Reconstitution of a minimal mtDNA replisome in vitro

We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.     

A Model to Induce Low Temperature Trauma for <Emphasis Type="Italic">in vitro</Emphasis> Astrogliosis Study

Astrogliosis is an inevitable and rapid response of astrocytes to physical, chemical and pathological injuries. To study astrogliosis, we developed a reproducible in vitro model in which low temperature injury to cultured astrocytes could be induced by placing the culture dish onto a copper pipe pre-cooled by liquid nitrogen. Using this model, the relationship between the temperature decline and the severity of cellular damage was analyzed. An increase in the expression of some known injury-related proteins, such as glial fibrillary acidic protein (GFAP), immediate early response genes (IEGs), and heat shock proteins 70 (HSP70), was demonstrated in astrocytes after low temperature trauma. With the use of this low temperature trauma model, the flexibility in the temperature control and injury area may allow researchers to evaluate cryotherapy and cryosurgery, which could be applicable to future development of quality health care.Special issue dedicated to Lawrence F. Eng.     

EP4 emerges as a novel regulator of bile acid synthesis and its activation protects against hypercholesterolemia

Prostaglandin E receptor subtype 4 (EP4) knockout mice develops spontaneous hypercholesterolemia but the detailed mechanisms by which EP4 affects cholesterol homeostasis remains unexplored. We sought to determine the cause of hypercholesterolemia in EP4 knockout mice, focusing on the role of EP4 in regulating the synthesis and elimination of cholesterol. Deficiency of EP4 significantly decreased total bile acid levels in the liver by 26.2% and the fecal bile acid content by 27.6% as compared to wild type littermates, indicating that the absence of EP4 decreased hepatic bile acid synthesis and their subsequent excretion in stools. EP4 deficiency negatively regulate bile acid synthesis through repression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK)-mediated cholesterol 7α-hydroxylase (CYP7A1) expression and that the hypercholesterolemia in EP4 knockout mice is due to a defect in cholesterol conversion into bile acids. Deficiency of EP4 also increased de novo cholesterol synthesis and altered cholesterol fluxes in and out of the liver. Treating high fat diet-challenged mice with the pharmacological EP4 agonist, CAY10580 (200?μg/kg body weight/day i.p) for three weeks effectively prevented diet-induced hypercholesterolemia, enhanced endogenous bile acid synthesis and their fecal excretion. In summary, EP4 plays a critical role in maintaining cholesterol homeostasis by regulating the synthesis and elimination of bile acids. Activation of EP4 serves as an effective novel strategy to promote cholesterol disposal in the forms of bile acids in order to lower plasma cholesterol levels.     

Karyotypes of Malayan rats (Rodentia-Muridae,genus Rattus Fischer)

The karyotypes of 18 species of Malayan rats, genus Rattus Fischer, are reported. Present studies reveal that the karyotype of rats is not species-specific. The variability in diploid number (2n) within the genus Rattus is reaffirmed. A range of 36 to 52 is reported, with 2n=42 having the highest frequency. The affinity of Malayan rats is discussed on the basis of chromosomal evidence.     

TRIP13 Functions in the Establishment of the Spindle Assembly Checkpoint by Replenishing O-MAD2

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Egg burial in penduline tits, Remiz pendulinus: its role in mate desertion and female polyandry

One important component in the mating strategy of an already-matedindividual is the decision to remain with the partner and carefor the offspring or to desert. Almost all research on nestdesertion has focused on the costs and benefits of continuedparental care versus desertion of both parents. However, ifit pays both parents to desert, the timing of desertion is mostimportant. In birds, where all eggs are fertilized well beforethe last egg is laid, males should be the first to desert. Evenif females try to hide their fertile period, it is likely thatthe appearance of eggs acts as a cue that males can use to calculatethe timing of their desertion. Here we examine egg burial behaviorin penduline tits and its possible effects on parental behaviorand desertion. Penduline tits perform uniparental care fromthe earliest point of breeding, and both sexes try to becomepolygamous. We found that 36% of investigated males were polygynousand deserted as soon as the first egg appeared in their nest,and 12.5% of females became polyandrous. About 27% of the nestswere deserted by both sexes, which means high costs for femalesin terms of wasted energy in eggs and for males in terms ofwasted energy and time in building elaborate nests. Femalescover the eggs, and several facts indicate that egg coveringis a deceptive behavior of females: (1) females cover the eggsin the morning before leaving the nest for the first time, (2)females are more aggressive toward their mates during the layingperiod than before laying, (3) females try to prevent malesfrom entering the nest when eggs have been experimentally uncovered,and (4) females uncover the eggs as soon as males are experimentallyremoved. Finally, we found that a female can only desert thenest before the male deserts when she covers the eggs. We concludethat the higher the proportion of eggs a female can hide, thegreater her chance of becoming polyandrous     

An alternative route to coloniality in the bearded tit: females pursue extra-pair fertilizations

We combine behavioral studies with DNA-fingerprinting of a populationof bearded tits Panurus biarmicus and test the idea that thepursuit of extra-pair copulations by females acts as selectivepressure that may contribute to the formation of colonies inthis socially monogamous species. We show that the rate of extra-pairpaternity in colonial breeding pairs is very high, whereas noextra-pair chicks were detected in solitary nests. We foundno cases of extra-pair maternity and intraspecific brood parasitismin solitary nests, whereas it did occur in dense breeding situations.We further found no difference in the quality of males betweencolonial and solitary individuals. However, colonial femaleswere of significantly higher quality in terms of body size andcondition. We propose that high-quality females settle in coloniesto increase their opportunities to adjust the choice of theirsocial partner by obtaining extra-pair copulations. We suggestthat colony formation enables high-quality females to incitemale-male competition for extra-pair copulations and consequentlysecure extra-pair fertilizations by high-quality males and thathigh-density nesting is controlled by female bearded tits     

Nest Predation Avoidance: an Alternative Explanation for Male Incubation in Acrocephalus melanopogon

In the monogamous moustached warbler, male incubation changes from predictably variable (it is dependent on ambient temperature and time of day in April) to high average levels across the day (with no predictor variables in May) as the season progresses. In contrast, females contribute the constant incubation component from April to May. This paper investigates possible explanations for the change in male incubation effort involving changing risks to either (1) embryonic survival within the egg, and/or (2) egg predation. Using egg temperature readouts, the probability of reaching the 25 °C thermal threshold (below which embryonic development ceases) across the season against the probability of predator sightings 0–15 m from the nest was calculated. The results show an inverse relationship between these two risks. During April, male incubation correlates with egg cold stress and changeovers between males and females occur prior to egg cooling below the thermal stress line. During May, the risk of predation increases. The results show increased predator encounter rates from April to May and active nest defense by the incubating parent. Furthermore, high male incubation reduces brood predation. Selection for reduction of the costs of laying replacement clutches (after predation) is suggested given high male incubation and infrequent male-female changeovers during midday, when egg temperatures are highest, with direct benefits to females of increased foraging. Thus, the shift in male incubation across the season may be explained by minimization of changing risk to offspring survival.     

Parental Investment and Territorial/Sexual Behavior in Male and Female Reed Warblers: Are they Mutually Exclusive?

The behavioral and endocrine changes from prenesting to feeding phases of reproduction were examined in free-living reed warblers. Apart from the onset of feeding behavior, these changes in males are particularly evident in song, territorial behavior and circulating levels of testosterone. The interactions between an individual's physiology and behavior during these behavioral transitions were tested first by correlating song and feeding rates with circulating levels of testosterone and secondly by implanting feeding males with steroids and documenting the resulting effects on song, territoriality and parental behavior. The physiological mechanisms associated with the transition from the sexual phase (nest-building and egg-laying) to the parental (incubation and feeding) in females were also examined. In this case, estrogens were implanted during the parental phase to enhance sexual behavior. The results demonstrate an antagonistic relationship between sexual/territorial behavior and parental care in males which is mediated by changes in circulating androgens and no such antagonism in females. The effects of parental manipulation on the feeding rates of the partners and the growth rates of offspring have been examined to shed light on the necessity for male parental input.