A possible explanation of the low-level brightness–contrast illusions in the light of an extended classical receptive field model of retinal ganglion cells

The low-level brightness–contrast illusions constitute a special class within visual illusions. Speculations exist that these illusions may be processed through the filtering action of the retinal ganglion cells without necessitating much intervention from higher order processes of visual perception. Concept of the classical receptive field of the ganglion cell, derived from early physiological studies, prompted the idea that a Difference of Gaussian (DoG) model might explain the low-level illusions. In spite of its many successes, the DoG model fails to explain some of these illusions. It has been shown in this paper that it is possible to simulate those illusions with a model that takes into cognizance the role of the extended classical receptive field     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

Histochemical studies on the distribution of β-glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of the rat

Summary The present study compares the distribution of -glucuronidase and succinic dehydrogenase in young and old spinal ganglion cells of rat. In young cells there are indications of cyclic activity of these enzymes, i.e., in some stages there are perinuclear concentrations of the enzymes, at other times -glucuronidase and succinic dehydrogenase are uniformly distributed throughout the cytoplasm. These stages have been discussed with the identical distribution of mitochondria. However, in old spinal ganglion cells both -glucuronidase and succinic dehydrogenase become mainly concentrated in the pigment areas, suggesting thereby their possible role in the production of pigment, through the medium of the mitochondria.     

A non-receptor-type protein phosphotyrosine phosphatase is enriched in secretory vesicles of glucagon – and pancreatic polypeptide – secreting cells of the endocrine pancreas

 The secretory vesicles of some cells of the islets of Langerhans of the pancreas contain high amounts of immunoreactive tyrosine phosphatase of the PTP1B/TCPTP subfamily. The cells are located in the peripheral parts of the islets and were identified as glucagon- and pancreatic polypeptide-forming cells. The tyrosine phosphatase is also enriched in some of the somatostatin-producing cells but is not elevated either in insulin-producing B-cells or in the exocrine pancreas. Virtually the same patterns were found in pancretic tissues of rats, guinea pigs, pigs, and mice. High levels of detergent-soluble tyrosine phosphatase were measured in the particular fraction of pancreatic islets with a substrate preferred by PTP1B/TCPTP-type protein tyrosine phosphatases. Accepted: 6 November 1998     

Expression of the cystine-glutamate exchanger (xc −) in retinal ganglion cells and regulation by nitric oxide and oxidative stress

The cystine-glutamate exchanger, system x_c ⁻, mediates the Na⁺-independent exchange of cystine into cells, coupled to the efflux of intracellular glutamate. System x_c ⁻ plays a critical role in glutathione homeostasis. Early studies of brain suggested that system x_c ⁻ was present primarily in astrocytes but not neurons. More recent work indicates that certain brain neurons have an active system x_c ⁻. In the retina, system x_c ⁻ has been demonstrated in Müller and retinal pigment epithelial cells. We have recently suggested that two protein components of system x_c ⁻, xCT and 4F2hc, are present in ganglion cells of the intact retina. Here, we have used (1) molecular and immunohistochemical assays to determine whether system x_c ⁻ is present in primary ganglion cells isolated from neonatal mouse retinas and (2) functional assays to determine whether its activity is regulated by oxidative stress in a retinal ganglion cell line (RGC–5). Primary mouse ganglion cells and RGC–5 cells express xCT and 4F2hc. RGC–5 cells take up [³H]glutamate in the absence of Na⁺, and this uptake is blocked by known substrates of system x_c ⁻ (glutamate, cysteine, cystine, quisqualic acid). Treatment of RGC–5 cells with NO and reactive oxygen species donors leads to increased activity of system x_c ⁻ associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. This is the first report of system x_c ⁻ in primary retinal ganglion cells and RGC–5 cells. Oxidative stress upregulates this transport system in RGC–5 cells, and the process is associated with an increase in xCT mRNA and protein but no change in 4F2hc mRNA or protein. This work was supported by National Institutes of Health grants EY014560 and EY012830.     

Role of the GNOM gene in Arabidopsis apical-basal patterning – From mutant phenotype to cellular mechanism of protein action

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.     

The nucleo-junctional interplay of the cellular prion protein: A new partner in cancer-related signaling pathways?

The cellular prion protein PrP^c plays important roles in proliferation, cell death and survival, differentiation and adhesion. The participation of PrP^c in tumor growth and metastasis was pointed out, but the underlying mechanisms were not deciphered completely. In the constantly renewing intestinal epithelium, our group demonstrated a dual localization of PrP^c, which is targeted to cell-cell junctions in interaction with Src kinase and desmosomal proteins in differentiated enterocytes, but is predominantly nuclear in dividing cells. While the role of PrP^c in the dynamics of intercellular junctions was confirmed in other biological systems, we unraveled its function in the nucleus only recently. We identified several nuclear PrP^c partners, which comprise γ-catenin, one of its desmosomal partners, β-catenin and TCF7L2, the main effectors of the canonical Wnt pathway, and YAP, one effector of the Hippo pathway. PrP^c up-regulates the activity of the β-catenin/TCF7L2 complex and its invalidation impairs the proliferation of intestinal progenitors. We discuss how PrP^c could participate to oncogenic processes through its interaction with Wnt and Hippo pathway effectors, which are controlled by cell-cell junctions and Src family kinases and dysregulated during tumorigenesis. This highlights new potential mechanisms that connect PrP^c expression and subcellular redistribution to cancer.     

Angiotensin IV upregulates the activity of protein phosphatase 1α in Neura-2A cells

The peptide angiotensin IV (Ang IV) is a derivative of angiotensin II. While insulin regulated amino peptidase (IRAP) has been proposed as a potential receptor for Ang IV, the signalling pathways of Ang IV through IRAP remain elusive. We applied high-resolution mass spectrometry to perform a systemic quantitative phosphoproteome of Neura-2A (N2A) cells treated with and without Ang IV using sta ble-isotope labeling by amino acids in cell culture (SILAC), and identified a reduction in the phosphorylation of a major Ser/Thr protein phosphorylase 1 (PP1) upon Ang IV treatment. In addition, spinophilin (spn), a PP1 regulatory protein that plays important functions in the neural system, was expressed at higher levels. Immunoblotting revealed decreased phosphorylation of p70S6 kinase (p70^S6K) and the major cell cycle modulator retinoblastoma protein (pRB). These changes are consistent with an observed decrease in cell proliferation. Taken together, our study suggests that Ang IV functions via regulating the activity of PP1.     

VIS – A database on the distribution of fishes in inland and estuarine waters in Flanders,Belgium

The Research Institute for Nature and Forest (INBO) has been performing standardized fish stock assessments in Flanders, Belgium. This Flemish Fish Monitoring Network aims to assess fish populations in public waters at regular time intervals in both inland waters and estuaries. This monitoring was set up in support of the Water Framework Directive, the Habitat Directive, the Eel Regulation, the Red List of fishes, fish stock management, biodiversity research, and to assess the colonization and spreading of non-native fish species. The collected data are consolidated in the Fish Information System or VIS. From VIS, the occurrence data are now published at the INBO IPT as two datasets: ‘VIS - Fishes in inland waters in Flanders, Belgium’ and ‘VIS - Fishes in estuarine waters in Flanders, Belgium’. Together these datasets represent a complete overview of the distribution and abundance of fish species pertaining in Flanders from late 1992 to the end of 2012. This data paper discusses both datasets together, as both have a similar methodology and structure. The inland waters dataset contains over 350,000 fish observations, sampled between 1992 and 2012 from over 2,000 locations in inland rivers, streams, canals, and enclosed waters in Flanders. The dataset includes 64 fish species, as well as a number of non-target species (mainly crustaceans). The estuarine waters dataset contains over 44,000 fish observations, sampled between 1995 and 2012 from almost 50 locations in the estuaries of the rivers Yser and Scheldt (“Zeeschelde”), including two sampling sites in the Netherlands. The dataset includes 69 fish species and a number of non-target crustacean species. To foster broad and collaborative use, the data are dedicated to the public domain under a Creative Commons Zero waiver and reference the INBO norms for data use.     

Differential partitioning of Gαi1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells

Background

Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance.

Results

Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Gαi1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the α-subunit of the G protein have been identified and the Gαi (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Gαi1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Gαi1 in these cells. Moreover, Gαi1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Gαi1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol.

Conclusion

Based on the opposing effects of taxol and the Gαi1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Gαi1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.     

A recombinant trans-membrane protein hMnSOD–R9 inhibits the proliferation of cervical cancer cells in vitro

Human manganese superoxide dismutase (hMnSOD) is a new type of cancer suppressor. Nonamer of arginine (R9) is an efficient protein transduction domain (PTD). The aim of the study was to improve the transduction efficiency of hMnSOD and investigate its activity in vitro. In this study, we designed, constructed, expressed, and purified a novel fusion protein containing the hMnSOD domain and R9 PTD (hMnSOD–R9). The DNA damaged by Fenton’s reagent was found to be significantly reduced when treated with hMnSOD–R9. hMnSOD–R9 fusion protein was successfully delivered into HeLa cells. The MTT assay showed that proliferation of various cancer cell lines were inhibited by hMnSOD–R9 in a dose-dependent manner. In addition, the cell cycle of HeLa cells was arrested at the sub-G0 phase by hMnSOD–R9. hMnSOD–R9 induced apoptosis of HeLa cells in a dose-dependent manner. With hMnSOD–R9 treatment, Bax, JNK, TBK1 gene expression was increased and STAT3 gene expression was gradually down-regulated in HeLa cells. We also found that apoptosis was induced by hMnSOD–R9 in HeLa cells via up-regulation of cleaved caspase-3 and down-regulation phospho-STAT3 pathway. These results indicated that hMnSOD–R9 may provide benefits to cervical cancer treatment.     

Distribution of mitochondria within Muller cells – II. Post-natal development of the rabbit retinal periphery in vivo and in vitro: dependence on oxygen supply

The occurrence and localization of mitochondria within glial (Muller) cells and neurons of the peripheral (avascular) rabbit retina was studied electron microscopically and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). Post-natal development in vivo was compared with development of organ cultures from neonatal rabbit retinae, grown over 2 weeks in vitro. The adult pattern of mitochondrial localization (restriction to the sclerad end of the cells) was observed from the beginning of enzyme expression at early post-natal stages. However, when neonatal retinal pieces were grown in vitro with their vitread surface exposed to the air, their Muller cells contained mitochondria along most of their length. When functionally developed retinae from postnatal day 14 were explanted in vitro, they retained their sclerad mitochondrial distribution for almost 24 h but thereafter the inner portions of their cytoplasm became occupied by mitochondria within a few hours. This was achieved mainly by mitochondrial migration rather than by formation of new mitochondria because it was not prevented by cycloheximide-induced inhibition of protein synthesis. These data support the following hypotheses: (1) the mitochondrial distribution in Muller cells is determined by the local cytoplasmic O2 pressure (pO2), (2) existing mitochondria move towards cytoplasmic regions of sufficient pO2 by rather rapid migration and (3) the start of this migration is delayed by almost 24 h due to the action of as yet unknown control mechanisms. In contrast, the mitochondrial content of retinal ganglion and amacrine cells in the vitread retinal layers was virtually independent of the source and level of oxygen supply.     

Effects of 5-bromo-2′-deoxyuridine on arachidonic acid metabolism of neuroblastoma and leukemia cells in culture: A possible role of endogenous prostaglandins in tumor cell proliferation and differentiation

Effects of 5-bromo-2′-deoxyuridine (BrdU) were studied on two neuroblastoma and two leukemia cell lines, in terms of the relationship between prostaglandin (PG) synthesis and cell growth/differentiation. After treatment with BrdU (5 μg/ml), cell growth of the 4 cell lines was inhibited and one neuroblastoma cell line (GOTO) showed flattened morphology with positive S-100 protein, one of the differentiation markers for Schwann or glial cells. In the 4 cell lines, BrdU treatment reduced [1-¹⁴C]-arachidonic acid incorporation into phosphatidylinositol and phosphatidylethanolamine and was associated with an increase into phosphatidylcholine and triglyceride. BrdU treatment also increased fractions of 6-keto-PGF_1α and PGF_2α , with a decreased TXB₂ fraction. The decreased ratio of TXB₂ /6-keto-PGF_1α or increased 6-keto-PGF_1α fraction correlated significantly with cell growth inhibition, suggesting that the changes in the balance of endogenous PGs might be associated with BrdU-induced cell growth inhibition with or without differentiation of neuroblastoma and leukemia cells in culture.     

Cholesterol enhances amyloid β deposition in mouse retina by modulating the activities of Aβ-regulating enzymes in retinal pigment epithelial cells

Cancer patients frequently develop autoantibodies. To test the hypothesis that the appearance of autoantibodies precedes the clinical diagnosis of cancer, we applied an immunoproteomic approach to compare autoantibody profiles before and after appearance of malignances. Proteins from A549 cells, a lung adenocarcinoma cell line, were separated by two dimensional electrophoresis and then immunoblotted with serum samples from 8 individuals who were eventually diagnosed with lung cancer. Compared with autoantibody profiles from 3 years prior to the appearance of malignances, 21 immunoreactive spots newly appeared or presented with stronger staining intensity when clinical diagnoses were made. Among them, 10 matched spots on 2-DE gels were identified by mass spectrometry analysis as 5 proteins. With immunoprecipitation analysis, the antigenicity of protein cathepsin D was confirmed, and notably, in lung cancer sera, the occurrences of autoantibodies against the specific forms of cathepsin D differed significantly from the control groups (p<0.05). Our findings suggest that harnessing immunity may have utility for early cancer marker discovery, and that comparing autoantibodies to specific forms of cathepsin D may be a promising early marker of lung cancer.     

A preliminary study on the induction of dioestrous ovulation in the mare – a possible method for inducing prolonged luteal phase

Background  

Strong oestrous symptoms in the mare can cause problems with racing, training and handling. Since long-acting progesterone treatment is not permitted in mares at competition (e.g. according to FEI rules), there is a need for methods to suppress unwanted cyclicity. Spontaneous dioestrous ovulations in the late luteal phase may cause a prolongation of the luteal phase in mares.     

Ultrastructural localization of calcitonin in control and stimulated thyroid C cells of the rat using protein A — gold immunocytochemical technique

Summary Using anti-human calcitonin serum and a protein !-gold technique, calcitonin was localized at the ultrastructural level in control and calcium gluconate-stimulated thyroid C cells of the rat. In control rats calcitonin was dedected within a majority of the secretory granules while in experimental animals it was demonstrated also within prosecretory granules present in Golgi apparatus.The study was partially supported by a grant from the Committee of Cytobiology, Polish Academy of Sciences     

cAMP increases the sensitivity of exocytosis to Ca²+ primarily through protein kinase A in mouse pancreatic beta cells

Cyclic AMP regulates the late step of Ca2+-dependent exocytosis in many secretory cells through two major mechanisms: a protein kinase A-dependent and a cAMP-GEF/Epac-dependent pathway. We designed a protocol to characterize the role of these two cAMP-dependent pathways on the Ca2+ sensitivity and kinetics of regulated exocytosis in mouse pancreatic beta cells, using a whole-cell patch-clamp based capacitance measurements. A train of depolarizing pulses or slow photo-release of caged Ca2+ were stimuli for the exocytotic activity. In controls, due to exocytosis after slow photo-release, the C(m) change had typically two phases. We observed that the Ca2+-dependency of the rate of the first C(m) change follows saturation kinetics with high cooperativity and half-maximal rate at 2.9±0.2 μM. The intracellular depletion of cAMP did not change amp1, while rate1 and amp2 were strongly reduced. This manipulation pushed the Ca2+-dependency of the exocytotic burst to significantly lower [Ca2+](i). To address the question of which of the cAMP-dependent mechanisms regulates the observed shifts in Ca2+ dependency we included regulators of PKA and Epac2 activity in the pipette solution. PKA activation with 100 μM 6-Phe-cAMP or inhibition with 500 μM Rp-cAMPs in beta cells significantly shifted the EC(50) in the opposite directions. Specific activation of Epac2 did not change Ca2+ sensitivity. Our findings suggest that cAMP modulates Ca2+-dependent exocytosis in mouse beta cells mainly through a PKA-dependent mechanism by sensitizing the insulin releasing machinery to [Ca2+](i); Epac2 may contribute to enhance the rates of secretory vesicle fusion.