The formation and positioning of cilia in Ciona intestinalis embryos in relation to the generation and evolution of chordate left-right asymmetry

In the early mouse embryo monocilia on the ventral node rotate to generate a leftward flow of fluid. This nodal flow is essential for the left-sided expression of nodal and pitx2, and for subsequent asymmetric organ patterning. Equivalent left fluid flow has been identified in other vertebrates, including Xenopus and zebrafish, indicating it is an ancient vertebrate mechanism. Asymmetric nodal and Pitx expression have also been identified in several invertebrates, including the vertebrates' nearest relatives, the urochordates. However whether cilia regulate this asymmetric gene expression remains unknown, and previous studies in urochordates have not identified any cilia prior to the larval stage, when asymmetry is already long established. Here we use Scanning and Transmission Electron Microscopy and immunofluorescence to investigate cilia in the urochordate Ciona intestinalis. We show that single cilia are transiently present on each ectoderm cell of the late neurula/early tailbud stage embryo, a time point just before onset of asymmetric nodal expression. Mapping the position of each cilium on these cells shows they are posteriorly positioned, something also described for mouse node cilia. The C. intestinalis cilia have a 9+0 ring ultrastructure, however we find no evidence of structures associated with motility such as dynein arms, radial spokes or nexin. Furthermore the 9+0 ring structure becomes disorganised immediately after the cilia have exited the cell, indicative of cilia which are not capable of motility. Our results indicate that although cilia are present prior to molecular asymmetries, they are not motile and hence cannot be operating in the same way as the flow-generating cilia of the vertebrate node. We conclude that the cilia may have a role in the development of C. intestinalis left-right asymmetry but that this would have to be in a sensory capacity, perhaps as mechanosensors as hypothesised in two-cilia physical models of vertebrate cilia-driven asymmetry.     

The effect of variation in parameters of the male calling song of the katydid,Amblycorypha parvipennis (Orthoptera: Tettigoniidae), on female phonotaxis and phonoresponse

After being placed equidistant from playbacks of two computer-generated male calls, females of the katydidAmblycorypha parvipennis preferentially moved toward call phrases that were louder, longer, and leading (initiated without being overlapped by other call phrase endings). Because earlier tests with calling males had indicated that male mating success was related to these call parameters, we suggest that mating success is partly a result of female choice. Females also preferentially responded to the initial, rather than the latter, half of male phrases. Results of other tests suggested that females were responding to increased phonatome rate characteristic of the first half of the phrase. Males may compete to lead in order to prevent jamming of initial phrase information. Females also preferentially phonoresponded (ticked) in response to longer phrases. In earlier studies of male mating success and female phonotaxis using live males, male weight, sound level, and leading were intercorrelated; however, none of these parameters were correlated with phrase length. We suggest that females may respond to different call parameters under different environmental conditions.     

Immunofluorescent visualisation of focal adhesion kinase in human skeletal muscle and its associated microvasculature

Within animal skeletal muscle, focal adhesion kinase (FAK) has been associated with load-dependent molecular and metabolic adaptation including the regulation of insulin sensitivity. This study aimed to generate the first visual images of the localisation of FAK within human skeletal muscle fibres and its associated microvasculature using widefield and confocal immunofluorescence microscopy. Percutaneous muscle biopsies, taken from five lean, active males, were frozen and 5-μm cryosections were incubated with FAK antibodies for visualisation in muscle fibres and the microvasculature. Anti-myosin heavy chain type I was used for fibre-type differentiation. Muscle sections were also incubated with anti-dihydropyridine receptor (DHPR) to investigate co-localisation of FAK with the t-tubules. FITC-conjugated Ulex europaeus Agglutinin I stained the endothelium of the capillaries, whilst anti-smooth muscle actin stained the vascular smooth muscle of arterioles. Fibre-type differences in the intensity of FAK immunofluorescence were determined with image analysis software. In transversely and longitudinally orientated fibres, FAK was localised at the sarcolemmal regions. In longitudinally orientated fibres, FAK staining also showed uniform striations across the fibre and co-staining with DHPR suggests FAK associates with the t-tubules. There was no fibre-type difference in sarcoplasmic FAK content. Within the capillary endothelium and arteriolar smooth muscle, FAK was distributed heterogeneously as clusters. This is the first study to visualise FAK in human skeletal muscle microvasculature and within the (sub)sarcolemmal and t-tubule regions using immunofluorescence microscopy. This technique will be an important tool for investigating the role of FAK in the intracellular signalling of human skeletal muscle and the endothelium of its associated microvasculature.     

Isolation and phenotypic characterisation of stem cells from late stage osteoarthritic mesenchymal tissues

Introduction: Osteoarthritis (OA) represents an increasing health issue worldwide. Regenerative medicine (RM) has raised the hope for introducing revolutionary therapies in clinical practice. Detection of autologus cell sources can improve accessibility to RM strategies. Objectives: To assess the presence and biological potential of mesehchymal stem cells in three tissues (subchondral bone, synovial layer, periarticular adipose tissue) in late stages osteoarthritic patients. Material and Methods: Samples were collected from subjects undergoing total knee replacement (TKR). MSCs were isolated and cultured in complete αMEM with β FGF. Cell morphology and growth potential was assessed. Flow cytometry was used for detection of several relevant cell surface markers. Quantitative and qualitative assessment of differentiation potential towards three mesenchymal lineages (osteogenesis adipogenesis chondrogenesis) was performed. Time lapse life cell imaging of nondiferentiated cells over 24 hours period was used to determine cell kinetics. Results: Mesenchymal cells derived from all donors and tissue types showed morphology, growth and surface cell markers associated with stemness. All cell types underwent differentiation toward three mesenchymal lineages with significant differences between tissues of origin, not between donors. Cell kinetics, as derived from life imaging records, was variable with tissue of origin, significant higher for adipose derived MSCS. Conclusion: Human late stage OA mesenchymal tissues, contain progenitors with proliferative and differentiation potential of MSCs. These populations can be used for research and autologus regenerative therapies. Further comparative studies with age matched non OA samples has the potential of contributing to deepening knowledge about disease occurrence and progression.     

High glucose augments the angiotensin II-induced activation of JAK2 in vascular smooth muscle cells via the polyol pathway

Angiotensin II (Ang II), protein kinase C (PKC), reactive oxygen species (ROS) generated by NADPH oxidase, the activation of Janus kinase 2 (JAK2), and the polyol pathway play important parts in the hyperproliferation of vascular smooth muscle cells (VSMC), a characteristic feature of diabetic macroangiopathy. The precise mechanism, however, remains unclear. This study investigated the relation between the polyol pathway, PKC-beta, ROS, JAK2, and Ang II in the development of diabetic macroangiopathy. VSMC cultured in high glucose (HG; 25 mm) showed significant increases in the tyrosine phosphorylation of JAK2, production of ROS, and proliferation activities when compared with VSMC cultured in normal glucose (5.5 mm (NG)). Both the aldose reductase specific inhibitor (zopolrestat) or transfection with aldose reductase antisense oligonucleotide blocked the phosphorylation of JAK2, the production of ROS, and proliferation of VSMC induced by HG, but it had no effect on the Ang II-induced activation of these parameters in both NG and HG. However, transfection with PKC-beta antisense oligonucleotide, preincubation with a PKC-beta-specific inhibitor (LY379196) or apocynin (NADPH oxidase-specific inhibitor), or electroporation of NADPH oxidase antibodies blocked the Ang II-induced JAK2 phosphorylation, production of ROS, and proliferation of VSMC in both NG and HG. These observations suggest that the polyol pathway hyperactivity induced by HG contributes to the development of diabetic macroangiopathy through a PKC-beta-ROS activation of JAK2.     

The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood

The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4.), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood. Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase. We have arbitarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37°C with a saturating concentration of exogenous thymidine.

In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 ± 11 units (mean ± S.D., n =20, range 45–89). The apparent Michaelis constant for thymidine was of the order to 10⁻⁴ M but varied nearly 5-fold between different individuals. Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors. When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets. Whole-blood thymidine phosphorylase activity correlated well with platelet parameters. Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin.

Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity ± S.D. was 96 ± 27 units; range 58–140, n = 8).

Thymine formation from thymidine was temperature- and pH-depdendent in whole blood. 2′-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3′-azido-3′-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature. Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously.

In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.     

Continuous Stroke Volume Estimation from Aortic Pressure Using Zero Dimensional Cardiovascular Model: Proof of Concept Study from Porcine Experiments

Introduction

Accurate, continuous, left ventricular stroke volume (SV) measurements can convey large amounts of information about patient hemodynamic status and response to therapy. However, direct measurements are highly invasive in clinical practice, and current procedures for estimating SV require specialized devices and significant approximation.

Method

This study investigates the accuracy of a three element Windkessel model combined with an aortic pressure waveform to estimate SV. Aortic pressure is separated into two components capturing; 1) resistance and compliance, 2) characteristic impedance. This separation provides model-element relationships enabling SV to be estimated while requiring only one of the three element values to be known or estimated. Beat-to-beat SV estimation was performed using population-representative optimal values for each model element. This method was validated using measured SV data from porcine experiments (N = 3 female Pietrain pigs, 29–37 kg) in which both ventricular volume and aortic pressure waveforms were measured simultaneously.

Results

The median difference between measured SV from left ventricle (LV) output and estimated SV was 0.6 ml with a 90% range (5^th–95^th percentile) −12.4 ml–14.3 ml. During periods when changes in SV were induced, cross correlations in between estimated and measured SV were above R = 0.65 for all cases.

Conclusion

The method presented demonstrates that the magnitude and trends of SV can be accurately estimated from pressure waveforms alone, without the need for identification of complex physiological metrics where strength of correlations may vary significantly from patient to patient.     

Growth of B95-8 cells and expression of Epstein-Barr virus lytic phase in serum-free medium.

Epstein-Barr virus (EBV)-transformed tamarin (Saguinus oedipus) cells (B95-8) were selected for growth in medium with reduced serum and then transferred to serum-free medium which consisted of RPMI 1640 supplemented with insulin, transferrin, and selenium. Serum-free cells in continuous passage for 1 year had a morphology, growth rate, and culture density which approached those of B95-8 cells grown with serum. The cells expressed virus-induced antigens, including the EBV-associated DNA polymerase. Cells exposed to EBV-inducing agents, n-butyric acid and phorbol 12-myristate-13-acetate, produced transforming virus with titers comparable to those of cultures grown with serum. These findings demonstrate that serum is neither required for the growth of B95-8 cells nor necessary for induction or full expression of the EBV lytic phase in these cells.     

Identification of CpG islands in a physical map encompassing the Friedreich's ataxia locus

The Friedreich's ataxia locus has been previously assigned to chromosome 9q 13-21.1 by the demonstration of tight linkage to two anonymous DNA markers. MCT112 (Z greater than 80, theta = 0) and DR47 (Z greater than 50, theta = 0). The absence of recombination between these three loci has prevented the resolution of gene/probe order in this region, impeding strategies for gene isolation. We report physical mapping over a 4-Mb genomic interval, linking the markers MCT112 and DR47 on a common 460-kb NotI fragment and identifying 11 CpG islands in the 1.7-Mb interval most likely to contain the Friedreich's ataxia locus. Four of these islands were detected only by analysis of three YAC clones spanning a 700-kb interval including the MCT112/DR47 cluster. Without clear evidence of the precise location of the disease locus from recombination events, each of these regions must be considered as specifying a potential "candidate" sequence for the mutated gene.