γ-Synuclein Interacts with Phospholipase Cβ2 to Modulate G Protein Activation

Phospholipase Cβ2 (PLC β2) is activated by G proteins and generates calcium signals in cells. PLCβ2 is absent in normal breast tissue, but is highly expressed in breast tumors where its expression is correlated with the progression and migration of the tumor. This pattern of expression parallels the expression of the breast cancer specific gene protein 1 which is also known as γ-synuclein. The cellular function of γ-synuclein and the role it plays in proliferation are unknown. Here, we determined whether γ-synuclein can interact with PLCβ2 and affect its activity. Using co-immunprecitation and co-immunofluorescence, we find that in both benign and aggressive breast cancer cell lines γ-synuclein and PLCβ2 are associated. In solution, purified γ-synuclein binds to PLCβ2 with high affinity as measured by fluorescence methods. Protease digestion and mass spectrometry studies show that γ-synuclein binds to a site on the C-terminus of PLCβ2 that overlaps with the Gαq binding site. Additionally, γ-synuclein competes for Gαq association, but not for activators that bind to the N-terminus (i.e. Rac1 and Gβγ). Binding of γ-synuclein reduces the catalytic activity of PLCβ2 by mechanism that involves inhibition of product release without affecting membrane interactions. Since activated Gαq binds more strongly to PLCβ2 than γ-synuclein, addition of Gαq(GTPγS) to the γ-synuclein -PLCβ2 complex allows for relief of enzyme inhibition along with concomitant activation. We also find that Gβγ can reverse γ-synuclein inhibition without dissociating the γ-synuclein- PLCβ2- complex. These studies point to a role of γ-synuclein in promoting a more robust G protein activation of PLCβ2.     

Emerging roles of the tumor-associated stroma in promoting tumor metastasis

The stroma in human carcinomas consists of extracellular matrix and various types of non-carcinoma cells, mainly leukocytes, endothelial cells, fibroblasts, myofibroblasts and bone marrow-derived progenitors. The tumor-associated stroma actively supports tumor growth by stimulating neo-angiogenesis, as well as proliferation and invasion of apposed carcinoma cells. It has long been accepted that alterations within carcinoma cells mediate metastasis in a cell-autonomous fashion. Recent studies have, however, suggested an additional notion that cancer cells instigate local and systemic changes in the tumor microenvironment and contribute to niche formation for metastasis. Research, aiming to establish the roles of the tumor-associated stroma in facilitating the spread of carcinoma cells into distant organs, has provided an abundance of data and greater knowledge of the biology of metastatic carcinoma cells and associated stromal cells. This has stimulated further advances in the development of novel therapeutic approaches targeting tumor metastasis.     

CacyBP/SIP phosphatase activity in neuroblastoma NB2a and colon cancer HCT116 cells

Recently, we have reported that CacyBP/SIP could be a novel phosphatase for ERK1/2 kinase. In this work, we analyzed the CacyBP/SIP phosphatase activity toward ERK1/2 in 2 cell lines of different origin. We showed that overexpression of CacyBP/SIP in NB2a cells resulted in a lower level of phosphorylated ERK1/2 (P-ERK1/2) in the nuclear fraction while such overexpression in HCT116 cells had no effect on the level of P-ERK1/2. Moreover, we found that overexpression of CacyBP/SIP resulted in higher phosphatase activity in the nuclear fraction obtained from NB2a cells when compared with HCT116 cells. Using 2-D electrophoresis we showed that the pattern of spots representing CacyBP/SIP differed in these 2 cell lines and was probably due to a different phosphorylation state of this protein. We also established that after overexpression of CacyBP/SIP in NB2a cells, the amount of nuclear β-catenin was low, while it remained high in HCT116 cells. Since NB2a cells have differentiation potential and HCT116 cells do not, our data suggest that different activity of CacyBP/SIP in these 2 cell lines might affect the ERK1/2 pathway in the differentiation or proliferation processes.     

MTHFR 677T is a strong determinant of the degree of hearing loss among Polish males with postlingual sensorineural hearing impairment

Hearing impairment (HI) is the most common sensory handicap. Congenital HI often has a genetic basis, whereas the etiology of nonsyndromic postlingual HI (npHI) usually remains unidentified. Our purpose was to test whether the MTHFR C677T (rs1801133) polymorphism affecting folate metabolism is associated with the occurrence or severity of npHI. We studied rs1801133 genotypes in 647 npHI patients (age <40, sudden sensorineural loss excluded, HI characterized as mean of better ear hearing thresholds for 0.5-8 kHz) and 3273 adult controls from the background population. Genotype distribution among patients and controls was similar, but among male cases (n = 302) we found a dose-dependent correlation of MTHFR 677T with the degree of HI (mean thresholds in dB: 38.8, 44.9, and 53.3, for CC, CT, and TT genotypes, respectively; p = 0.0013, p(cor.) = 0.017). Among male patients rs1801133 TT significantly increased the risk of severe/profound HI (odds ratio = 4.88, p = 0.001). Among controls the known effect of MTHFR 677T on plasma total homocysteine was more pronounced in men than in women (p<0.00004 for genotype-sex interaction) suggesting that in Poland folate deficiency is more prevalent in males. In conclusion, we report a novel strong effect of MTHFR 677T among males with npHI. The functional significance of rs1801133 suggests that these patients may benefit from folate supplementation-an intervention which is simple, cheap, and devoid of side effects.     

Functioning of the TA cassette of streptococcal plasmid pSM19035 in various Gram-positive bacteria

Toxin-antitoxin (TA) systems are common in microorganisms and are frequently found in the chromosomes and low-copy number plasmids of bacterial pathogens. One such system is carried by the low copy number plasmid pSM19035 of the pathogenic bacterium Streptococcus pyogenes. This plasmid encodes an omega-epsilon-zeta cassette that ensures its stable maintenance by post-segregational killing of plasmid-free cells. In this study, the activity of the ω-ε-ζ cassette was examined in various Gram-positive bacteria with a low G/C content in their DNA. The broad host range of pSM19035 was confirmed and the copy number of a truncated derivative in transformed strains was determined by real-time qPCR.     

The Effect of Contrast Medium SonoVue<Superscript>®</Superscript> on the Electric Charge Density of Blood Cells

The effect of contrast medium SonoVue® on the electric charge density of blood cells (erythrocytes and thrombocytes) was measured using a microelectrophoretic method. We examined the effect of adsorbed H⁺ and OH^? ions on the surface charge of erythrocytes or thrombocytes. Surface charge density values were determined from electrophoretic mobility measurements of blood cells performed at various pH levels. The interaction between solution ions and the erythrocyte’s or thrombocyte’s surface was described by a four-component equilibrium model. The agreement between the experimental and theoretical charge variation curves of the erythrocytes and thrombocytes was good at pH 2–9. The deviation observed at a higher pH may be caused by disregarding interactions between the functional groups of blood cells.     

Changes of activity and kinetics of certain liver and heart enzymes of hypothyroid and T(3)-treated rats

Thyroid diseases are one of the most common metabolic disorders in the human population. In this work, we present data concerning changes in the activity and kinetic parameters of several enzymes associated with both anabolic (glucose-6-phosphate dehydrogenase-G6PDH, EC 1.1.1.49; 6-phosphogluconate dehydrogenase-6PGDH, EC 1.1.1.44; malic enzyme-ME, EC 1.1.1.40; and isocitrate dehydrogenase-IDH, EC 1.1.1.42) and catabolic (NAD-dependent malate dehydrogenase-NAD-MDH, EC 1.1.1.37; and lactate dehydrogenase-LDH, EC 1.1.1.27) processes under conditions of hypothyroidism and T(3) treatment. Hypothyroidism was induced in rats by the surgical removal of the thyroid gland. T(3)-treated rats were injected by T(3) (0.5 mg T(3)/kg body weight daily during 10 days). We have found that T(3) treatment caused an increase of NAD-MDH activity as well as heart hypertrophy whereas the activity of LDH increased in the direction of pyruvate reduction. Moreover, we observed increased activity of both enzymes in the liver. These results confirm earlier observation concerning the relevance of oxidative metabolism in the heart under T(3) treatment. Hypothyroidism resulted in changes in the LDH activity in the heart whereas NAD-MDH activity did not change. Moreover, our data show that T(3) treatment caused an increase of G6PDH, 6PGDH, and ME activities in the liver. We also observed a decrease of IDH activity in both organs, whereas hypothyroidism caused the opposite effect. This data indicate that either deficiency or excess of thyroid hormones can prove to be particularly dangerous for the physiology of the heart muscle by disturbing bioenergetic and anabolic processes.     

The combined effect of temperature and salinity changes on osmoregulation and haemocyanin concentration in Saduria entomon (Linnaeus, 1758)

Global warming is having an impact on the temperature and salinity of Baltic Sea waters. Therefore, it is important to determine the conditions in which animals can exist and how these changes may influence their functioning. Hence, the purpose of this research was to determine the broad tolerance limits of temperature and salinity of the glacial relict Saduria entomon by studying its behaviour, osmoregulatory ability and haemocyanin concentration. This effect of temperature was confirmed in the laboratory for individuals acclimated to different salinity and temperature regimes. Changes in the physiological parameters of S. entomon at various temperatures (5.5–21.5°C) and salinity levels (1–15 PSU) were recorded. There were statistically significant differences in haemolymph osmotic pressure under the influence of salinity and temperature. The mean haemolymph osmotic pressures were the lowest at 1 PSU at all the temperatures examined and the highest at 15 PSU and high temperatures 16.5 and 21.5°C. The haemocyanin concentration decreased significantly with increasing temperature at 1 PSU. There was a significant difference in haemocyanin concentration due to salinity at temperatures of 5.5 and 10.0°C (the haemocyanin concentration decreased with increasing salinity). The results showed that, although S. entomon is classified as a cold-water animal, it can survive at high temperatures above 16.5°C at least for a short time, as it is capable of osmoregulation. The tolerance to temperature changes was better than expected.