The ATPase activity of Fml1 is essential for its roles in homologous recombination and DNA repair

In fission yeast, the DNA helicase Fml1, which is an orthologue of human FANCM, is a key component of the machinery that drives and governs homologous recombination (HR). During the repair of DNA double-strand breaks by HR, it limits the occurrence of potentially deleterious crossover recombinants, whereas at stalled replication forks, it promotes HR to aid their recovery. Here, we have mutated conserved residues in Fml1’s Walker A (K99R) and Walker B (D196N) motifs to determine whether its activities are dependent on its ability to hydrolyse ATP. Both Fml1^K99R and Fml1^D196N are proficient for DNA binding but totally deficient in DNA unwinding and ATP hydrolysis. In vivo both mutants exhibit a similar reduction in recombination at blocked replication forks as a fml1Δ mutant indicating that Fml1’s motor activity, fuelled by ATP hydrolysis, is essential for its pro-recombinogenic role. Intriguingly, both fml1^K99R and fml1^D196N mutants exhibit greater sensitivity to genotoxins and higher levels of crossing over during DSB repair than a fml1Δ strain. These data suggest that without its motor activity, the binding of Fml1 to its DNA substrate can impede alternative mechanisms of repair and crossover avoidance.     

The Socioeconomic and Institutional Determinants of Participation in India’s Health Insurance Scheme for the Poor

The Rashtriya Swasthya Bima Yojana (RSBY), which was introduced in 2008 in India, is a social health insurance scheme that aims to improve healthcare access and provide financial risk protection to the poor. In this study, we analyse the determinants of participation and enrolment in the scheme at the level of districts. We used official data on RSBY enrolment, socioeconomic data from the District Level Household Survey 2007–2008, and additional state-level information on fiscal health, political affiliation, and quality of governance. Results from multivariate probit and OLS analyses suggest that political and institutional factors are among the strongest determinants explaining the variation in participation and enrolment in RSBY. In particular, districts in state governments that are politically affiliated with the opposition or neutral parties at the centre are more likely to participate in RSBY, and have higher levels of enrolment. Districts in states with a lower quality of governance, a pre-existing state-level health insurance scheme, or with a lower level of fiscal deficit as compared to GDP, are significantly less likely to participate, or have lower enrolment rates. Among socioeconomic factors, we find some evidence of weak or imprecise targeting. Districts with a higher share of socioeconomically backward castes are less likely to participate, and their enrolment rates are also lower. Finally, districts with more non-poor households may be more likely to participate, although with lower enrolment rates.     

Effect of TGF-Alpha on growth of normal human breast epithelial cells in serum-free primary culture using 3-dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.     

Cdk5-mediated Acn/Acinus phosphorylation regulates basal autophagy independently of metabolic stress

In neurons, autophagy counteracts consequences of aging. It is therefore of interest how basal rates of macroautophagy/autophagy can be controlled independently of metabolic stress. We recently investigated the regulation of basal, starvation-independent autophagy by Acn/Acinus, a multifunctional nuclear protein with proposed roles in apoptosis, alternative RNA splicing, and basal autophagy. We found that Acn is stabilized by phosphorylation of the conserved serine 437. The phosphomimetic Acn^S437D mutation causes no overt developmental phenotypes, but significantly elevates levels of basal autophagy and extends life spans. An RNAi screen identified Cdk5 as a kinase targeting S437, a role confirmed by gain- and loss-of-function mutants of Cdk5 or its obligatory cofactor Cdk5r1/p35. Flies lacking Cdk5 function display reduced basal autophagy and a shortened life span. Both of these phenotypes are suppressed by the phosphomimetic Acn^S437D mutation, indicating that phosphorylating serine 437 of Acn, and thereby maintaining basal levels of autophagy, is critical for Cdk5's function in maintaining neuronal health.     

A Route to Direct Fitness: Natural and Experimentally Induced Queen Succession in the Tropical Primitively Eusocial Wasp <Emphasis Type="Italic">Ropalidia marginata</Emphasis>

Insect societies are hallmarks of cooperation because one or a few queens monopolize reproduction and several non-reproductive workers cooperatively raise brood. However, the loss of the queen exposes a colony to potential reproductive conflict, which is resolved only after a new queen takes over. We studied queen succession in natural and experimental colonies of the primitively eusocial wasp Ropalidia marginata to understand the proximate behavioral strategies involved in the resolution of this conflict. Previous work has shown that in this species, experimental queen removal always results in only one worker becoming hyper-aggressive and taking over the colony as its next queen, without ever being challenged. Here we show that even during natural queen turnover, one and only one worker becomes hyper-aggressive and takes over as the next queen, without being challenged. During natural queen turn-over, aggression of the successor may sometimes begin before the loss of the old queen and may sometimes decline more rapidly, unlike in the case of experimental queen removal. The successor begins to lay eggs sooner after a natural queen turn-over as compared to experimental queen removal. This is expected because workers might detect the gradual decline of the queen preceding her disappearance. Because queen succession is expected to be more prevalent in tropical perennial species, we expect natural selection to have favored such an orderly queen succession so that a route to direct fitness is available without significant reduction in cooperation.     

Metabolism of Adenine and Hypoxanthine in a Hormone Autonomous Genetic Tumour Line of Tobacco

Genetic tumour tissues of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grow on auxin and cytokinin-free medium, were incubated with [14C]-/[3H]-adenine or [3H]-hypoxanthine to investigate cytokinin biosynthesis. Adenine was supplied to tissues of two different ages (2- and 3.5-week-old) for 8, 24 or 30 h. The uptake was over 91.0 % (of "supplied radioactivity") by 2-week-old tissues as compared to around 50.0 % uptake by 3.5-week-old tissues. Incorporation into cytokinins could not be detected. While unmetabolized adenine accounted for only about 24.0 and 13.4 % of "extracted radioactivity" (following 8 and 30 h incubation, respectively) in 2-week-old tissues, relatively higher levels, i.e. 36.0 and 34.5 % (following 8 and 24 h incubation, respectively) were present in 3.5-week-old tissues. The metabolites formed were adenosine and its nucleotides (4.5 - 16.5 % and 37.4 - 60.2 % of the extracted radioactivity, respectively). Hypoxanthine was supplied to 3.5-week-old tissues for 8 and 24 h. While the uptake was low (<28.0 % of supplied radioactivity), the major proportion of extracted radioactivity was due to unmetabolized hypoxanthine (79.8 % and 85.9 % after 8 and 24 h incubation periods, respectively); the minor metabolites were inosine and adenosine (both <0.5 %) and their nucleotides (< 3.5 %). Radioactivity incorporation into cytokinins from hypoxanthine was not detected. Thus in the present investigations precursor incorporation from either adenine or hypoxanthine into cytokinins could not be demonstrated. It is possible that this may be due to slow rate of cytokinin turnover in these tissues.     

Metabolism of zeatin riboside in a hormone autonomous genetic tumour line of tobacco

The metabolic fate of externally applied [³H]-zeatin riboside ([9R]Z) was studied in a cultured genetic tumour line of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grows on auxin and cytokinin free medium. Metabolism by 3.5-week-old tissues showed enhanced stability of supplied [9R]Z; unmetabolized [9R]Z accounted for 48.7 and 37.5% of extracted radioactivity following 8 and 24 h incubation, respectively; tissues of different ages (1–10 weeks following subculture) also indicated high cytokinin stability following 8 h incubation (unmetabolized [9R]Z accounted for 32.5–53.0% of extracted radioactivity). All analyses were performed by thin layer chromatography (TLC) and the results subsequently confirmed by high performance liquid chromatography (HPLC). Side-chain cleavage and modification of the purine ring were the major forms of metabolism; metabolites with an intact cytokinin moiety included zeatin (Z), [9R]Z nucleotides and glucosyl derivatives. Detailed analysis of metabolites carried out in the experiments using 3.5-week-old tissues indicated that both dihydro-derivatives as well as cis isomers of Z and [9R]Z were not formed. Adenine, adenosine and its nucleotide(s) were the main degradative metabolites; in 3.5-week-old tissues these metabolites accounted for about 5.9 and 7.8% (of ³H extracted) following 8 and 24 h incubation, respectively. In tissues of different ages (1–10 weeks following subculture), these metabolites accounted for about 7.6–22.9% of the extracted ³H. Some metabolites (zeatin, adenine and adenosine) were also detected in the staled incubation media. The observed high [9R]Z stability in this tissue may reflect low levels of cytokinin oxidase activity and/or some form of compartmentation.