds were unearthed, they were post‐scarified for 0, 2, 6 and 10 min and their germination percentages compared to the germination of a control batch of laboratory‐stored seeds. For both control and unearthed seeds, the protein pattern was determined in the enriched storage protein fraction in SDS‐PAGE gels stained with Coomassie blue. Percentage germination increased as the scarification time increased. Pre‐scarification significantly increased percentage germination of post‐scarified seeds in relation to the control and non‐pre‐scarified seeds. In seeds unearthed from the forest site, the buried pre‐scarified seeds had relatively high percentage germination, even in the absence of post‐scarification treatment. A 48‐kDa protein was not found in unearthed, pre‐scarified seeds nor in the control germinated seeds, indicating that mobilisation of this protein occurred during soil priming. Burying seeds for a short period, including the beginning of the rainy season, promoted natural priming, which increased protein mobilisation. Functionally, priming effects were reflected in high percentage seedling survival in both the shade house and the field. Seed burial also reduced the requirement for acidic post‐scarification.     

Ocular toxoplasmosis: evaluation of lacrimal-specific secretory IgA levels in both patients with active and inactive phases of the disease

Ocular toxoplasmosis can result in recurrent uveitis. Studies have shown that a correlation between active ocular toxoplasmosis and the presence of anti-Toxoplasma gondii secretory IgA (SIgA) in tears. This study compares anti-T. gondii SIgA levels in patients' tears during the acute and inactive phases of toxoplasmic uveitis. Twenty-nine positive tear specific SIgA for T. gondii patients with acute toxoplasmic uveitis were selected and were followed-up for at least two years, when the anti-T. gondii SIgA tears levels were determined. Specific SIgA for T. gondii was negative in 22 patients (75.86%) and positive in seven patients (24.13%) of whom six (85.7%) were followed over three years. Average SIgA levels during the acute phase are 1.54 and decrease significantly to 0.72 (p = 0.0001) during the inactive phase of disease. Because anti-T. gondii SIgA in the tear is negative in 75.86% of patients after the acute phase of infection, T. gondii SIgA levels may be used as a complementary diagnostic marker for active ocular toxoplasmosis.     

Schistosoma mansoni: SmLIMPETin, a member of a novel family of invertebrate-only regulatory proteins

Eukaryotic LIM domain proteins contain zinc finger forming motifs rich in cysteine and histidine that enable them to interact with other proteins. A cDNA clone isolated from an adult schistosome cDNA library revealed a sequence that coded for a novel class of proteins bearing 6 LIM domains and an N-terminal PET domain, SmLIMPETin. Phylogeny reconstruction of SmLIMPETin and comparison of its sequence to invertebrate homologues and to the vertebrate four-and-a-half LIM domains protein family (FHLs), uncovered a novel LIM domain protein family, the invertebrate LIM and PET domain protein family (LIMPETin). Northern blots, RT-PCR and Western blot showed that SmLIMPETin gene was less expressed in sexually mature adult females compared to sexually immature adult females and sexually mature and immature adult males, and not expressed in schistosomula.     

Hormonal induction of ovulation and artificial insemination in suckled beef cows under nutritional stress

The objective was to develop a program for inducing estrus (followed by insemination) of suckled beef cows under nutritional stress (poor body condition). A total of 123 cows, from 60 to 75 days postpartum, were classified according to their body condition score (BCS; range from 1 to 5, in increments of 0.5) and allocated into two groups. On Day 0 (without regard to stage of the estrous cycle), cows (n = 59) in the hormone induction (HI) treatment group were given an intravaginal device (IVD) containing 250 mg of medroxiprogesterone acetate (MAP) and an i.m. injection of 2.5 mg estradiol benzoate (EB). On Day 6, these cows were given 500 IU eCG i.m. and calves were weaned for 96 h. The IVD were removed on Day 7. Cows detected in estrus by 45 h after IVD removal were inseminated 12 h after standing estrus; cows not in estrus by 45 h after IVD removal received an i.m. injection of 100 microg gonadorelin (GnRH) and were inseminated 16-18 h later. In the control group (C), cows (n = 64) only had their calves weaned at Day 6 (for 96 h), with estrus detection and AI from Days 6 to 11. Overall, the BCS ranged from 2.0 to 3.0. In the treatment group, estrus and pregnancy rates in cows with BCS 2.0 (20 and 30%, respectively) was lower (P < 0.05) than those with BCS 3.0 (50 and 66.6%, respectively), but did not differ (P > 0.05) from BCS 2.5 (23.3 and 47.6%). In C group, only 2 of 66 cows were detected in estrus and bred (neither was pregnant). In conclusion, the program for induction of ovulation using MAP, EB, eCG and GnRH increased the pregnancy rate in beef cows in poor body condition, enabling AI to be done in a 63-h interval.     

Low seroprevalence of Neospora caninum infection associated with the limousin breed in cow-calf herds in Andorra, Europe

Neospora caninum seroprevalence and risk factors affecting seroprevalence in beef cattle in Andorra were investigated. Antibodies to N. caninum were evaluated by enzyme-linked immunosorbent assay performed on a yearly basis in 1,758 animals older than 6 mo, belonging to 26 herds. Mean seroprevalence of antibodies to N. caninum for the herds was 7.4 +/- 1.2% (130/1,758). Logistic regression analyses were performed on data from each animal, considering N. caninum seropositivity as the dependent variable, and herd, grazing area, year of sampling, repeat-test animal (animals sampled twice or more), sex, breed, age (animals <4 yr old or > or =5 yr old), and country of birth as possible risk factors. Based on the odds ratio, the prevalence of infection was 2.1 times higher (P < 0.01) in animals from the Ordino grazing area, 1.64 times higher in animals older than 5 yr (P < 0.01), and 6.7 times (1/0.15) lower in Limousin-mixed Limousin cattle (P < 0.002). The results suggest that the particular grazing location could promote the horizontal transmission of this parasite and that certain breeds are less susceptible to N. caninum infection than others.     

Cell cycle inhibitor, p19INK4d, promotes cell survival and decreases chromosomal aberrations after genotoxic insult due to enhanced DNA repair

Genome integrity and cell proliferation and survival are regulated by an intricate network of pathways that includes cell cycle checkpoints, DNA repair and recombination, and programmed cell death. It makes sense that there should be a coordinated regulation of these different processes, but the components of such mechanisms remain unknown. In this report, we demonstrate that p19INK4d expression enhances cell survival under genotoxic conditions. By using p19INK4d-overexpressing clones, we demonstrated that p19INK4d expression correlates with the cellular resistance to UV treatment with increased DNA repair activity against UV-induced lesions. On the contrary, cells transfected with p19INK4d antisense cDNA show reduced ability to repair DNA damage and increased sensitivity to genotoxic insult when compared with their p19INK4d-overexpressing counterparts. Consistent with these findings, our studies also show that p19INK4d-overexpressing cells present not only a minor accumulation of UV-induced chromosomal aberrations but a lower frequency of spontaneous chromosome abnormalities than p19INK4d-antisense cells. Lastly, we suggest that p19INK4d effects are dissociated from its role as CDK4/6 inhibitor. The results presented herein support a crucial role for p19INK4d in regulating genomic stability and overall cell viability under conditions of genotoxic stress. We propose that p19INK4d would belong to a protein network that would integrate DNA repair, apoptotic and checkpoint mechanisms in order to maintain the genomic integrity.     

Clinical trials of Shigella vaccines: two steps forward and one step back on a long, hard road

More than 50 years of research has yielded numerous Shigella vaccine candidates that have exemplified both the promise of vaccine-induced prevention of shigellosis and the impediments to developing a safe and effective vaccine for widespread use, a goal that has yet to be attained. This Review discusses the most advanced strategies for Shigella vaccine development, the immune responses that are elicited following disease or vaccination, the factors that have accelerated or impeded Shigella vaccine development and our ideas for the way forward.