Cystine storage in cultured myotubes from patients with nephropathic cystinosis.

Sorted muscle cells, cultured from a patient with nephropathic cystinosis, stored 100 times normal amounts of cystine. Subcellular fractionation and density-gradient centrifugation confirmed that the cystine was located in a lysosomal compartment. 2. Myoblasts from cystinotic patients in culture underwent fusion to myotubes in a normal fashion. 3. The free thiol cysteamine effectively depleted cystinotic-muscle cells of cystine. 4. Cultured myoblast and myotubes offered a unique system for investigating the effects of lysosomal storage on differentiated cell functions.     

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.     

Pairing centers recruit a Polo-like kinase to orchestrate meiotic chromosome dynamics in C. elegans

Faithful segregation of homologous chromosomes during meiosis requires pairing, synapsis, and crossing-over. In C.?elegans, homolog pairing and synapsis depend on pairing centers (PCs), special regions near one end of each chromosome that interact with the nuclear envelope (NE) and cytoplasmic microtubules. Here, we report that PCs are required for nuclear reorganization at the onset of meiosis. We demonstrate that PCs recruit the Polo-like kinase PLK-2 to induce NE remodeling, chromosome pairing, and synapsis. Recruitment of PLK-2 is also required to mediate a cell cycle delay and selective apoptosis of nuclei containing unsynapsed chromosomes, establishing a molecular link between these two quality control mechanisms. This work reveals unexpected functions for the conserved family of Polo-like kinases, and advances our understanding of how meiotic processes are properly coordinated to ensure transmission of genetic information from parents to progeny.     

Dysregulated zinc and sphingosine-1-phosphate signaling in pulmonary hypertension: Potential effects by targeting of bone morphogenetic protein receptor type 2 in pulmonary microvessels

Recently identified molecular targets in pulmonary artery hypertension (PAH) include sphingosine-1-phosphate (S1P) and zinc transporter ZIP12 signaling. This study sought to determine linkages between these pathways, and with BMPR2 signaling. Lung tissues from a rat model of monocrotaline-induced PAH and therapeutic treatment with bone marrow–derived endothelial-like progenitor cells transduced to overexpress BMPR2 were studied. Multifluorescence quantitative confocal microscopy (MQCM) was applied for analysis of protein expression and localization of markers of vascular remodeling (αSMA and BMPR2), parameters of zinc homeostasis (zinc transporter SLC39A/ZIP family members 1, 10, 12 and 14; and metallothionein MT3) and S1P extracellular signaling (SPHK1, SPNS2, S1P receptor isoforms 1, 2, 3, 5) in 20–200 µm pulmonary microvessels. ZIP12 expression in whole lung tissue lysates was assessed by western blot. Spearman nonparametric correlations between MQCM readouts and hemodynamic parameters, Fulton index (FI), and right ventricular systolic pressure (RVSP) were measured. In line with PAH status, pulmonary microvessels in monocrotaline-treated animals demonstrated significant (p < .05, n = 6 per group) upregulation of αSMA (twofold) and downregulation of BMPR2 (20%). Upregulated ZIP12 (92%), MT3 (57.7%), S1PR2 (54.8%), and S1PR3 (30.3%) were also observed. Significant positive and negative correlations were demonstrated between parameters of zinc homeostasis (ZIP12, MT3), S1P signaling (S1PRs, SPNS2), and vascular remodeling (αSMA, FI, RVSP). MQCM and western blot analysis showed that monocrotaline-induced ZIP12 upregulation could be partially negated by BMPR2-targeted therapy. Our results indicate that altered zinc transport/storage and S1P signaling in the monocrotaline-induced PAH rat model are linked to each other, and could be alleviated by BMPR2-targeted therapy.     

Genome-Wide Association Study Dissects the Genetic Architecture of Seed Weight and Seed Quality in Rapeseed (Brassica napus L.)

Association mapping can quickly and efficiently dissect complex agronomic traits. Rapeseed is one of the most economically important polyploid oil crops, although its genome sequence is not yet published. In this study, a recently developed 60K Brassica Infinium^® SNP array was used to analyse an association panel with 472 accessions. The single-nucleotide polymorphisms (SNPs) of the array were in silico mapped using ‘pseudomolecules’ representative of the genome of rapeseed to establish their hypothetical order and to perform association mapping of seed weight and seed quality. As a result, two significant associations on A8 and C3 of Brassica napus were detected for erucic acid content, and the peak SNPs were found to be only 233 and 128 kb away from the key genes BnaA.FAE1 and BnaC.FAE1. BnaA.FAE1 was also identified to be significantly associated with the oil content. Orthologues of Arabidopsis thaliana HAG1 were identified close to four clusters of SNPs associated with glucosinolate content on A9, C2, C7 and C9. For seed weight, we detected two association signals on A7 and A9, which were consistent with previous studies of quantitative trait loci mapping. The results indicate that our association mapping approach is suitable for fine mapping of the complex traits in rapeseed.     

Chromosomal mapping of Xenopus 5S genes: somatic-type versus oocyte-type.

Xenopus 5S RNA genes exhibit a pattern of differential expression during development in which some members (oocyte-type) are transcribed only in oocytes, while others (somatic-type) are expressed in both oocytes and somatic cells. Using cloned DNA probes specific for each gene type, we determined the positions of these genes on Xenopus metaphase chromosomes by in situ hybridization. Somatic-type 5S genes in both X. laevis and X. borealis are located at the distal end of the long arm of only one chromosome (number 9). The oocyte-type 5S RNA genes are found at the distal ends of the long arms of most Xenopus chromosomes, including chromosome 9. Thus, large scale differences in chromosomal location cannot explain the selective expression of these genes, as suggested previously.     

Proprotein convertase cleavage liberates a fibrillogenic fragment of a resident glycoprotein to initiate melanosome biogenesis

Lysosome-related organelles are cell type-specific intracellular compartments with distinct morphologies and functions. The molecular mechanisms governing the formation of their unique structural features are not known. Melanosomes and their precursors are lysosome-related organelles that are characterized morphologically by intralumenal fibrous striations upon which melanins are polymerized. The integral membrane protein Pmel17 is a component of the fibrils and can nucleate their formation in the absence of other pigment cell-specific proteins. Here, we show that formation of intralumenal fibrils requires cleavage of Pmel17 by a furin-like proprotein convertase (PC). As in the generation of amyloid, proper cleavage of Pmel17 liberates a lumenal domain fragment that becomes incorporated into the fibrils; longer Pmel17 fragments generated in the absence of PC activity are unable to form organized fibrils. Our results demonstrate that PC-dependent cleavage regulates melanosome biogenesis by controlling the fibrillogenic activity of a resident protein. Like the pathologic process of amyloidogenesis, the formation of other tissue-specific organelle structures may be similarly dependent on proteolytic activation of physiological fibrillogenic substrates.     

A relict Ordovician brachiopod fauna from the Parakidograptus acuminatus Biozone (lower Silurian) of the English Lake District

At Yewdale Beck in northern England, a brachiopod fauna dominated by species of Hindella, Kinnella, Mirorthis, Paromalomena and Plectothyrella , occurs interbedded with a graptolite fauna which includes Akidograptus ascensus , Atavograptus ceryx , Persculptograptus parvulus and Normalograptus spp. (including probable examples of N. normalis , N. angustus and N. medius ) within the lower part of the Skelgill Formation. This suggests that taxa of the terminal Ordovician Hirnantia fauna occur within the lower P. acuminatus Biozone, representing the youngest documented occurrence of the Hirnantia brachiopod fauna. Biostratigraphy, brachiopods, graptolites, Ordovician-Silurian boundary.     

Functional linkages for the pace of life, life-history, and environment in birds

For vertebrates, body mass underlies much of the variation in metabolism, but among animals of the same body mass, metabolism varies six-fold. Understanding how natural selection can influence variation in metabolism remains a central focus of Physiological Ecologists. Life-history theory postulates that many physiological traits, such as metabolism, may be understood in terms of key maturational and reproductive characteristics over an organism's life-span. Although it is widely acknowledged that physiological processes serve as a foundation for life-history trade-offs, the physiological mechanisms that underlie the diversification of life-histories remain elusive. Data show that tropical birds have a reduced basal metabolism (BMR), field metabolic rate, and peak metabolic rate compared with temperate counterparts, results consistent with the idea that a low mortality, and therefore increased longevity, and low productivity is associated with low mass-specific metabolic rate. Mass-adjusted BMR of tropical and temperate birds was associated with survival rate, in accordance with the view that animals with a slow pace of life tend to have increased life spans. To understand the mechanisms responsible for a reduced rate of metabolism in tropical birds compared with temperate species, we summarized an unpublished study, based on data from the literature, on organ masses for both groups. Tropical birds had smaller hearts, kidneys, livers, and pectoral muscles than did temperate species of the same body size, but they had a relatively larger skeletal mass. Direct measurements of organ masses for tropical and temperate birds showed that the heart, kidneys, and lungs were significantly smaller in tropical birds, although sample sizes were small. Also from an ongoing study, we summarized results to date on connections between whole-organism metabolism in tropical and temperate birds and attributes of their dermal fibroblasts grown in cell culture. Cells derived from tropical birds had a slower rate of growth, consistent with the hypothesis that these cells have a slower metabolism. We found that dermal fibroblasts from tropical birds resisted chemical agents that induce oxidative and non-oxidative stress better than do cells from temperate species, consistent with the hypothesis that birds that live longer invest more in self-maintenance such as antioxidant properties of cells.