A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

The frequency of "rare" protein variants in Marshall islanders and other Micronesians.

Blood specimens from a sample of 373 Marshall Islanders were studied with reference to variants of 23 serum proteins and erythrocyte enzymes. Six of the traits studied exhibited genetic polymorphisms (adenosine deaminase, phosphoglucomutase1, acid phosphatase, 6-phosphogluconate dehydrogenase, haptoglobin, and group specific component). There were in addition four "rare" variants (albumin, transferrin, lactate dehydrogenase, and galactose-1-phosphate uridylyltransferase) involving nine persons, among 8,503 determinations. The frequency of rare variants in Micronesians was compared with the frequencies in West European Caucasians and Amerindians. There are many difficulties in such comparisons, and although the observed values for the three ethnic groups differ by a factor of three (the Micronesians exhibiting the lowest frequency), it is felt that no firm conclusions concerning differences between ethnic groups can be drawn at this time.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Ancient DNA analyses reveal high mitochondrial DNA sequence diversity and parallel morphological evolution of late pleistocene cave bears

Cave bears (Ursus spelaeus) existed in Europe and western Asiauntil the end of the last glaciation some 10,000 years ago.To investigate the genetic diversity, population history, andrelationship among different cave bear populations, we havedetermined mitochondrial DNA sequences from 12 cave bears thatrange in age from about 26,500 to at least 49,000 years andoriginate from nine caves. The samples include one individualfrom the type specimen population, as well as two small-sizedhigh-Alpine bears. The results show that about 49,000 yearsago, the mtDNA diversity among cave bears was about 1.8-foldlower than the current species-wide diversity of brown bears(Ursus arctos). However, the current brown bear mtDNA gene poolconsists of three clades, and cave bear mtDNA diversity is similarto the diversity observed within each of these clades. The resultsalso show that geographically separated populations of the high-Alpinecave bear form were polyphyletic with respect to their mtDNA.This suggests that small size may have been an ancestral traitin cave bears and that large size evolved at least twice independently.     

PHYSIOLOGICAL CONTROL OF CHAMISE SHOOT GROWTH AFTER FIRE

Five controlled burns (ca. 2 ha each) were conducted in the Coast Range of northern California near Hopland, California, between November 18, 1975 and September 15, 1976 to determine the effect of shrub phenology on the sprouting response of chamise (Adenostoma fasciculatum H&A) following fire. The time of burn had little effect on the amount of shoot growth that occurred after fire, although the pattern of growth was altered. Shrubs burned on June 22 or August 2, 1976 grew continually until August 1977, while unburned shrubs or those burned at other times ceased seasonal growth during the first summer after fire. Neither the amount nor the pattern of shoot growth was influenced by the shrub water status before the fire occurred. The relationship between chamise shoot growth after fire, ¹⁴CO₂ fixation, xylem sap tension, and air temperature was also studied. The growth rate of regrowing shrubs was highly correlated with ¹⁴CO₂ fixation prior to the growth measurement. Water status (pre-dawn xylem sap tension) had a negative correlation with ¹⁴CO₂ fixation. The abundance of carbohydrate at the shoot apex is believed to influence the seasonal pattern and rate of chamise shoot growth following fire.     

Establishment and Assessment of a New Human Embryonic Stem Cell‐Based Biomarker Assay for Developmental Toxicity Screening

A metabolic biomarker‐based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery‐phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure‐based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9‐point dose–response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.