Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

The Hitchhiking Effect on the Site Frequency Spectrum of DNA Polymorphisms

The level of DNA sequence variation is reduced in regions of the Drosophila melanogaster genome where the rate of crossing over per physical distance is also reduced. This observation has been interpreted as support for the simple model of genetic hitchhiking, in which directional selection on rare variants, e.g., newly arising advantageous mutants, sweeps linked neutral alleles to fixation, thus eliminating polymorphisms near the selected site. However, the frequency spectra of segregating sites of several loci from some populations exhibiting reduced levels of nucleotide diversity and reduced numbers of segregating sites did not appear different from what would be expected under a neutral equilibrium model. Specifically, a skew toward an excess of rare sites was not observed in these samples, as measured by Tajima's D. Because this skew was predicted by a simple hitchhiking model, yet it had never been expressed quantitatively and compared directly to DNA polymorphism data, this paper investigates the hitchhiking effect on the site frequency spectrum, as measured by Tajima's D and several other statistics, using a computer simulation model based on the coalescent process and recurrent hitchhiking events. The results presented here demonstrate that under the simple hitchhiking model (1) the expected value of Tajima's D is large and negative (indicating a skew toward rare variants), (2) that Tajima's test has reasonable power to detect a skew in the frequency spectrum for parameters comparable to those from actual data sets, and (3) that the Tajima's Ds observed in several data sets are very unlikely to have been the result of simple hitchhiking. Consequently, the simple hitchhiking model is not a sufficient explanation for the DNA polymorphism at those loci exhibiting a decreased number of segregating sites yet not exhibiting a skew in the frequency spectrum.     

Strategies for imaging mitophagy in high-resolution and high-throughput

The selective autophagic removal of mitochondria called mitophagy is an essential physiological signaling for clearing damaged mitochondria and thus maintains the functional integrity of mitochondria and cells. Defective mitophagy is implicated in several diseases, placing mitophagy as a target for drug development. The identification of key regulators of mitophagy as well as chemical modulators of mitophagy requires sensitive and reliable quantitative approaches. Since mitophagy is a rapidly progressing event and sub-microscopic in nature, live cell image-based detection tools with high spatial and temporal resolution is preferred over end-stage assays. We describe two approaches for measuring mitophagy in mammalian cells using stable cells expressing EGFP-LC3 – Mito-DsRed to mark early phase of mitophagy and Mitochondria-EGFP – LAMP1-RFP stable cells for late events of mitophagy. Both the assays showed good spatial and temporal resolution in wide-field, confocal and super-resolution microscopy with high-throughput adaptable capability. A limited compound screening allowed us to identify a few new mitophagy inducers. Compared to the current mitophagy tools, mito-Keima or mito-QC, the assay described here determines the direct delivery of mitochondrial components to the lysosome in real time mode with accurate quantification if monoclonal cells expressing a homogenous level of both probes are established. Since the assay described here employs real-time imaging approach in a high-throughput mode, the platform can be used both for siRNA screening or compound screening to identify key regulators of mitophagy at decisive stages.     

Identification and characterization of the nonphosphorylated precursor of pp17, a phosphoprotein associated with phorbol ester induction of growth arrest and monocytic differentiation in HL-60 promyelocytic leukemia cells

Treatment of HL-60 promyelocytic leukemia cells with the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), causes rapid phosphorylation and dephosphorylation of pp17, a 17-20-kDa, pI 5.5 cytosolic protein, as an early event in a response sequence leading to growth arrest and terminal differentiation into monocytes (Feuerstein, N., and Cooper, H. L., (1984) J. Biol. Chem. 259, 2782-2788). In the present study, we have identified the nonphosphorylated precursor to pp17 by tryptic peptide mapping of single proteins recovered from two-dimensional gels. The pI of the precursor, p17, was 5.9, and the apparent Mr of both p17 and pp17 was 18,400 by SDS-polyacrylamide gel electrophoresis (one dimension). p17 was shown to be a major cytosolic protein, comprising about 0.5% of steady state labeled protein in that fraction. Both p17 and pp17 were found exclusively in the cytosol (detergent-released SOL) and were not detected in membranes, cytoskeleton, or nuclei. In untreated cells, about 90% of the protein was present in the nonphosphorylated form. Upon TPA treatment, pre-existing p17 was rapidly phosphorylated to pp17. After 15 min, the two forms were nearly equal in quantity. This corresponds to phosphorylation, within that period, of about 0.2% of total cytosolic protein, represented by a single species. The maximum level of pp17 was reached within 1 h, with pp17 exceeding p17 by about 25%. Quantitatively, therefore, the phosphorylation of p17 to pp17 is one of the most prominent early biochemical responses to TPA treatment. Available data indicate that p17 predominates in rapidly proliferating cells, while phosphorylation to pp17 occurs where cell growth is modified by TPA or other agents. Thus, the p17/pp17 system is potentially a major mechanism for intracellular propagation of growth regulatory signals. We propose the name, prosolin, for this prominent cytosolic protein.     

Human dermatosparaxis: a form of Ehlers-Danlos syndrome that results from failure to remove the amino-terminal propeptide of type I procollagen.

Dermatosparaxis is a recessively inherited connective-tissue disorder that results from lack of the activity of type I procollagen N-proteinase, the enzyme that removes the amino-terminal propeptides from type I procollagen. Initially identified in cattle more than 20 years ago, the disorder was subsequently characterized in sheep, cats, and dogs. Affected animals have fragile skin, lax joints, and often die prematurely because of sepsis following avulsion of portions of skin. We recently identified two children with soft, lax, and fragile skin, which, when examined by transmission electron microscopy, contained the twisted, ribbon-like collagen fibrils characteristic of dermatosparaxis. Skin extracts from one child contained collagen precursors with amino-terminal extensions. Cultured fibroblasts from both children failed to cleave the amino-terminal propeptides from the pro alpha 1(I) and pro alpha 2(I) chains in type I procollagen molecules. Extracts of normal cells cleaved to collagen, the type I procollagen synthesized by cells from both children, demonstrating that the enzyme, not the substrate, was defective. These findings distinguish dermatosparaxis from Ehlers-Danlos syndrome type VII, which results from substrate mutations that prevent proteolytic processing of type I procollagen molecules.     

Hepatic conversion of thyroxine to triiodothyronine in obese and lean Zucker rats

The in vitro conversion of thyroxine (T4) to triiodothyronine (T3) was studied in liver homogenates from fed and fasted lean and obese Zucker rats. T3 generation was decreased in fed young (2 month) obese rats as compared to values in fed lean controls. This was not corrected by the addition of dithiothreitol (DTT), suggesting a deficiency in 5'-deiodinase activity in young obese rats. Both lean and obese 2 month old rats responded to a 2 day fast by decreasing hepatic T3 generation as is always observed in other strains of rats. The hepatic conversion rate was not decreased in older (5 month) fed obese rats when compared to age-matched lean controls. Hepatic conversion of T4 to T3 was markedly decreased in 5 month old lean Zucker rats fasted for 4 days. In contrast, a 4 day fast had no effect on the hepatic conversion rate in the 5 month old obese rats. The hepatic conversion rate was assessed in 5 month old obese rats fasted for up to 28 days and hepatic conversion still did not decrease. This paradoxical response of the 5 month old obese rat may provide a new model to further evaluate the control of hepatic T3 generation from T4.     

Zellweger spectrum disorder patient–derived fibroblasts with the PEX1-Gly843Asp allele recover peroxisome functions in response to flavonoids

Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5′,7′-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of import-rescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843D–associated ZSD.     

Variants associated with common disease are not unusually differentiated in frequency across populations

Genetic variants that contribute to risk of common disease may differ in frequency across populations more than random variants in the genome do, perhaps because they have been exposed to population-specific natural selection. To assess this hypothesis empirically, we analyzed data from two groups of single-nucleotide polymorphisms (SNPs) that have shown reproducible (n = 9) or reported (n = 39) associations with common diseases. We compared the frequency differentiation (between Europeans and Africans) of the disease-associated SNPs with that of random SNPs in the genome. These common-disease-associated SNPs are not significantly more differentiated across populations than random SNPs. Thus, for the data examined here, ethnicity will not be a good predictor of genotype at many common-disease-associated SNPs, just as it is rarely a good predictor of genotype at random SNPs in the genome.     

1,25-Dihydroxycholecalciferol modulates 3H-thymidine incorporation in FRTL5 cells.

1,25-dihydroxycholecalciferol (1,25(OH)2D3) possesses proliferation and differentiation modulating effects in many cell types in vitro. We studied the effect of 1,25(OH)2D3 on 3H-thymidine incorporation in FRTL5 cells, a cultured rat thyroid follicular cell line. 1,25(OH)2D3 alone at 10(-11) and 10(-9) M exerted no effect on 3H-thymidine incorporation. However, at 10(-7) M, 1,25(OH)2D3 slightly enhanced 3H-thymidine incorporation. In the presence of 5% calf serum, 1,25(OH)2D3 increased 3H-thymidine incorporation induced by calf serum in a dose-dependent manner. 1,25(OH)2D3 also enhanced 3H-thymidine incorporation induced by PMA, an extrinsic stimulator of protein kinase C, without directly affecting PMA-induced protein kinase C translocation. In contrast to the stimulatory effects of 1,25(OH)2D3 on the calf serum and PMA-induced 3H-thymidine incorporation, 1,25(OH)2D3 inhibited the increase in 3H-thymidine incorporation induced by TSH in a dose-dependent manner. This effect of 1,25(OH)2D3 on TSH-induced 3H-thymidine incorporation may be, in part, due to post-cAMP pathways since 1,25(OH)2D3 also inhibited the increase in 3H-thymidine incorporation induced by Bu2cAMP without affecting the TSH-induced increase in cAMP. The stimulatory effect of insulin on 3H-thymidine incorporation, a cAMP-independent process, was also inhibited by 1,25(OH)2D3. We conclude that 1,25(OH)2D3 affects 3H-thymidine incorporation in FRTL5 cells raising the possibility of a physiologic role for 1,25(OH)2D3 in the growth and function of thyroid follicular cells.