Electrophysiological characteristics of the unicellular green alga Micrasterias torreyi

The electrophysiological characteristics of the unicellular green alga Micrasterias torreyi Bail. are studied here for the first time using microelectrode techniques. The resting potential of the plasma membrane varied between –39.5 and –42.2 mV for different developmental stages of the dividing cell and was –41.7 mV ( se = 3.2, n = 9) in the interphase cells. The resting potential of the chloroplast envelope was lower, –53.9 mV ( se = 3.6, n = 15). Supraoptimal K⁺ (20 m M ) had no clear effects on the plasma membrane but caused a depolarization of 10 mV in the chloroplast. Additional external Ca²⁺ (10 m M ) depolarized the membrane potential quite strongly (by 23 mV). Low external pH did not affect the resting potential of the cell. There is a marked difference in the resting potential values between non-vacuolated cells (about –40 mV), to which Micrasterias belongs, and vacuolated plant cells (–100 to –250 mV). This indicates the participation of the tonoplast in the transport of ions and charged molecules in vacuolated cells. Na⁺ and Cl⁻, which play an important role in ion metabolism in most plant cells, are not needed by Micrasterias .     

Localization of nonerythroid spectrin and actin in mouse oocytes and preimplantation embryos

Mouse oocytes, cleavage-stage embryos, and blastocyst-stage embryos were studied to show the distribution of both an immunoanalog to nonerythroid spectrin (p 230) and F-actin. Using antibodies to nonerythroid spectrin, diffuse, positive cytoplasmic fluorescence was regularly seen in oocytes and embryo cells. The presence of nonerythroid spectrin in oocytes was confirmed by immunoblotting. Oocytes usually exhibited an inconspicuous submembranous layer of nonerythroid spectrin, which was more pronounced in the area of the polar body. Oocytes regularly exhibited a peripheral concentration of actin. Throughout the cleavage and blastocyst stages, a cortical layer of nonerythroid spectrin and actin was usually observed in embryo cells. These submembranous layers on the outer surface of the embryo were relatively thin as compared to those in areas of intercellular contact. The contact areas regularly showed distinct positive staining, including a concentration of label at the most peripheral region of each contact area. This resulted in the presence of ring-like fluorescence around each blastomere. Nonerythroid spectrin and actin showed concentration to the contact area between the oocyte and the polar body. Although the general localization patterns of nonerythroid spectrin and actin were similar, double-staining experiments revealed that slightly different planes of focus were necessary to obtain sharp definition of the fluorescence of these components in areas of intercellular contact: the ring-like concentration of nonerythroid spectrin appeared to be localized more peripherally than that of actin. The cells of preimplantation embryos show motile features that include actual cell movements and striking changes in cell shape (e.g., during compaction). The submembraneous layers of nonerythroid spectrin and actin may contribute to the regulation of the deformability and thus the shape of embryo cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiplex PCR and Next Generation Sequencing for the Non-Invasive Detection of Bladder Cancer

Background

Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.

Methods

DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).

Results

Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).

Conclusion

This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.     

Phenotypic characters of static homology increase phylogenetic stability under direct optimization of otherwise dynamic homology characters

Direct optimization of unaligned sequence characters provides a natural framework to explore the sensitivity of phylogenetic hypotheses to variation in analytical parameters. Phenotypic data, when combined into such analyses, are typically analyzed with static homology correspondences unlike the dynamic homology sequence data. Static homology characters may be expected to constrain the direct optimization and thus, potentially increase the similarity of phylogenetic hypotheses under different cost sets. However, whether a total-evidence approach increases the phylogenetic stability or not remains empirically largely unexplored. Here, I studied the impact of static homology data on sensitivity using six empirical data sets composed of several molecular markers and phenotypic data. The inclusion of static homology phenotypic data increased the average stability of phylogenetic hypothesis in five out of the six data sets. To investigate if any static homology characters would have similar effect, the analyses were repeated with randomized phenotypic data, and with one of the molecular markers fixed as static homology characters. These analyses had, on average, almost no effect on the phylogenetic stability, although the randomized phenotypic data sometimes resulted in even higher stability than empirical phenotypic data. The impact was related to the strength of the phylogenetic signal in the phenotypic data: higher average jackknife support of the phenotypic tree correlated with stronger stabilizing effect in the total-evidence analysis. Phenotypic data with a strong signal made the total-evidence trees topologically more similar to the phenotypic trees, thus, they constrained the dynamic homology correspondences of the sequence data. Characters that increase phylogenetic stability are particularly valuable for phylogenetic inference. These results indicate an important role and additive value of phenotypic data in increasing the stability of phylogenetic hypotheses in total-evidence analyses.     

Action of cytochalasin B on cytoplasmic streaming systems and morphogenesis in Micrasterias torreyi (Conjugatophyceae)

The effects of cytochalasin B on the development and morphology of the Micrasterias cell vary according to different developmental stages. CB (especially 5–6 μm/ml) does not stop the growth of a developing semicell during early developmental stages and allows the formation of a three–lobed form, which is a kind of a basic form preceding the actual morphogenesis, further growth and development still being totally prevented by the same concentration. This is due to the occurrence of two separate cytoplasmic streaming systems in the developing semicell, one being cortical, more CB sensitive, the other being more central, not as sensitive to CB, both being microfilament based. The cortical streaming system supports tip growth, and many observations indicate that this system is also associated with the intermediation of the nuclear control regulating morphogenesis in Micrasterias. The effects of CB are specific: it has not been found to have distinct effects on the ultrastructure of the cell, on cell wall material production, or on the turgor pressure of the cell.     

Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis.

We previously demonstrated that the intracellular third loop (i3 loop) of angiotensin II type 2 receptor (AT2) plays a key role in mediating the biological functions of this receptor. To determine which residues are important for AT2 signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 within the intermediate portion of the i3 loop resulted in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases (ERK), and SHP-1 activation. In contrast to well characterized heptahelical receptors, the AT2 functions were not affected by deletions of the amino- or carboxyl-terminal portions of the i3 loop. Alanine substitutions further demonstrated that lysine 240, asparagine 242, and serine 243 are key residues for AT2-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT2-induced ERK inhibition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT2 function and that SHP-1 is a proximal effector of the AT2 receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.     

Teratocarcinoma stem cells as a model for differentiation in the mouse embryo

Embryonal carcinoma (EC) cells, which are the malignant stem cells of teratocarcinomas, are considered similar to early embryo cells. The EC cells can be grown in vitro, and many of them can be experimentally induced to differentiate; upon differentiation, the cells become benign. Here we review some of the changes that take place in the cellular and molecular characteristics of murine F9 EC cells as they differentiate into endodermal cells. Upon differentiation of F9 cells, distinct changes occur in their cell surface molecules, cytoskeleton-associated proteins and cell adhesion properties. Simultaneously, the rate of cell proliferation decreases due to a dramatic increase in duration of G1 and S phases of the cell cycle. The changes in gene expression and cell behavior occurring during endodermal differentiation of EC cells closely resemble those occurring when the endoderm differentiates in the embryo. Teratocarcinoma stem cell lines may thus be exploited to enhance understanding of both teratoma-type neoplasms and embryonic development.