Differential effects of c-Ras upon transformation, adipocytic differentiation, and apoptosis mediated by the simian virus 40 large tumor antigen.

To investigate the functional relationship between the ability of the simian virus 40 large tumor antigen (TAg) to transform and its ability to block adipocytic differentiation and induce apoptosis, we expressed TAg in C3H10T1/2 (10T1/2)-derived preadipocytes. The results demonstrated that differentiation could be suppressed at lower TAg levels than at the levels required for full neoplastic conversion. Progressively higher TAg levels were accompanied by apoptosis induction in this system. To further examine the role of the cellular Ras protooncogene product (Ras) in TAg function, TAg was expressed in 10T1/2-derived preadipocytes rendered deficient in Ras activity by transfection with inducible or constitutive antisense ras gene constructs. The results indicated that Ras is required for TAg-mediated transformation and for suppression of adipocytic differentiation, while TAg-mediated apoptosis following serum starvation was independent from Ras action. Unexpectedly, our results further demonstrated a dramatic reduction in the levels of the TAg protein itself as differentiation progressed in Ras-knockdown cells, with a concomitant reduction in TAg's ability to induce apoptosis as a result. These findings suggest that Ras, although cytoplasmic, is an integral component of the pathway whereby TAg, an oncoprotein believed to have primarily nuclear targets, suppresses differentiation or induces neoplastic conversion of murine preadipocytes.     

A method to probe the cytoplasmic osmolality and osmotic water and solute fluxes across the cell membrane of cyanobacteria with chlorophyll a fluorescence: Experiments with Synechococcus sp. PCC7942

We present a method with which osmotic properties of the cytoplasm of cyanobacterial cells and the osmotic permeability of plasma membranes to water and solutes can be assessed from measurements of chlorophyll a fluorescence. When the electron transport of photosystem II is inhibited, the quantum yield of chlorophyll a fluorescence in cyanobacterial cells varied between a low yield limit that was attained after acclimation to darkness (state 2) and a high yield limit that was attained after acclimation to light (state 1). It was shown recently that the difference between chlorophyll a fluorescence of light‐acclimated and of dark‐acclimated cells relates quantitatively to the internal osmolality of cyanobacteria (G. C. Papageorgiou and A. Alygizaki‐Zorba. 1997. Biochim. Biophys. Acta 1335: 1‐4). In the present work we employed rapid mixing of Synechococcus sp. PCC7942 (strain PAMCOD) suspensions with solutions of defined osmolality in order to measure cell osmolality and turgor threshold, as well as water and solute fluxes across cell membranes. Concentration upshocks with sorbitol, glycine betaine, Na⁺ and K⁺ salts caused rapid (t_1/2 < 10 ms) depression of fluorescence that was correlated to osmotic water outflow from the cells. The fluorescence remained depressed in all cases except for NaCl. With NaCl, the depression was transient and fluorescence recovered with an apparent time constant of 200 ms. The fluorescence rise correlates to inflows of NaCl and water.     

Affective touch and attachment style modulate pain: a laser-evoked potentials study

Affective touch and cutaneous pain are two sub-modalities of interoception with contrasting affective qualities (pleasantness/unpleasantness) and social meanings (care/harm), yet their direct relationship has not been investigated. In 50 women, taking into account individual attachment styles, we assessed the role of affective touch and particularly the contribution of the C tactile (CT) system in subjective and electrophysiological responses to noxious skin stimulation, namely N1 and N2-P2 laser-evoked potentials. When pleasant, slow (versus fast) velocity touch was administered to the (non-CT-containing) palm of the hand, higher attachment anxiety predicted increased subjective pain ratings, in the same direction as changes in N2 amplitude. By contrast, when pleasant touch was administered to CT-containing skin of the arm, higher attachment anxiety predicted attenuated N1 and N2 amplitudes. Higher attachment avoidance predicted opposite results. Thus, CT-based affective touch can modulate pain in early and late processing stages (N1 and N2 components), with the direction of effects depending on attachment style. Affective touch not involving the CT system seems to affect predominately the conscious perception of pain, possibly reflecting socio-cognitive factors further up the neurocognitive hierarchy. Affective touch may thus convey information about available social resources and gate pain responses depending on individual expectations of social support.This article is part of the themed issue ‘Interoception beyond homeostasis: affect, cognition and mental health’.     

Light-induced and osmotically-induced changes in chlorophyll a fluorescence in two Synechocystis sp. PCC 6803 strains that differ in membrane lipid unsaturation

Membranes of wild-type (WT) cells of the cyanobacterium Synechocystis sp. PCC 6803 are abundant in polyunsaturated fatty acids in membrane lipids and thus more fluid than membranes of desA^-/desD^- mutant cells which contain no polyunsaturated fatty acids. Using intact cells we examined the effects of normal and chilling temperatures on membrane fluidity-dependent properties. We probed the thylakoid membranes by inducing light/dark acclimative changes in chlorophyll a (Chl a) fluorescence; and we probed the plasma membranes either by suppressing the Chl a fluorescence of light-acclimated cells under hyper-osmotic conditions, or by measuring the electric conductivity of cell suspensions. Thylakoid membranes of mutant cells undergo reversible thermotropic transition between 19 °C and 22 °C (midpoint at 20.5 °C). No analogous transition was detected in the thylakoid membranes of WT cells in the temperature range from 2 to 34 °C. Plasma me mbranes of both WT and mutant cells did not experience thermotropic transition in the temperature range from 2 °C to 34 °C as detected either fluorimetrically or by means of electric conductivity. Hyper-osmotic conditions caused fast transient fluorescence quenching in WT cells at 34 °C, but not at 14 °C, and not in mutant cells at either 34 °C or 14 °C. This transient quenching sensed probably the higher fluidity of the plasma membranes of WT cells. Hyper-osmotic media and dark acclimation had similar effects on the 77 K fluorescence of Synechocystis cells: they suppressed the ratio of photosystem II fluorescence to photosystem I fluorescence.     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Vaginal fold prolapse during the last third of pregnancy, followed by normal parturition, in a bitch

This article describes a 1.5-year-old female, Greek Hound dog, weighing 16 kg, presented with a type III vaginal prolapse which occurred during the last third of pregnancy. Trans-abdominal ultrasonography revealed four live foetuses in the uterine horns. The animal was hospitalized and 4 days later gave birth without any interference. Three days later, resection of the prolapsed tissue was performed and the bitch recovered completely. Recurrence of a type I vaginal prolapse was observed 4 months later, during subsequent oestrus. This case is unusual because, although vaginal fold prolapse is mainly seen during proestrus/oestrus or during parturition, it was first noticed 47 days after mating and 13 days before parturition. Furthermore, even though the prolapse of vaginal fold was of type III and of considerable size, parturition proceeded normally. Finally, even though resection of the prolapsed tissue was performed 3 days after parturition, recurrence of vaginal fold oedema (type I) was observed in the subsequent oestrus.     

Fibroblast growth factor-2 modulates melanoma adhesion and migration through a syndecan-4-dependent mechanism

Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells’ ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration.These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.     

HAX-1: a family of apoptotic regulators in health and disease

HAX-1 comprises a family of ubiquitously expressed proteins that play important roles in the regulation of programmed cell death. Herein, we provide a comprehensive review of the expression profile of HAX-1 and its functional implications during health and disease, highlighting its direct involvement in the development of congenital neutropenia and neural abnormalities, when absent, and its contribution to the progression of psoriasis and cancer, when overexpressed. Moreover, we provide new information on the differential expression of the HAX-1 subfamily in three distinct types of epithelial cancers, including breast, skin, and colon. Our results demonstrate a significant up-regulation of the anti-apoptotic HAX-1 variant 001 in skin and colon, but not in breast and cancer cells, indicating tissue-specific differences in its expression pattern and properties during cancer formation and progression. Our findings further reveal a considerable down-regulation, if not abrogation, of three distinct, yet to be characterized, HAX-1 isoforms in breast cancer cells, suggesting that they may function in an opposite manner to the anti-apoptotic variant 001. This study aims to summarize our current knowledge on the physiological implications of the expression profile of the HAX-1 subfamily in health and disease, and provide new information on the differential expression and activities of HAX-1 members in three distinct types of cancer.