The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila.

The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus. A mutant in which the replication properties of this molecule were altered was isolated and characterized. This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D. L. Larson, E. H. Blackburn, P. C. Yaeger, and E. Orias, Cell 47:229-240, 1986). The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion. This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1. Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion. In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA. The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage. We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T. thermophilia.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Trypanosoma cruzi: a considerable phylogenetic divergence indicates that the agent of Chagas disease is indigenous to the native fauna of the United States.

Thirty U.S. Trypanosoma cruzi stocks isolated mainly from wild mammals were characterized by multilocus enzyme electrophoresis at 22 genetic loci and random amplification of polymorphic DNA for 10 primers. Two main phylogenetic clusters, separated by large genetic distances, were discriminated by both methods, corresponding, respectively, to the formerly described zymodemes I and III. Two stocks isolated from indigenous human cases were identified as zymodeme I. Genetic diversity of the U.S. T. cruzi isolates was considerable, comparable to that scored in similarly sized samples from South America. These results favor the hypothesis that T. cruzi U.S. stocks were not imported at a historical time and are indigenous to the native fauna of the United States. The population structure of these stocks appeared to be basically clonal, as previously reported in South America, and no evidence of hybrid genotypes was found in the United States.     

Accelerated formation of α-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations

A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. α-Synuclein (αSyn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves αSyn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length αSyn. Three point mutations in αSyn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of αSyn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of αSyn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype αSyn. These truncations accelerated fibrillization of all α-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of αSyn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of αSyn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated α-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.     

Which population level environmental factors are associated with asthma,rhinoconjunctivitis and eczema? Review of the ecological analyses of ISAAC Phase One

The International Study of Asthma and Allergies in Childhood (ISAAC) Phase One showed large worldwide variations in the prevalence of symptoms of asthma, rhinoconjunctivitis and eczema, up to 10 to 20 fold between countries. Ecological analyses were undertaken with ISAAC Phase One data to explore factors that may have contributed to these variations, and are summarised and reviewed here.In ISAAC Phase One the prevalence of symptoms in the past 12 months of asthma, rhinoconjunctivitis and eczema were estimated from studies in 463,801 children aged 13 - 14 years in 155 centres in 56 countries, and in 257,800 children aged 6-7 years in 91 centres in 38 countries. Ecological analyses were undertaken between symptom prevalence and the following: Gross National Product per capita (GNP), food intake, immunisation rates, tuberculosis notifications, climatic factors, tobacco consumption, pollen, antibiotic sales, paracetamol sales, and outdoor air pollution.Symptom prevalence of all three conditions was positively associated with GNP, trans fatty acids, paracetamol, and women smoking, and inversely associated with food of plant origin, pollen, immunisations, tuberculosis notifications, air pollution, and men smoking. The magnitude of these associations was small, but consistent in direction between conditions. There were mixed associations of climate and antibiotic sales with symptom prevalence.The potential causality of these associations warrant further investigation. Factors which prevent the development of these conditions, or where there is an absence of a positive correlation at a population level may be as important from the policy viewpoint as a focus on the positive risk factors. Interventions based on small associations may have the potential for a large public health benefit.     

Characterization of proliferating human skeletal muscle-derived cells in vitro: differential modulation of myoblast markers by TGF-beta2

Adult human skeletal muscle-derived cells (HuSkMC) propagated in vitro are under investigation as a cell-based therapy for the treatment of myocardial infarction. We have characterized HuSkMC with respect to cell identity and state of differentiation as a prerequisite to their clinical use. Flow cytometric analysis of propagated HuSkMC revealed a population of cells that expressed the myoblast markers CD56 and desmin. The presence of myoblasts in these cultures was further confirmed by their capacity to form myotubes and increase creatine kinase activity when cultured in low serum conditions. The non-myoblast fraction of these propagated cells expressed TE7, a marker associated with the fibroblast phenotype. Spontaneous differentiation of myoblasts occurred during serial propagation of HuSkMC, as judged by myotube formation, thereby reducing the myoblast representative fraction with continued cell expansion. We examined transforming growth factor beta2 (TGF-beta2) for its utility in controlling this spontaneous differentiation of adult human myoblasts in vitro. Propagation of HuSkMC in the presence of 1 ng/ml TGF-beta2 for 5 days decreased desmin expression within the myoblast population and caused a parallel reduction of creatine kinase activity. CD56 expression was unaffected, indicating a differential regulation of these myoblast markers. The reduction in desmin expression and creatine kinase activity was, however, reversible upon the removal of TGF-beta. These data collectively indicate that TGF-beta2 restrained differentiation of adult human skeletal myoblasts during propagation without causing irreversible loss of the myoblast phenotype, demonstrating the potential utility of using TGF-beta2 during cultivation and expansion of HuSkMC intended for therapeutic implantation.