Comparison of functional aspects of the coagulation cascade in human and sea turtle plasmas

Functional hemostatic pathways are critical for the survival of all vertebrates and have been evolving for more than 400 million years. The overwhelming majority of studies of hemostasis in vertebrates have focused on mammals with very sparse attention paid to reptiles. There have been virtually no studies of the coagulation pathway in sea turtles whose ancestors date back to the Jurassic period. Sea turtles are often exposed to rapidly altered environmental conditions during diving periods. This may reduce their blood pH during prolonged hypoxic dives. This report demonstrates that five species of turtles possess only one branch of the mammalian coagulation pathway, the extrinsic pathway. Mixing studies of turtle plasmas with human factor-deficient plasmas indicate that the intrinsic pathway factors VIII and IX are present in turtle plasma. These two factors may play a significant role in supporting the extrinsic pathway by feedback loops. The intrinsic factors, XI and XII are not detected which would account for the inability of reagents to induce coagulation via the intrinsic pathway in vitro. The analysis of two turtle factors, factor II (prothrombin) and factor X, demonstrates that they are antigenically/functionally similar to the corresponding human factors. The turtle coagulation pathway responds differentially to both pH and temperature relative to each turtle species and relative to human samples. The coagulation time (prothrombin time) increases as the temperature decreases between 37 and 15 °C. The increased time follows a linear relationship, with similar slopes for loggerhead, Kemps ridley and hawksbill turtles as well as for human samples. Leatherback turtle samples show a dramatic nonlinear increased time below 23 °C, and green turtle sample responses were similar but less dramatic. All samples also showed increased prothrombin times as the pH decreased from 7.8 to 6.4, except for three turtle species. The prothrombin times decreased, to varying extents, in a linear fashion relative to reduced pH with the rate of change greatest in leatherbacks>greenloggerhead turtles. All studies were conducted with reagents developed for human samples which would impact on the quantitative results with the turtle samples, but are not likely to alter the qualitative results. These comparative studies of the coagulation pathway in sea turtles and humans could enhance our knowledge of structure/function relationships and evolution of coagulation factors.     

Poly(dG)-poly(dC) DNA appears shorter than poly(dA)-poly(dT) and possibly adopts an A-related conformation on a mica surface under ambient conditions

Three types of DNA: approximately 2700 bp polydeoxyguanylic olydeoxycytidylic acid [poly(dG)-poly(dC)], approximately 2700 bp polydeoxyadenylic polydeoxythymidylic acid [poly(dA)-poly(dT)] and 2686 bp linear plasmid pUC19 were deposited on a mica surface and imaged by atomic force microscopy. Contour length measurements show that the average length of poly(dG)-poly(dC) is approximately 30% shorter than that of poly(dA)-poly(dT) and the plasmid. This led us to suggest that individual poly(dG)-poly(dC) molecules are immobilized on mica under ambient conditions in a form which is likely related to the A-form of DNA in contrast to poly(dA)-poly(dT) and random sequence DNA which are immobilized in a form that is related to the DNA B-form.     

Clinical Impact of the Temporal Relationship between Depression and Type 2 Diabetes: The Fremantle Diabetes Study Phase II

Background

The clinical features of type 2 diabetes may differ depending on whether first depression episode precedes or follows the diagnosis of diabetes.

Methods

Type 2 patients from the observational community-based Fremantle Diabetes Study Phase II underwent assessment of lifetime depression using the Brief Lifetime Depression Scale (developed and validated for this study) supplemented by information on current depression symptoms (Patient Health Questionnaire, 9-item version) and use of antidepressants. Patients were categorized as never depressed (Group 1), having had depression before diabetes diagnosis (Group 2), diagnosed with depression and diabetes within 2 years of each other (Group 3) and having depression after diabetes diagnosis (Group 4).

Results

Of 1391 patients, 20.8% were assigned to Group 2, 6.0% to Group 3 and 14.5% to Group 4. In Group 2, depression occurred a median 15.6 years before diabetes onset at age 37.2±14.7 years. These patients had similar clinical characteristics to never depressed patients except for reduced self-care behaviours and having more symptomatic peripheral arterial disease. In Group 4, depression occurred a median 9.9 years after diabetes onset at age 59.8±13.0 years. These patients had long duration diabetes, poor glycaemic control, more intensive management and more diabetic complications. Group 4 patients had more current depression than Group 2 but were less likely to be receiving antidepressants.

Conclusions/Interpretation

The clinical features of depression and type 2 diabetes are heterogeneous depending on their temporal relationship. There may be corresponding differences in the pathogenesis of depression in diabetes that have implications for diagnosis and management.     

Design of riboflavin-presenting PAMAM dendrimers as a new nanoplatform for cancer-targeted delivery

This communication describes the synthesis and in vitro biological evaluation of novel generation 5 PAMAM dendrimers conjugated with riboflavin as a targeting ligand. Cell-based experiments demonstrated that a dendrimer conjugated with riboflavin is able to undergo cellular binding and uptake in KB cells, and when the dendrimer is also conjugated with methotrexate, the riboflavin dendrimer conjugate can potently inhibit cell growth.     

Mutation analysis of a Sandhoff disease patient in the Maronite community in Cyprus

Sandhoff disease occurs in the Christian Maronite community in Cyprus, a community that established over a thousand years ago. Nowadays, this community comprises less than 1% of the whole population, and has been culturally and socially isolated. Cultured fibroblasts from a patient from this inbred group showed a -hexosaminidase subunit mRNA of apparently the normal size but of reduced quantity. A mutational analysis of cDNA obtained by polymerase chain reaction amplification of mRNA showed a deletion of A at nt 76 (counted from A of the initiation codon, ATG). The deletion results in a frame shift and a premature termination within 20 amino acids from the N-terminus of the normal mature enzyme protein. The patient was homozygous for the deletion. The 5-end of the gene showed many discrepancies from the previously published sequence. We consider that these differences are probably polymorphisms of little functional significance, because the patient's fibroblasts generate decreased but stable mRNA and because some of these base changes were also found in the genes from control fibroblasts. An extensive evaluation of the prevalence of this mutant allele in this community is being initiated.     

Temperature dependence of DNA persistence length

We have determined the temperature dependence of DNA persistence length, a, using two different methods. The first approach was based on measuring the j-factors of short DNA fragments at various temperatures. Fitting the measured j-factors by the theoretical equation allowed us to obtain the values of a for temperatures between 5°C and 42°C. The second approach was based on measuring the equilibrium distribution of the linking number between the strands of circular DNA at different temperatures. The major contribution into the distribution variance comes from the fluctuations of DNA writhe in the nicked circular molecules which are specified by the value of a. The computation-based analysis of the measured variances was used to obtain the values of a for temperatures up to 60°C. We found a good agreement between the results obtained by these two methods. Our data show that DNA persistence length strongly depends on temperature and accounting for this dependence is important in quantitative comparison between experimental results obtained at different temperatures.     

Histidyl-tRNA synthetase urzymes: Class I and II aminoacyl tRNA synthetase urzymes have comparable catalytic activities for cognate amino acid activation

Four minimal (119-145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine activation as fusion proteins and after TEV cleavage, using the (32)PP(i) exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (～0.1-0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered K(m) values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ～10(9) times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate amino acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.     

The Ancestral Carnivore Karyotype As Substantiated by Comparative Chromosome Painting of Three Pinnipeds,the Walrus,the Steller Sea Lion and the Baikal Seal (Pinnipedia,Carnivora)

Karyotype evolution in Carnivora is thoroughly studied by classical and molecular cytogenetics and supplemented by reconstructions of Ancestral Carnivora Karyotype (ACK). However chromosome painting information from two pinniped families (Odobenidae and Otariidae) is noticeably missing. We report on the construction of the comparative chromosome map for species from each of the three pinniped families: the walrus (Odobenus rosmarus, Odobenidae–monotypic family), near threatened Steller sea lion (Eumetopias jubatus, Otariidae) and the endemic Baikal seal (Pusa sibirica, Phocidae) using combination of human, domestic dog and stone marten whole-chromosome painting probes. The earliest karyological studies of Pinnipedia showed that pinnipeds were characterized by a pronounced karyological conservatism that is confirmed here with species from Phocidae, Otariidae and Odobenidae sharing same low number of conserved human autosomal segments (32). Chromosome painting in Pinnipedia and comparison with non-pinniped carnivore karyotypes provide strong support for refined structure of ACK with 2n = 38. Constructed comparative chromosome maps show that pinniped karyotype evolution was characterized by few tandem fusions, seemingly absent inversions and slow rate of genome rearrangements (less then one rearrangement per 10 million years). Integrative comparative analyses with published chromosome painting of Phoca vitulina revealed common cytogenetic signature for Phoca/Pusa branch and supports Phocidae and Otaroidea (Otariidae/Odobenidae) as sister groups. We revealed rearrangements specific for walrus karyotype and found the chromosomal signature linking together families Otariidae and Odobenidae. The Steller sea lion karyotype is the most conserved among three studied species and differs from the ACK by single fusion. The study underlined the strikingly slow karyotype evolution of the Pinnipedia in general and the Otariidae in particular.     

Genomic signatures of fifth autotrophic carbon assimilation pathway in bathypelagic Crenarchaeota

Marine Crenarchaeota, ubiquitous and abundant organisms in the oceans worldwide, remain metabolically uncharacterized, largely due to their low cultivability. Identification of candidate genes for bicarbonate fixation pathway in the Cenarchaeum symbiosum A was an initial step in understanding the physiology and ecology of marine Crenarchaeota. Recent cultivation and genome sequencing of obligate chemoautotrophic Nitrosopumilus maritimus SCM1 were a major breakthrough towards understanding of their functioning and provide a valuable model for experimental validation of genomic data. Here we present the identification of multiple key components of 3-hydroxipropionate/4-hydroxybutyrate cycle, the fifth pathway in carbon fixation, found in data sets of environmental sequences representing uncultivated superficial and bathypelagic Crenarchaeota from Sargasso sea (GOS data set) and KM3 (Mediterranean Sea) and ALOHA (Atlantic ocean) stations. These organisms are likely to use acetyl-CoA/propionyl-CoA carboxylase(s) as CO₂-fixing enzyme(s) to form succinyl-CoA, from which one molecule of acetyl-CoA is regenerated via 4-hydroxybutyrate cleavage and another acetyl-CoA to be the pathway product. The genetic distinctiveness and matching sympatric abundance imply that marine crenarchaeal genotypes from the three different geographic sites share similar ecophysiological properties, and therefore may represent fundamental units of marine ecosystem functioning. To couple results of sequence comparison with the dark ocean primary production, dissolved inorganic carbon fixation rates were measured at KM3 Station (3000 m depth, Eastern Mediterranean Sea), i.e. at the same site and depth used for metagenomic library construction.     

Changes in the miRNA-mRNA Regulatory Network Precede Motor Symptoms in a Mouse Model of Multiple System Atrophy: Clinical Implications

Multiple system atrophy (MSA) is a fatal rapidly progressive α-synucleinopathy, characterized by α-synuclein accumulation in oligodendrocytes. It is accepted that the pathological α-synuclein accumulation in the brain of MSA patients plays a leading role in the disease process, but little is known about the events in the early stages of the disease. In this study we aimed to define potential roles of the miRNA-mRNA regulatory network in the early pre-motor stages of the disease, i.e., downstream of α-synuclein accumulation in oligodendroglia, as assessed in a transgenic mouse model of MSA. We investigated the expression patterns of miRNAs and their mRNA targets in substantia nigra (SN) and striatum, two brain regions that undergo neurodegeneration at a later stage in the MSA model, by microarray and RNA-seq analysis, respectively. Analysis was performed at a time point when α-synuclein accumulation was already present in oligodendrocytes at neuropathological examination, but no neuronal loss nor deficits of motor function had yet occurred. Our data provide a first evidence for the leading role of gene dysregulation associated with deficits in immune and inflammatory responses in the very early, non-symptomatic disease stages of MSA. While dysfunctional homeostasis and oxidative stress were prominent in SN in the early stages of MSA, in striatum differential gene expression in the non-symptomatic phase was linked to oligodendroglial dysfunction, disturbed protein handling, lipid metabolism, transmembrane transport and altered cell death control, respectively. A large number of putative miRNA-mRNAs interaction partners were identified in relation to the control of these processes in the MSA model. Our results support the role of early changes in the miRNA-mRNA regulatory network in the pathogenesis of MSA preceding the clinical onset of the disease. The findings thus contribute to understanding the disease process and are likely to pave the way towards identifying disease biomarkers for early diagnosis of MSA.