Identification of the PRMT1v1 and PRMT1v2 specific interactomes by quantitative mass spectrometry in breast cancer cells

Arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). The PRMT1 gene generates at least seven distinct alternatively spliced isoforms (PRMT v1–v7), which together contribute a significant portion of the cellular arginine methylome. The distinct biochemical and biological functions of these PRMT1 isoforms have not been well characterized. Previously we have shown that while both PRMT1v1 and PRMT1v2 are overexpressed in breast cancer cells, PRMT1v2 specifically promotes breast cancer cell survival and invasion. These isoforms also have distinct subcellular localizations, PRMT1v1 is mainly nuclear and PRMT1v2 cytosolic. To gain further knowledge into their isoform‐specific roles within cells we used a SILAC‐based quantitative affinity purification/MS approach to identify their individual protein interactomes in breast cancer cells. This analysis has uncovered distinct interactomes for PRMT1v1 and PRMT1v2. Consistent with their distinct subcellular localizations, PRMT1v1 enriched a mainly nuclear protein interactome, while PRMT1v2 enriched predominantly cytoplasmic interactors from whole‐cell extracts. Furthermore, these interactomes revealed that PRMT1v1 has a role in regulating gene expression, while PRMT1v2 functions in cytoskeletal dynamics. These results highlight the unique functions of these isoforms and the distinct roles they may play within cells, with potential implications for breast cancer and other diseases.     

Calls,colours, shape,and genes: a multi‐trait approach to the study of geographic variation in the Amazonian frog Allobates femoralis

Evolutionary divergence in behavioural traits related to mating may represent the initial stage of speciation. Direct selective forces are usually invoked to explain divergence in mate‐recognition traits, often neglecting a role for neutral processes or concomitant differentiation in ecological traits. We adopted a multi‐trait approach to obtain a deeper understanding of the mechanisms behind allopatric divergence in the Amazonian frog, Allobates femoralis. We tested the null hypothesis that geographic distance between populations correlates with genetic and phenotypic divergence, and compared divergence between mate‐recognition (acoustic) and ecological (coloration, body‐shape) traits. We quantified geographic variation in 39 phenotypic traits and a mitochondrial DNA marker among 125 individuals representing eight populations. Geographic variation in acoustic traits was pronounced and tracked the spatial genetic variation, which appeared to be neutral. Thus, the evolution of acoustic traits tracked the shared history of the populations, which is unexpected for pan‐Amazonian taxa or for mate‐recognition traits. Divergence in coloration appeared uncorrelated with genetic distance, and might be partly attributed to local selective pressures, and perhaps to Batesian mimicry. Divergence in body‐shape traits was low. The results obtained depict a complex evolutionary scenario and emphasize the importance of considering multiple traits when disentangling the forces behind allopatric divergence. ©2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 826–838.     

Strain-specific kinetics of prion protein formation in vitro and in vivo

The molecular basis of prion strain diversity is proposed to be encoded by distinct conformations of the abnormal scrapie isoform of the prion protein (PrP(Sc)). PrP(Sc) formation for the hyper (HY) and drowsy (DY) strains of the transmissible mink encephalopathy (TME) agent was investigated using the cell-free PrP conversion reaction to determine the role of distinct PrP(Sc) conformations in the rate of in vitro conversion of cellular PrP into protease-resistant PrP. PrP conversion increased at an exponential rate for both TME strains until peak levels were reached at 72-96 h of reaction time. The amount and rate of PrP conversion for HY TME was greater than those for DY TME between 48 h and the peak level of PrP conversion. Between 96 and 120 h, there was a negative rate of PrP conversion; and between 120 and 168 h, the net rate of HY and DY PrP conversion approached zero. These findings suggest that PrP conversion can occur in three distinct stages: an elongation phase, a depolymerization phase, and a steady-state phase. Strain-specific properties between the TME strains were identified only during the elongation phase. The steady-state phase could be disrupted by the addition of PrP(Sc) to, or by sonication of, the cell-free PrP conversion reaction. These treatments resulted in an increase in the amount of PrP conversion that was equal to or greater than that found during the peak level of PrP conversion for both TME strains, indicating that the steady-state phase was in dynamic equilibrium. In a related study, the rate of accumulation of HY and DY PrP(Sc) in hamster brain exhibited a strain-specific pattern that had similarities to the strain-specific PrP conversion reaction during the elongation phase. These results suggest that strain-specific conformations of PrP(Sc) have the ability to influence the rate of additional PrP(Sc) formation from cellular PrP both in vitro and in vivo.     

Small subunit ribosomal DNA characterization of an unidentified aurantiactinomyxon form and its oligochaete host Tubifex ignotus

In this study, the small subunit (18S) ribosomal DNA gene from an aurantiactinomyxon form of unknown taxonomic position (A1) and from its aquatic oligochaete host (Tubifex ignotus) were characterized. Molecular sequence information on A1 was obtained to allow comparisons of this gene with known sequences from known myxosporean forms, and therefore to investigate possible relationships between this organism and its alternate myxosporean stage. Sequence data for the oligochaete host, together with morphological features, will allow reliable identification of this species in the future. Sequence data derived from the 18S DNA gene and data from other related or non-related organisms were analyzed and used to construct a phylogenetic tree. Phylogenetic studies provided an insight into the taxonomic position of A1. Sequence similarities within the 18S rDNA A1 gene and compared organisms indicated that A1 was most closely related to members of the sub-order Variisporina (Myxidium lieberkuehni [Ml] and Sphaerospora oncorhynchi [So]). Clustering of the 3 organisms in the same branch was well supported by high bootstrap values (81%). A1 showed higher similarities with sequences of Ml (approximately 80%) than with So (approximately 79%). Myxosporean sequence analysis indicated that phylogenetic arrangements do not support traditional classification based on morphological criteria of the spores, but rather support arrangement by tissue location. Marine actinosporeans Triactinomyxon sp. and Tetraspora discoidea were found to be associated with Platysporinid myxosporeans, supporting previous findings. In this study, 18S rDNA sequence data are generated for first time for the aquatic oligochaete T. ignotus. Phylogenetic 18S rDNA gene analyses performed with T. ignotus support and confirm existing morphological and molecular phylogenetic studies. Paraphyly of the Tubificidae family was noticed.     

Long term follow up of severely ill patients who underwent urgent cardiac transplantation.

OBJECTIVE--To assess long term survival (> 5 years) and quality of life in severely ill patients referred for urgent cardiac transplantation. SETTING--Tertiary referral centres: before transplantation at the National Heart Hospital (late 1984 to end 1986); after transplantation at Harefield Hospital. SUBJECTS--Eighteen patients (15 men; three women) who had required intensive support in hospital before cardiac transplantation and were alive at short term follow up. INTERVENTIONS--Intravenous infusions of cardiac drugs (mean 2.2 infusions), intravenous diuretics (17 patients), and many other drugs before transplantation. Intra-aortic balloon counterpulsation (four patients), temporary pacing (two), and resuscitation from cardiac arrest (three). Patients had specialised nursing care on a medical intensive care unit in almost every case. MAIN OUTCOME MEASURES--Long term survival in patients after urgent cardiac transplantation and perceived quality of life. RESULTS--Of 18 patients who were alive at short term follow up (mean (range) 19.4 (10-33) months), 14 were still alive in 1992 (69 (61-83) months). Ten still worked full time, and 11 reported no restrictions in their daily activities. Three of four patients who died in the intervening period survived > 5 years after transplantation. Overall, 17 of 18 patients survived at least 5 years. CONCLUSIONS--In severely ill patients who undergo urgent cardiac transplantation and survive in the short term, long term (5-7 year) survival and quality of life seem good.     

Evolution of base composition in the insulin and insulin-like growth factor genes

The genomes of homeothermic (warm-blooded) vertebrates are mosaic interspersions of homogeneously GC-rich and GC-poor regions (isochores). Evolution of genome compartmentalization and GC-rich isochores is hypothesized to reflect either selective advantages of an elevated GC content or chromosome location and mutational pressure associated with the timing of DNA replication in germ cells. To address the present controversy regarding the origins and maintenance of isochores in homeothermic vertebrates, newly obtained as well as published nucleotide sequences of the insulin and insulin-like growth factor (IGF) genes, members of a well-characterized gene family believed to have evolved by repeated duplication and divergence, were utilized to examine the evolution of base composition in nonconstrained (flanking) and weakly constrained (introns and fourfold degenerate sites) regions. A phylogeny derived from amino acid sequences supports a common evolutionary history for the insulin/IGF family genes. In cold- blooded vertebrates, insulin and the IGFs were similar in base composition. In contrast, insulin and IGF-II demonstrate dramatic increases in GC richness in mammals, but no such trend occurred in IGF- I. Base composition of the coding portions of the insulin and IGF genes across vertebrates correlated (r = 0.90) with that of the introns and flanking regions. The GC content of homologous introns differed dramatically between insulin/IGF-II and IGF-I genes in mammals but was similar to the GC level of noncoding regions in neighboring genes. Our findings suggest that the base composition of introns and flanking regions is determined by chromosomal location and the mutational pressure of the isochore in which the sequences are embedded. An elevated GC content at codon third positions in the insulin and the IGF genes may reflect selective constraints on the usage of synonymous codons.