Conversion of midbodies into germ cell intercellular bridges

Whereas somatic cell cytokinesis resolves with abscission of the midbody, resulting in independent daughter cells, germ cell cytokinesis concludes with the formation of a stable intercellular bridge interconnecting daughter cells in a syncytium. While many proteins essential for abscission have been discovered, until recently, no proteins essential for mammalian germ cell intercellular bridge formation have been identified. Using TEX14 as a marker for the germ cell intercellular bridge, we show that TEX14 co-localizes with the centralspindlin complex, mitotic kinesin-like protein 1 (MKLP1) and male germ cell Rac GTPase-activating protein (MgcRacGAP) and converts these midbody matrix proteins into stable intercellular bridge components. In contrast, septins (SEPT) 2, 7 and 9 are transitional proteins in the newly forming bridge. In cultured somatic cells, TEX14 can localize to the midbody in the absence of other germ cell-specific factors, suggesting that TEX14 serves to bridge the somatic cytokinesis machinery to other germ cell proteins to form a stable intercellular bridge essential for male reproduction.     

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.     

Alcohol regulates eukaryotic elongation factor 2 phosphorylation via an AMP-activated protein kinase-dependent mechanism in C2C12 skeletal myocytes

Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.     

Sequencing complete mitochondrial and plastid genomes

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing" protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing" protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.     

Induced prion protein controls immune-activated retroviruses in the mouse spleen

The prion protein (PrP) is crucially involved in transmissible spongiform encephalopathies (TSE), but neither its exact role in disease nor its physiological function are known. Here we show for mice, using histological, immunochemical and PCR-based methods, that stimulation of innate resistance was followed by appearance of numerous endogenous retroviruses and ensuing PrP up-regulation in germinal centers of the spleen. Subsequently, the activated retroviruses disappeared in a PrP-dependent manner. Our results reveal the regular involvement of endogenous retroviruses in murine immune responses and provide evidence for an essential function of PrP in the control of the retroviral activity. The interaction between PrP and ubiquitous endogenous retroviruses may allow new interpretations of TSE pathophysiology and explain the evolutionary conservation of PrP.     

Evaluation of risk profiles by subcutaneous adipose tissue topography in obese juveniles

Objective: To compare subcutaneous adipose tissue topography (SAT‐top) in obese juveniles with age‐matched normal‐weight controls. Research Methods and Procedures: The optical device LIPOMETER (European Patent EP 0516251) enables the non‐invasive, rapid, safe, and precise measurement of the thickness of subcutaneous adipose tissue. Fifteen defined body sites (1 = neck to 15 = calf) characterize the individual SAT‐top like an individual fingerprint. SAT‐top of 1351 juveniles (obese: 42 boys, 59 girls, normal weight: 680 boys, 570 girls) from 7 to 19 years of age were measured. For visual comparison, the 15‐dimensional SAT‐top information was condensed by factor analysis into a two‐dimensional factor plot. Results: Both female and male obese juveniles had markedly increased adipose tissue layers at 7 = upper abdomen, 8 = lower abdomen, 5 = front chest, and 6 = lateral chest. The pubertal changes of body shape and fat distribution of the normal‐weight boys and girls (boys show thinner adipose tissue layers on their legs, whereas girls had thicker adipose tissue layers at the extremities) were not seen in the obese group. Independently of age and sex, all of the obese juveniles showed a similar, more android body fat distribution with increased trunk fat. Discussion: SAT‐top of the obese juveniles is similar to that of patients with type 2 diabetes, polycystic ovary syndrome, and coronary heart disease. Patients with these metabolic disorders and obese juveniles are located in the factor plot in the same area. This body shape may indicate a risk profile for developing polycystic ovary syndrome (women), type 2 diabetes, and early atherosclerosis (both sexes).     

Micropearls and other intracellular inclusions of amorphous calcium carbonate: an unsuspected biomineralization capacity shared by diverse microorganisms

An unsuspected biomineralization process, which produces intracellular inclusions of amorphous calcium carbonate (ACC), was recently discovered in unicellular eukaryotes. These mineral inclusions, called micropearls, can be highly enriched with other alkaline-earth metals (AEM) such as Sr and Ba. Similar intracellular inclusions of ACC have also been observed in prokaryotic organisms. These comparable biomineralization processes involving phylogenetically distant microorganisms are not entirely understood yet. This review gives a broad vision of the topic in order to establish a basis for discussion on the possible molecular processes behind the formation of the inclusions, their physiological role, the impact of these microorganisms on the geochemical cycles of AEM and their evolutionary relationship. Finally, some insights are provided to guide future research.