cAMP elevating agents suppress secretory phospholipase A(2)-induced matrix metalloproteinase-2 activation

Phospholipase A2 proteins are major regulators of the arachidonic acid cascade and are involved in various cellular responses. Previously, we reported that group IB PLA2 proteins stimulate MMP-2 activation and subsequent cell migration. Here, we describe a novel mechanism whereby sPLA2-induced proMMP-2 activation is regulated by intracellular cAMP in HT1080 cells, although sPLA2 itself had no effect on the regulation of cAMP levels. Exogenous dibutyryl cAMP (a cAMP analogue) strongly inhibited proMMP-2 activation, and cAMP elevating agents, namely, cholera toxin (a Gs activator) and forskolin (an adenylyl cyclase activator), abrogated basal and sPLA2-induced proMMP-2 activation. We also found that the down-regulation of TIMP-2 expression and extracellular signal-regulated kinase (ERK)1/2 activation by sPLA2 were blocked by increasing the intracellular cAMP level. Taken together, our data indicate that sPLA2-induced proMMP-2 activation is influenced by intracellular cAMP levels via the modulations of TIMP-2 expression and ERK1/2 activation.     

Classification methods for the development of genomic signatures from high-dimensional data

Personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies. We present Classification by Ensembles from Random Partitions (CERP) for class prediction and apply CERP to genomic data on leukemia patients and to genomic data with several clinical variables on breast cancer patients. CERP performs consistently well compared to the other classification algorithms. The predictive accuracy can be improved by adding some relevant clinical/histopathological measurements to the genomic data.     

SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination

The p53 tumour suppressor has a key role in the control of cell growth and differentiation, and in the maintenance of genome integrity. p53 is kept labile under normal conditions, but in response to stresses, such as DNA damage, it accumulates in the nucleus for induction of cell-cycle arrest, DNA repair or apoptosis. Mdm2 is an ubiquitin ligase that promotes p53 ubiquitination and degradation. Mdm2 is also self-ubiquitinated and degraded. Here, we identified a novel cascade for the increase in p53 level in response to DNA damage. A new SUMO-specific protease, SUSP4, removed SUMO-1 from Mdm2 and this desumoylation led to promotion of Mdm2 self-ubiquitination, resulting in p53 stabilization. Moreover, SUSP4 competed with p53 for binding to Mdm2, also resulting in p53 stabilization. Overexpression of SUSP4 inhibited cell growth, whereas knockdown of susp4 by RNA interference (RNAi) promoted of cell growth. UV damage induced SUSP4 expression, leading to an increase in p53 levels in parallel with a decrease in Mdm2 levels. These findings establish a new mechanism for the elevation of cellular p53 levels in response to UV damage.     

Failure of catecholamines to shift T-cell cytokine responses toward a Th2 profile in patients with rheumatoid arthritis

To further understand the role of neuro-immunological interactions in the pathogenesis of rheumatoid arthritis (RA), we studied the influence of sympathetic neurotransmitters on cytokine production of T cells in patients with RA. T cells were isolated from peripheral blood of RA patients or healthy donors (HDs), and stimulated via CD3 and CD28. Co-incubation was carried out with epinephrine or norepinephrine in concentrations ranging from 10(-5) M to 10(-11) M. Interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-4, and IL-10 were determined in the culture supernatant with enzyme-linked immunosorbent assay. In addition, IFN-gamma and IL-10 were evaluated with intracellular cytokine staining. Furthermore, basal and agonist-induced cAMP levels and catecholamine-induced apoptosis of T cells were measured. Catecholamines inhibited the synthesis of IFN-gamma, TNF-alpha, and IL-10 at a concentration of 10(-5) M. In addition, IFN-gamma release was suppressed by 10(-7) M epinephrine. Lower catecholamine concentrations exerted no significant effect. A reduced IL-4 production upon co-incubation with 10(-5) M epinephrine was observed in RA patients only. The inhibitory effect of catecholamines on IFN-gamma production was lower in RA patients as compared with HDs. In RA patients, a catecholamine-induced shift toward a Th2 (type 2) polarised cytokine profile was abrogated. Evaluation of intracellular cytokines revealed that CD8-positive T cells were accountable for the impaired catecholaminergic control of IFN-gamma production. The highly significant negative correlation between age and catecholamine effects in HDs was not found in RA patients. Basal and stimulated cAMP levels in T-cell subsets and catecholamine-induced apoptosis did not differ between RA patients and HDs. RA patients demonstrate an impaired inhibitory effect of catecholamines on IFN-gamma production together with a failure to induce a shift of T-cell cytokine responses toward a Th2-like profile. Such an unfavorable situation is a perpetuating factor for inflammation.     

Feasibility of supercritical CO₂ treatment for controlling biofouling in the reverse osmosis process

Physical cleaning and/or chemical cleaning have been generally used to control biofouling in the reverse osmosis (RO) process. However, conventional membrane cleaning methods to control biofouling are limited due to the generation of by-products and the potential for damage to the RO membranes. In this study, supercritical carbon dioxide (SC CO(2)) treatment, an environmentally friendly technique, was introduced to control biofouling in the RO process. SC CO(2) (100 bar at 35°C) treatment was performed after biofouling was induced on a commercial RO membrane using Pseudomonas aeruginosa PA01 GFP as a model bacterial strain. P. aeruginosa PA01 GFP biofilm cells were reduced on the RO membrane by >8 log within 30 min, and the permeate flux was sufficiently recovered in a laboratory-scale RO membrane system without any significant damage to the RO membrane. These results suggest that SC CO(2) treatment is a promising alternative membrane cleaning technique for biofouling in the RO process.     

TNF-α mediates the stimulation of sclerostin expression in an estrogen-deficient condition

Obesity consists in fat accumulation leading to increase in adipose cells number and size. Adipocyte membrane biophysical properties are critical to maintain cellular viability in metabolically healthy obesity. This study investigated the effect of the genetic background and dietary protein restriction on fat tissue lipid composition, adipocyte membrane fluidity and water permeability using the pig as experimental model. Twenty-four male pigs from distinct genotypes, lean and obese, were fed on normal and reduced protein diets within a 2 × 2 factorial arrangement (two genotypes and two diets). Backfat thickness was twofold higher in obese than in lean pigs but unrelated to dietary protein level. In contrast, total fatty acids in the subcutaneous adipose tissue were dependent on both breed and diet, with increased lipid content promoted by the fatty genotype and by the restriction of dietary protein. Adipose membranes isolated from obese pig's subcutaneous fat tissue showed higher permeability to water, in line with an increased fluidity. Moreover, the reduced content of dietary protein influenced positively the fluidity of adipose membranes. Neither genotype nor diet affected total cholesterol concentration in the adipose membranes. Membrane-saturated fatty acids' content was influenced by genotype, while membrane-polyunsaturated fatty acids, particularly from the n-6 family, was influenced by diet. The ratio of oleic (18:1c9)/linoleic (18:2n-6) acids was positively correlated with membrane fluidity. All together, these findings reinforce the genetic background as a determinant player on adipose membrane biophysical properties and point to the dietary protein level as an important factor for subcutaneous lipid deposition as well as for regulation of membrane function, factors that may have impact on human obesity and metabolic syndrome.     

Cadmium inhibits the protein degradation of Sml1 by inhibiting the phosphorylation of Sml1 in Saccharomyces cerevisiae

Cadmium is a toxic metal, and the mechanism of cadmium toxicity in living organisms has been well studied. Here, we used Saccharomyces cerevisiae as a model system to examine the detailed molecular mechanism of cell growth defects caused by cadmium. Using a plate assay of a yeast deletion mutant collection, we found that deletion of SML1, which encodes an inhibitor of Rnr1, resulted in cadmium resistance. Sml1 protein levels increased when cells were treated with cadmium, even though the mRNA levels of SML1 remained unchanged. Using northern and western blot analyses, we found that cadmium inhibited Sml1 degradation by inhibiting Sml1 phosphorylation. Sml1 protein levels increased when cells were treated with cadmium due to disruption of the dependent protein degradation pathway. Furthermore, cadmium promoted cell cycle progression into the G2 phase. The same result was obtained using cells in which SML1 was overexpressed. Deletion of SML1 delayed cell cycle progression. These results are consistent with Sml1 accumulation and with growth defects caused by cadmium stress. Interestingly, although cadmium treatment led to increase Sml1 levels, intracellular dNTP levels also increased because of Rnr3 upregulation due to cadmium stress. Taken together, these results suggest that cadmium specifically affects the phosphorylation of Sml1 and that Sml1 accumulates in cells.     

Regulatory role of mouse epidermal growth factor-like protein 8 in thymic epithelial cells

S100A7 (psoriasin) is a calcium-binding protein that is upregulated in many types of cancer and often associated with poor prognosis. Its role in carcinogenesis has been associated with the stimulation of VEGF and EGF activity. The recent research showed that psoriasin directly interacts with αvβ6 integrin, a protein related to the invasive phenotype of cancer. Moreover, this interaction promotes the αvβ6-dependent invasive activity. The important function of S100A7 in carcinoma development determines a great need for valuable tools enabling its detection, quantification and also activity inhibition. Here, we show the selection of S100A7 specific antibody fragments from the human scFv phage library ETH-2 Gold. We have selected antibody fragments specific for psoriasin, purified them and analyzed by BIAcore affinity measurements. The best clone was subjected to affinity maturation procedure yielding molecule with a subnanomolar affinity towards human S100A7 protein. Selected clone was expressed in a bivalent format and applied for immunostaining analysis, which confirmed the ability of the antigen recognition in physiological conditions. We therefore propose that obtained antibody, that is the first phage display-derived human antibody against psoriasin, can serve as a useful psoriasin binding platform in research, diagnostics and therapy of cancer.     

Gliosis in the Mice Hippocampus Without Neuronal Death After Systemic Administration of High Dosage of Tetanus Toxin

Tetanus toxin (TeT), an exotoxin, has been studied to cause tetanus in mammalian brains, and it can block the release of some neurotransmitters and affect seizure propagation. In the present study, we investigated neuronal damage/death and glial changes in the mouse hippocampus after systemic administration (intraperitoneal injection) of TeT 10 and 100 ng/kg. In both the 10 and 100 ng/kg TeT-treated groups, no neuronal death occurred in any subregions of the mouse hippocampus until 24 h post-treatment; however, there were changes in glia in the hippocampus depending on time course and dosage. The morphology of GFAP-immunoreactive astrocytes and Iba-1-immunoreactive microglia was apparently changed in the 100 ng/kg TeT treated-group compared to the 10 ng/kg TeT treated-group. In the 100 ng/kg TeT treated-group, they were increased in size and their immunoreactivity was distinctively increased from 12 h post-treatment. We also found that their protein levels were increased in the hippocampus at 12 h post-treatment of 100 ng/kg TeT. In conclusion, these results indicate that the systemic administration of 100 ng/kg TeT induced a distinctive microglia changes in the mouse hippocampus without any neuronal death/damage.