Obesity and type 2 diabetes are associated with mitochondrial dysfunction in adipose tissue, but the role for adipose tissues mitochondria in the development of these disorders is currently unknown. To understand the impact of adipose tissue mitochondria on whole body metabolism, we have generated a mouse model with disruption of the mitochondrial transcription factor A (TFAM) specifically in fat. F-TFKO adipose tissue exhibit decreased mtDNA copy number, altered levels of proteins of the electron transport chain, and perturbed mitochondrial function with decreased Complex I activity and greater oxygen consumption and uncoupling. As a result, F-TFKO mice exhibit higher energy expenditure and are protected from age- and diet-induced obesity, insulin resistance and hepatosteatosis, despite a greater food intake. Thus, TFAM deletion in the adipose tissue increases mitochondrial oxidation that has positive metabolic effects suggesting that regulation of adipose tissue mitochondria may be a potential therapeutic target for the treatment of obesity.
Obesity; Brown adipose tissue; Mitochondrial function; mitochondrial bioenergetics; White adipose tissue; Insulin resistance; Diabetes
Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent “primer walking” combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.
Human pressures have put many top predator populations at risk of extinction. Recent years have seen alarming declines in sharks worldwide, while their resilience remains poorly understood. Studying the ecology of small populations of marine predators is a priority to better understand their ability to withstand anthropogenic and environmental stressors. In the present study, we monitored a naturally small island population of 40 adult sicklefin lemon sharks in Moorea, French Polynesia over 5 years. We reconstructed the genetic relationships among individuals and determined the population’s mating system. The genetic network illustrates that all individuals, except one, are interconnected at least through one first order genetic relationship. While this species developed a clear inbreeding avoidance strategy involving dispersal and migration, the small population size, low number of breeders, and the fragmented environment characterizing these tropical islands, limits its complete effectiveness.
The present study aimed to determine the effects of breed and sex on growth patterns and metabolic features of advanced-pregnancy foetuses exposed to the same environmental conditions. Thus, at Day 62 of pregnancy, swine foetuses from an obese breed with leptin resistance (Iberian breed) were compared to lean crossbred foetuses (25% Large White ×25% Landrace ×50% Pietrain). There were differential developmental patterns in foetuses with leptin resistance, mainly a higher relative weight of the brain resembling “brain-sparing effect”. Prioritization of brain growth may be protective for the adequate growth and postnatal survival of the Iberian individuals, an ancient breed reared in extensive semi-feral conditions for centuries. There were also clear sex-related differences in foetal development and metabolism in the Iberian breed. Female Iberian foetuses were similar in size and weight to male littermates but had a significantly higher relative liver to body weight ratio resembling “liver-sparing effect” and a trend for a higher relative intestine to body ratio. Moreover, the availability of triglycerides, cholesterol and IL-6 in female Iberian foetuses was similar to that of lean crossbred foetuses. Overall, these features may favour a better postnatal survival and development of females, the sex more critical for the species survival. These findings set the basis for future translational studies aimed at increasing the knowledge on the interaction between genetic and environmental factors in the early programming of the adult phenotype.
A genetic bottleneck explains the marked changes in mitochondrial DNA (mtDNA) heteroplasmy observed during the transmission of pathogenic mutations, but the precise timing remains controversial, and it is not clear whether selection plays a role. These issues are critically important for the genetic counseling of prospective mothers, and developing treatments aimed at disease prevention. By studying mice transmitting a heteroplasmic single base-pair deletion in the mitochondrial tRNAMet gene, we show that mammalian mtDNA heteroplasmy levels are principally determined prenatally within the developing female germ line. Although we saw no evidence of mtDNA selection prenatally, skewed heteroplasmy levels were observed in the offspring of the next generation, consistent with purifying selection. High percentage levels of the tRNAMet mutation were linked to a compensatory increase in overall mitochondrial RNAs, ameliorating the biochemical phenotype, and explaining why fecundity is not compromised.
The population dynamics of shark species are generally poorly described because highly mobile marine life is challenging to investigate. Here we investigate the genetic population structure of the blacktip reef shark (Carcharhinus melanopterus) in French Polynesia. Five demes were sampled from five islands with different inter-island distances (50–1500 km). Whether dispersal occurs between islands frequently enough to prevent moderate genetic structure is unknown. We used 11 microsatellites loci from 165 individuals and a strong genetic structure was found among demes with both F-statistics and Bayesian approaches. This differentiation is correlated with the geographic distance between islands. It is likely that the genetic structure seen is the result of all or some combination of the following: low gene flow, time since divergence, small effective population sizes, and the standard issues with the extent to which mutation models actually fit reality. We suggest low levels of gene flow as at least a partial explanation of the level of genetic structure seen among the sampled blacktip demes. This explanation is consistent with the ecological traits of blacktip reef sharks, and that the suitable habitat for blacktips in French Polynesia is highly fragmented. Evidence for spatial genetic structure of the blacktip demes we studied highlights that similar species may have populations with as yet undetected or underestimated structure. Shark biology and the market for their fins make them highly vulnerable and many species are in rapid decline. Our results add weight to the case that total bans on shark fishing are a better conservation approach for sharks than marine protected area networks.
Many Caenorhabditis elegans mutants with dysfunctional mitochondrial electron transport chain are surprisingly long lived. Both short-lived (gas-1(fc21)) and long-lived (nuo-6(qm200)) mutants of mitochondrial complex I have been identified. However, it is not clear what are the pathways determining the difference in longevity. We show that even in a short-lived gas-1(fc21) mutant, many longevity assurance pathways, shown to be important for lifespan prolongation in long-lived mutants, are active. Beside similar dependence on alternative metabolic pathways, short-lived gas-1(fc21) mutants and long-lived nuo-6(qm200) mutants also activate hypoxia-inducible factor –1α (HIF-1α) stress pathway and mitochondrial unfolded protein response (UPRmt). The major difference that we detected between mutants of different longevity, is in the massive loss of complex I accompanied by upregulation of complex II levels, only in short-lived, gas-1(fc21) mutant. We show that high levels of complex II negatively regulate longevity in gas-1(fc21) mutant by decreasing the stability of complex I. Furthermore, our results demonstrate that increase in complex I stability, improves mitochondrial function and decreases mitochondrial stress, putting it inside a “window” of mitochondrial dysfunction that allows lifespan prolongation.
Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.
One of the main functions of the mitochondrial network is to provide the energy currency ATP to drive a large array of cellular metabolic processes. The formation of the mitochondrial respiratory chain, which allows this energy supply, is under the control of two separate genetic systems, the nuclear and the mitochondrial genomes, whose expressions have to be tightly coordinated to ensure efficient mitochondrial function. The regulation of mitochondrial genome expression is still poorly understood despite the profound importance of this process in human physiology, disease, and aging. Here, we make one step forward by unraveling a new role for the mitochondrial transcription termination factor 3 (MTERF3), which was initially characterized as a factor able to decrease mitochondrial transcription. Using gene invalidation approaches, we show in two distinct model organisms, the fruit fly and the mouse, that MTERF3 is not only involved in mitochondrial transcription but also favors the assembly of the mitochondrial ribosome and thereby reinforces the coordination between transcription and translation events, two key steps in mitochondrial genome expression.
In 2010, a chimpanzee died at Copenhagen Zoo following an outbreak of respiratory disease among chimpanzees in the zoo. Identification of coxsackie B3 virus, a common human pathogen, as the causative agent, and its severe manifestation, raise questions about pathogenicity and transmissibility among humans and other primates.
viruses; coxsackie B3 virus; coxsackievirus; nonhuman primate; human; chimpanzee; myocarditis; Copenhagen Zoo; Denmark; transmission; zoonoses
Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.
Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.
Coccidian parasites have a major impact on human and animal health world-wide and are among the most successful and widespread parasitic protozoa. They include Neospora caninum which is a leading cause of abortion in cattle and one of its nearest relatives, Toxoplasma gondii. Despite its close phylogenetic relationship to Toxoplasma, Neospora has a far more restricted host range, does not infect humans and its epidemiology depends predominantly on efficient vertical transmission. The divergent biology of these two closely related species provides a unique opportunity to study the mechanisms of host specificity, pathogenesis and zoonotic potential not only in these, but other Coccidia. We have sequenced the genome of Neospora and the transcriptomes of both species to show that despite diverging some 28 million years ago, both genome and gene expression remain remarkably conserved. Evolution has focused almost exclusively on molecules which control the interaction of the parasite with the host cell. We show that some secreted invasion-related proteins and surface genes which are known to control virulence and host cell interactions in Toxoplasma are dramatically altered in their expression and functionality in Neospora and propose that evolution of these genes may underpin the ecological niches inhabited by coccidian parasites.
The majority of metabolic principles are evolutionarily conserved from nematodes to humans. Caenorhabditis elegans has widely accelerated the discovery of new genes important to maintain organismic metabolic homeostasis. Various methods exist to assess the metabolic state in worms, yet they often require large animal numbers and tend to be performed as bulk analyses of whole worm homogenates, thereby largely precluding a detailed studies of metabolic changes in specific worm tissues. Here, we have adapted well-established histochemical methods for the use on C. elegans fresh frozen sections and demonstrate their validity for analyses of morphological and metabolic changes on tissue level in wild type and various mutant strains. We show how the worm presents on hematoxylin and eosin (H&E) stained sections and demonstrate their usefulness in monitoring and the identification of morphological abnormalities. In addition, we demonstrate how Oil-Red-O staining on frozen worm cross-sections permits quantification of lipid storage, avoiding the artifact-prone fixation and permeabilization procedures of traditional whole-mount protocols. We also adjusted standard enzymatic stains for respiratory chain subunits (NADH, SDH, and COX) to monitor metabolic states of various C. elegans tissues. In summary, the protocols presented here provide technical guidance to obtain robust, reproducible and quantifiable tissue-specific data on worm morphology as well as carbohydrate, lipid and mitochondrial energy metabolism that cannot be obtained through traditional biochemical bulk analyses of worm homogenates. Furthermore, analysis of worm cross-sections overcomes the common problem with quantification in three-dimensional whole-mount specimens.
The bicoid stability factor (BSF) of Drosophila melanogaster has been reported to be present in the cytoplasm, where it stabilizes the maternally contributed bicoid mRNA and binds mRNAs expressed from early zygotic genes. BSF may also have other roles, as it is ubiquitously expressed and essential for survival of adult flies. We have performed immunofluorescence and cell fractionation analyses and show here that BSF is mainly a mitochondrial protein. We studied two independent RNAi knockdown fly lines and report that reduced BSF protein levels lead to a severe respiratory deficiency and delayed development at the late larvae stage. Ubiquitous knockdown of BSF results in a severe reduction of the polyadenylation tail lengths of specific mitochondrial mRNAs, accompanied by an enrichment of unprocessed polycistronic RNA intermediates. Furthermore, we observed a significant reduction in mRNA steady state levels, despite increased de novo transcription. Surprisingly, mitochondrial de novo translation is increased and abnormal mitochondrial translation products are present in knockdown flies, suggesting that BSF also has a role in coordinating the mitochondrial translation in addition to its role in mRNA maturation and stability. We thus report a novel function of BSF in flies and demonstrate that it has an important intra-mitochondrial role, which is essential for maintaining mtDNA gene expression and oxidative phosphorylation.
The majority of the cellular energy currency ATP is formed in a tubular network, termed mitochondria, present within virtually all eukaryotic cells. The mitochondria are unique among cellular organelles in that they contain their own genome, which encodes critical proteins necessary for cellular energy production. However, the vast majority of mitochondrial proteins are encoded in the nucleus and imported into mitochondria. Gene expression thus needs to be coordinated between the two genomes to ensure efficient mitochondrial function and sufficient adaptation to different physiological demands. The regulation of the mitochondrial genome is poorly understood, with many of the basic regulators not yet being characterized. We used RNAi in the fruit fly to study the in vivo function of the bicoid stability factor (BSF), previously thought to be a cytoplasmic and nuclear protein important for fly development. We show here that BSF is mainly localized to mitochondria, where it is essential for mtDNA gene expression, regulating the polyadenylation and maturation of specific mRNAs. Furthermore, BSF coordinates the translation and assembly of mitochondrial peptides in the inner mitochondrial membrane.
Piwi-associated RNAs (piRNAs) bind transcripts from retrotransposable elements (RTE) in mouse germline cells and seemingly act as guides for genomic methylation, thereby repressing the activity of RTEs. It is currently unknown if and how Piwi proteins distinguish RTE transcripts from other cellular RNAs. During germline development, the main target of piRNAs switch between different types of RTEs. Using the piRNA targeting of RTEs as an indicator of RTE activity, and considering the entire population of genomic RTE loci along with their age and location, this study aims at further elucidating the dynamics of RTE activity during mouse germline development.
Due to the inherent sequence redundancy between RTE loci, assigning piRNA targeting to specific loci is problematic. This limits the analysis, although certain features of piRNA targeting of RTE loci are apparent. As expected, young RTEs display a much higher level of piRNA targeting than old RTEs. Further, irrespective of age, RTE loci near protein-coding coding genes are targeted to a greater extent than RTE loci far from genes. During development, a shift in piRNA targeting is observed, with a clear increase in the relative piRNA targeting of RTEs residing within boundaries of protein-coding gene transcripts.
Reanalyzing published piRNA sequences and taking into account the features of individual RTE loci provide novel insight into the activity of RTEs during development. The obtained results are consistent with some degree of proportionality between what transcripts become substrates for Piwi protein complexes and the level by which the transcripts are present in the cell. A transition from active transcription of RTEs to passive co-transcription of RTE sequences residing within protein-coding transcripts appears to take place in postnatal development. Hence, the previously reported increase in piRNA targeting of SINEs in postnatal testis development does not necessitate widespread active transcription of SINEs, but may simply be explained by the prevalence of SINEs residing in introns.
Mitochondria are the structures that produce the bulk part of the cellular energy currency ATP, which drives numerous energy requiring processes in the cell. This process involves a series of large enzyme complexes—the respiratory chain—that couples the transfer of electrons to the creation of a concentration gradient of protons across the inner mitochondrial membrane, which drives ATP synthesis. Complex I (or NADH-quinone oxidoreductase) is the largest and by far the most complicated of the respiratory chain enzyme complexes. The molecular mechanism whereby it couples electron transfer to proton extrusion has remained mysterious until very recently. Low-resolution X-ray structures of complex I have, surprisingly, suggested that electron transfer in the hydrophilic arm, protruding into the mitochondrial matrix, causes movement of a coupling rod that influences three putative proton pumps within the hydrophobic arm embedded in the inner mitochondrial membrane. In this Primer, we will briefly introduce the recent progress made in this area and highlight the road ahead that likely will unravel the detailed molecular mechanisms of complex I function.
IL-2 has been reported to be critical for peripheral Treg survival in mouse models. Here, we examined Treg maintenance in a series of paediatric liver transplant recipients who received basiliximab, a therapeutic anti-CD25 monoclonal antibody.
FoxP3+ CD4 T cells were analyzed by flow cytometry before liver grafting and more than 9 months later. We found that in vivo CD25 blockade did not lead to Treg depletion: the proportion of FoxP3+ cells among CD4 T cells and the level of FoxP3 expression were both unchanged. IL-2Rβ expression was enhanced in FoxP3+ cells both before and after basiliximab treatment, while the level of IL-2Rγ expression was similar in Tregs and non-Tregs. No significant change in the weak or absent expression of IL-7Rα and IL-15Rα expression on FoxP3+ cells was observed. Although the proportion of FoxP3+ cells among CD4 T cells did not vary, food allergies occurred more rapidly after liver grafting in patients who received basiliximab, raising questions as to Treg functionality in vivo in the absence of functional CD25.
CD25 appears non essential for human Treg peripheral maintenance in vivo. However, our results raise questions as to Treg functionality after therapeutic CD25 targeting.
Retrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed - or near fixation - in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR) retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs.
Two independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes.
Presumably, the host organism negatively regulates proliferation of LTR retrotransposons. The finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. Alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. Our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
Eukaryotic genomes are scattered with retroelements that proliferate through retrotransposition. Although retroelements make up around 40 percent of the human genome, large regions are found to be completely devoid of retroelements. This has been hypothesised to be a result of genomic regions being intolerant to insertions of retroelements. The inadvertent transcriptional activity of retroelements may affect neighbouring genes, which in turn could be detrimental to an organism. We speculate that such retroelement transcription, or transcriptional interference, is a contributing factor in generating and maintaining retroelement-free regions in the human genome.
Based on the known transcriptional properties of retroelements, we expect long interspersed elements (LINEs) to be able to display a high degree of transcriptional interference. In contrast, we expect short interspersed elements (SINEs) to display very low levels of transcriptional interference. We find that genomic regions devoid of long interspersed elements (LINEs) are enriched for protein-coding genes, but that this is not the case for regions devoid of short interspersed elements (SINEs). This is expected if genes are subject to selection against transcriptional interference. We do not find microRNAs to be associated with genomic regions devoid of either SINEs or LINEs. We further observe an increased relative activity of genes overlapping LINE-free regions during early embryogenesis, where activity of LINEs has been identified previously.
Our observations are consistent with the notion that selection against transcriptional interference has contributed to the maintenance and/or generation of retroelement-free regions in the human genome.
The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes encode ~12,500 predicted proteins, a high proportion of which have long repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal rDNA element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal/fungal lineage after the plant/animal split, but Dictyostelium appears to have retained more of the diversity of the ancestral genome than either of these two groups.
a heparin preparation with low antithrombin activity and different disaccharide composition than mammalian heparin was isolated from the body of the ascidian Styela plicata (Chordata-Tunicata). The disaccharide composition and the effect of the invertebrate glycan on venous and arterial models of thrombosis was investigated.
Methods and Results
High performance liquid chromatography of the products formed by a mixture of heparin-lyases showed that the ascidian heparin is composed mainly by ΔUA(2SO4)-1→4-β-D-GlcN(SO4) (47.5%), ΔUA(2SO4)-1→4-β-D-GlcN(SO4)(6SO4) (38.3%) disaccharides. Smaller amounts of the disaccharides ΔUA(2SO4)-1→4-β-D-GlcN(SO4)(3SO4)(6SO4) (2.8%) and ΔUA(2SO4)-1→4-β-D-GlcN(SO4)(3SO4) (8.0%). The invertebrate heparin has an aPTT activity of 18 IU/mg and an antithrombin-mediated anti-thrombin and anti-factor Xa activities 10-fold lower than that of mammalian heparin. In a venous model of thrombosis in the vena cava, S.plicata heparin inhibits only 80% of thrombosis at a dose 10-fold higher than that of the mammalian heparin that inhibits 100% of thrombosis. However, in an arterio-shunt model of arterial thrombosis, both S.plicata and mammalian heparin possess equivalent antithrombotic activity. It is also shown that at equivalent doses, ascidian heparin has a lower bleeding effect than mammalian heparin.
the antithrombin-mediated anticoagulant activity of heparin polymers is not directly related to antithrombotic potency in the arterio-venous shunt. The results of the present work suggest that heparin preparations obtained from the body of S.plicata may have a safer therapeutic action in the treatment of arterial thrombosis than mammalian heparin.
Heparin; dermatan sulfate; antithrombotic effect; arterio-venous model; venous model; ascidian
colorectal cancer; CPT-11; efficacy; irinotecan; pharmacokinetics; safety