Receptor activator of nuclear factor-kappaB ligand (RANKL) as a novel prognostic marker in prostate carcinoma

Combined immunodetection of parathyroid hormone-related protein (PTHrP) and receptor activator of NF-kappaB ligand (RANKL) has shown to successfully distinguish poorly- and well-differentiated prostate carcinoma (PCa). In the present study, we aimed to assess whether immunohistochemical evaluation of these factors, and also osteoprotegerin (OPG) and Ki67, in radical prostatectomy specimens can predict biochemical recurrence. Fifty nine PCa cases undergoing radical prostatectomy between 1995 and 1998, without history of neoadjuvant hormonal therapy, were studied. Preoperative serum prostate-specific antigen (PSA), Gleason-sum score, pathologic stage, perineural invasion, seminal vesicle involvement, and positive surgical margins were assessed in these patients. Biochemical recurrence, defined by PSA > 0.4 ng/mL at 90 days or later after prostatectomy, occurred in 32/59 patients. In these patients, positivity for OPG and RANKL in the tumoral epithelium was higher than in those patients with no biochemical recurrence. Using univariate analysis, Gleason-sum score, surgical margins, and seminal vesicle involvement, as well as OPG and RANKL immunostaining (using a score value corresponding to moderate staining as cut-off) were significant predictors of biochemical recurrence (p<0.05). Using the multivariate Cox model, among the evaluated factors only RANKL expression (hazard ratio 11.6; p <0.001) was an independent prognostic indicator. Our findings suggest that immunohistochemical evaluation of RANKL in the primary tumor is a potential risk factor in PCa patients.     

High-Melting Lipid Mixtures and the Origin of Detergent-Resistant Membranes Studied with Temperature-Solubilization Diagrams

The origin of resistance to detergent solubilization in certain membranes, or membrane components, is not clearly understood. We have studied the solubilization by Triton X-100 of binary mixtures composed of egg sphingomyelin (SM) and either ceramide, diacylglycerol, or cholesterol. Solubilization has been assayed in the 4–50°C range, and the results are summarized in a novel, to our knowledge, form of plots, that we have called temperature-solubilization diagrams. Despite using a large detergent excess (lipid/detergent 1:20 mol ratio) and extended solubilization times (24–48 h) certain mixtures were not amenable to Triton X-100 solubilization at one or more temperatures. DSC of all the lipid mixtures, and of all the lipid + detergent mixtures revealed that detergent resistance was associated with the presence of gel domains at the assay temperature. Once the system melted down, solubilization could occur. In general adding high-melting lipids limited the solubilization, whereas the addition of low-melting lipids promoted it. Lipidomic analysis of Madin-Darby canine kidney cell membranes and of the corresponding detergent-resistant fraction indicated a large enrichment of the nonsolubilized components in saturated diacylglycerol and ceramide. SM-cholesterol mixtures were special in that detergent solubilization was accompanied, for certain temperatures and compositions, by an independent phenomenon of reassembly of the partially solubilized lipid bilayers. The temperature at which lysis and reassembly prevailed was ∼25°C, thus for some SM-cholesterol mixtures solubilization occurred both above and below 25°C, but not at that temperature. These observations can be at the origin of the detergent resistance effects observed with cell membranes, and they also mean that cholesterol-containing detergent-resistant membrane remnants cannot correspond to structures existing in the native membrane before detergent addition.     

Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells

Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.     

MONITORING THE TREND OF HARBOR SEALS IN PRINCE WILLIAM SOUND,ALASKA, AFTER THE EXXON VALDEZ OIL SPILL

We used aerial counts to monitor the trend in numbers of harbor seals, Phoca vitulina richardsi, in Prince William Sound, Alaska, following the 1989 Exxon Valdez oil spill. Repetitive counts were made at 25 haul-out sites during the annual molt period each year from 1990 through 1997. A generalized linear model indicated that time of day, date, and time relative to low tide significantly affected seal counts. When Poisson regression was used to adjust counts to a standardized set of survey conditions, results showed a highly significant decline of 4.6% per year. Unadjusted counts indicated a slight, but not statistically significant, decline in the number of seals. The number of harbor seals on the trend-count route in eastern and central PWS has been declining since at least 1984, with an overall population reduction of 63% through 1997. Programs to monitor long-term changes in animal population sizes should account for factors that can cause short-term variations in indices of abundance. The inclusion of such factors as covariates in models can improve the accuracy of monitoring programs.     

Chimaereicella alkaliphila gen. nov., sp. nov., a Gram-negative alkaliphilic bacterium isolated from a nonsaline alkaline groundwater

A Gram-negative bacterium designated AC-74(T) was isolated from a highly alkaline groundwater environment (pH 11.4). This organism formed rod-shaped cells, is strictly aerobic, catalase and oxidase positive, tolerates up to 3.0% NaCl, has an optimum growth temperature of 30 degrees C, but no growth occurs at 10 or 40 degrees C, and an optimum pH value of 8.0, but no growth occurs at pH 7.0 or 11.3. The predominant fatty acids are iso-15:0, iso-17:1 omega9c and 16:1 omega7c and or iso-15:2OH. The G+C content of DNA was 43.5mol%. The phylogenetic analyses of the sequences of the 16s RNA genes indicated that strain AC-74(T) belongs to the family "Flexibacteriaceae" and is phylogenetically equidistant ( approximately 94.5%) from the majority of the species of the genus Algoriphagus and from the genus Hongiella. Based on the phylogenetic analyses and distinct phenotypic characteristics, we are of the opinion that strain AC-74(T), represents a new species of the novel genus for which we propose the name Chimaereicella alkaliphila gen. nov., sp. nov.     

Piscirickettsia salmonis induces apoptosis in macrophages and monocyte‐like cells from rainbow trout

Piscirickettsia salmonis is the etiologic agent of the salmonid rickettsial septicemia (SRS) which causes significant losses in salmon production in Chile and other and in other regions in the southern hemisphere. As the killing of phagocytes is an important pathogenic mechanism for other bacteria to establish infections in vertebrates, we investigated whether P. salmonis kills trout macrophages by apoptosis. Apoptosis in infected macrophages was demonstrated by techniques based on morphological changes and host cell DNA fragmentation. Transmission electron microcopy showed classic apoptotic characteristics and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling showed fragmented DNA. Programmed cell death type I was further confirmed by increased binding of annexin V to externalized phosphatidylserine in infected macrophages. Moreover, significant increases of caspase 3 activation were detected in infected cells and treatment with caspase inhibitor caused a decrease in levels of apoptosis. This is the first evidence that P. salmonis induces cell death in trout macrophages. This could lead to bacterial survival and evasion of the host immune response and play an important role in the establishment of infection in the host. J. Cell. Biochem. 110: 468–476, 2010. © 2010 Wiley‐Liss, Inc.     

Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in Fermented Milk Enriched with Green Banana Pulp Under Acid Stress and in the Presence of Bile Salts

This study evaluated the in vitro effect of 3.0, 6.0, and 9.0% of green banana pulp (GBP) incorporation in fermented milk on the survival of Lactobacillus paracasei subsp. paracasei LBC 81 subjected to acid stress conditions and in the presence of bile salts. Tolerance to acid stress in pH 2.0 and in the presence of 0.30% of bile salts was evaluated right after the incorporation of the fermented milk in each of these conditions, and also during 3 and 4 h of exposure, respectively. The addition of GBP (3.0%) gives a protective effect on L. paracasei LBC 81 when exposed to stress conditions evaluated, while of 9.0% there is a marked decrease of L. paracasei LBC 81. In the absence of GBP, a decrease of L. paracasei LBC 81 is observed, but lower in the presence of GBP (9.0%).

     

Biological and genetic aspects of crosses between species of the genus Meccus (Hemiptera: Reduviidae Triatominae)

The degree of reproductive isolation between Meccus phyllosomus and the remaining five species of the genus Meccus, as well as between Meccus bassolsae and Meccus pallidipennis, Meccus longipennis and Meccus picturatus, was examined. Fertility and the segregation of morphological characteristics were examined in two generations of hybrids from crosses between these species. The percentage of couples with offspring (fertile) was high in the vast majority of sets of crosses, with the exception of that between ♀M. phyllosomus and ♂Meccus mazzottii. In sets of crosses involving M. bassolsae specimens, no first-generation (F1) individuals were morphologically similar to M. bassolsae, but instead shared the morphology of the other parental species. A similar phenomenon was observed in most sets of crosses involving M. phyllosomus. These results indicated that different degrees of reproductive isolation exist among the species of Meccus involved in this study. The biological evidence obtained in this study does not support the proposal that M. bassolsae is a full species. It could indicate that, on the contrary, it should be considered a subspecies of a single polytypic species. The biological evidence does support the proposal that M. phyllosomus is a full species.     

Depolarization-induced ERK phosphorylation depends on the cytosolic Ca2+ level rather than on the Ca2+ channel subtype of chromaffin cells

The contribution of Ca2+ entry through different voltage-activated Ca2+ channel (VACC) subtypes to the phosphorylation of extracellular signal regulated kinase (ERK) was examined in bovine adrenal-medullary chromaffin cells. High K+ depolarization (40 mM, 3 min) induced ERK phosphorylation, an effect that was inhibited by specific mitogen-activated protein kinase kinase inhibitors. By using selective inhibitors, we observed that depolarization-induced ERK phosphorylation completely depended on protein kinase C-alpha (PKC-alpha), but not on Ca2+/calmodulin-dependent protein kinase nor cyclic AMP-dependent protein kinase. Blockade of L-type Ca2+ channels by 3 microm furnidipine, or blockade of N channels by 1 micromomega-conotoxin GVIA reduced ERK phosphorylation by 70%, while the inhibition of P/Q channels by 1 micromomega-agatoxin IVA only caused a 40% reduction. The simultaneous blockade of L and N, or P/Q and N channels completely abolished this response, yet 23% ERK phosphorylation remained when L and P/Q channels were simultaneously blocked. Confocal imaging of cytosolic Ca2+ elevations elicited by 40 mm K+, showed that Ca2+ levels increased throughout the entire cytosol, both in the presence and the absence of Ca2+ channel blockers. Fifty-eight percent of the fluorescence rise depended on Ca2+ entering through N channels. Thus, ERK phosphorylation seems to depend on a critical level of Ca2+ in the cytosol rather than on activation of a given Ca2+ channel subtype.