Comparative proteome analysis identified CD44 as a possible serum marker for docetaxel resistance in castration‐resistant prostate cancer

Baseline or acquired resistance to docetaxel (DOC) represents a significant risk for patients with metastatic prostate cancer (PC). In the last years, novel therapy regimens have been approved providing reasonable alternatives for DOC‐resistant patients making prediction of DOC resistance of great clinical importance. We aimed to identify serum biomarkers, which are able to select patients who will not benefit from DOC treatment. DOC‐resistant PC3‐DR and DU145‐DR sublines and their sensitive parental cell lines (DU145, PC3) were comparatively analyzed using liquid chromatography‐coupled tandem mass spectrometry (LC‐MS/MS). Results were filtered using bioinformatics approaches to identify promising serum biomarkers. Serum levels of five proteins were determined in serum samples of 66 DOC‐treated metastatic castration‐resistant PC patients (mCRPC) using ELISA. Results were correlated with clinicopathological and survival data. CD44 was subjected to further functional cell culture analyses. We found at least 177 two‐fold significantly overexpressed proteins in DOC‐resistant cell lines. Our bioinformatics method suggested 11/177 proteins to be secreted into the serum. We determined serum levels of five (CD44, MET, GSN, IL13RA2 and LNPEP) proteins in serum samples of DOC‐treated patients and found high CD44 serum levels to be independently associated with poor overall survival (p = 0.001). In accordance, silencing of CD44 in DU145‐DR cells resulted in re‐sensitization to DOC. In conclusion, high serum CD44 levels may help identify DOC‐resistant patients and may thereby help optimize clinical decision‐making regarding type and timing of therapy for mCRPC patients. In addition, our in vitro results imply the possible functional involvement of CD44 in DOC resistance.     

Multimeric α-Lactalbumin from Human Milk Induces Apoptosis through a Direct Effect on Cell Nuclei

A fraction from human milk containing spf-multimer α-lactalbumin (MAL) induces apoptosis in tumor cells and immature cells but spares mature cells. The mechanism of apoptosis induction and the molecular basis for the difference in susceptibility between tumor cells and healthy cells have not been defined. In this study we examined the interaction of MAL with different cellular compartments, using confocal microscopy and subcellular fractionation. MAL was shown to accumulate in the nuclei of sensitive cells rather than in the cytosol, the vesicular fraction, or the ER–Golgi complex. Nuclear uptake occurred rapidly in cells that were susceptible to the apoptosis-inducing effect, but not in nuclei of resistant cells. Nuclear uptake was through the nuclear pore complex and was critical for the induction of DNA fragmentation, since inhibition of nuclear uptake with WGA rescued digitonin-permeabilized cells from induction of DNA fragmentation. Ca²⁺was required for MAL-induced DNA fragmentation but nuclear uptake of MAL was independent of Ca²⁺. This way MAL differs from most previously described agents in that it crosses the plasma membrane and cytosol, and enters cell nuclei where it induces DNA fragmentation through a direct effect at the nuclear level.     

Diversification of ectoparasite assemblages and climate: an example with fleas parasitic on small mammals

Aim We studied the relationships between the numbers of species and numbers of higher taxa (genera, tribes, subfamilies and families) in flea assemblages of small mammalian hosts with the aims of: (a) comparing these relationships across different regions, and (b) testing the hypothesis that flea assemblages in warmer regions diversify mainly via intrahost speciation, whereas those in colder regions diversify mainly via host switching. Location The study used previously published data on flea assemblages on small mammalian hosts from 25 different regions of the Holarctic. Methods The number of flea genera, tribes, subfamilies or families in an assemblage (host species) was plotted against the number of flea species in this assemblage for each region separately, and a power function was fitted to the resulting relationships. Then, the values of the exponent of the power function for a region were regressed against the mean annual temperature in this region, across all regions. Results The relationships between the number of flea species and the numbers of flea genera, tribes, subfamilies or families on a host species in each region were found to be well described by simple power functions. The exponent of the power function of the relationship between the number of flea species and the number of flea genera per host tended to decrease with increasing local mean annual temperature. When two apparent outliers from the trend (corresponding to regions where sampling was not performed as in other regions) were omitted from the analysis, the negative relationship between temperature and the exponent of the power function between the number of flea species and number of flea genera per host became highly significant. No relationship was found between the values of the exponents of the power functions between the number of flea species and the number of flea tribes, subfamilies or families per host, and the mean local annual temperature. Main conclusions The results suggest that the diversification of flea assemblages is associated with climatic variables. In warm regions, the greater number of congeneric species per flea assemblage, reflected by the lower exponent of the power function, may well be the outcome of intrahost speciation. This indicates that, as regional temperature increases, intrahost speciation becomes a relatively more important mode of diversification than acquisition of fleas via host switching.     

Moulting frequency and behavioural responses to salinity and diesel oil in Austromegabalanus psittacus (Molina) (Cirripedia: Balanidae)

Moulting frequency and behavioural responses to salinity and diesel oil concentration were studied in specimens of the giant barnacle Austromegabalanus psittacus (Molina). Moulting frequency and frequency and type of cirral beat, in addition to opercular valve closure time, were measured under controlled conditions. A binary factorial experimental design was carried out at salinities of 20 and 30‰ and diesel oil concentrations of 0.1 and 0.5% v/v. Moulting frequency was greater at 30‰ than at 20‰ salinity and at 0.1% oil concentration than at both 0.5% oil concentration and in controls; it is unlikely that this signified variations in growth. Diesel oil provoked lethal effects at 0.5%, with an average lethal time of 8 days; at 0.1%, only sublethal effects were generated. Cirral beat frequency was greater at 0.1 and 0.5% diesel oil concentration than in controls. This was probably associated with an increase in metabolism, since the most frequent cirral beats are associated with respiration and the active capture of plankton. The effects of the contaminant varied with time, as observed at 15 and 60 min. The opercular valve closure time was longer in controls and decreased as diesel oil concentration increased. Results suggest that this species is highly resistant to pollution, although contaminants could provoke changes in the feeding and growth of specimens. Local variations in salinity have only minor effects on barnacle behaviour.     

Force to Divide: Structural and Mechanical Requirements for Actomyosin Ring Contraction

One of the unresolved questions in the field of cell division is how the actomyosin cytoskeleton remains structurally organized while generating the contractile force to divide one cell into two. In analogy to the actomyosin-based force production mechanism in striated muscle, it was originally proposed that contractile stress in the actomyosin ring is generated via a sliding filament mechanism within an organized sarcomere-like array. However, over the last 30 years, ultrastructural and functional studies have noted important distinctions between cytokinetic structures in dividing cells and muscle sarcomeres. Myosin-II motor activity is not always required, and there is evidence that actin depolymerization contributes to contraction. In this Review, the architecture and contractile dynamics of the actomyosin ring at the cell division plane will be discussed. We will report the interdisciplinary advances in the field as well as their integration into a mechanistic understanding of contraction in cell division and in other biological processes that rely on an actomyosin-based force-generating system.     

Inhibitory properties of cystatin F and its localization in U937 promonocyte cells

Cystatin F is a recently discovered type II cystatin expressed almost exclusively in immune cells. It is present intracellularly in lysosome-like vesicles, which suggests a potential role in regulating papain-like cathepsins involved in antigen presentation. Therefore, interactions of cystatin F with several of its potential targets, cathepsins F, K, V, S, H, X and C, were studied in vitro. Cystatin F tightly inhibited cathepsins F, K and V with Ki values ranging from 0.17 nM to 0.35 nM, whereas cathepsins S and H were inhibited with 100-fold lower affinities (Ki approximately 30 nM). The exopeptidases, cathepsins C and X were not inhibited by cystatin F. In order to investigate the biological significance of the inhibition data, the intracellular localization of cystatin F and its potential targets, cathepsins B, H, L, S, C and K, were studied by confocal microscopy in U937 promonocyte cells. Although vesicular staining was observed for all the enzymes, only cathepsins H and X were found to be colocalized with the inhibitor. This suggests that cystatin F in U937 cells may function as a regulatory inhibitor of proteolytic activity of cathepsin H or, more likely, as a protection against cathepsins misdirected to specific cystatin F containing endosomal/lysosomal vesicles. The finding that cystatin F was not colocalized with cystatin C suggests distinct functions for these two cysteine protease inhibitors in U937 cells.     

LPS-induced bronchial hyperreactivity: interference by mIL-10 differs according to site of delivery

When administered to mice systemically or via the airways, LPS induces bronchoconstriction (BC) and/or bronchopulmonary hyperreactivity (BHR), associated with inflammation. Accordingly, a relationship between inflammation and allergic and nonallergic BHR can be hypothesized. We therefore studied the interference of the anti-inflammatory cytokine murine IL-10 (mIL-10) with LPS-induced lung inflammation, BC, and BHR. mIL-10 was administered directly into the airways by intranasal instillation or generated in vivo after muscle electrotransfer of mIL-10-encoding plasmid. Electrotransfer led to high mIL-10 circulating levels for a longer time than after the injection of recombinant mIL-10 (rmIL-10). rmIL-10 administered intranasally reduced lung inflammation and BHR after LPS administration into airways. It also reduced the ex vivo production of TNF-alpha by LPS-stimulated lung tissue explants. Two days after electrotransfer, mIL-10 blood levels were elevated, but lung inflammation, BC, and BHR persisted unaffected. Blood mIL-10 reaches the airways poorly, which probably accounts for the ineffectiveness of mIL-10-encoding plasmid electrotransfer. When LPS was aerosolized 15 days after electrotransfer, lung inflammation persisted but BHR was significantly reduced, an effect that may be related to the longer exposure of the relevant cells to mIL-10. The dissociation between inflammation and BHR indicates that both are not directly correlated. In conclusion, this study shows that mIL-10 is efficient against BHR when present in the airway compartment. Despite this, the muscle electrotransfer with mIL-10-encoding plasmid showed a protective effect against BHR after a delay of 2 wk that should be further investigated.     

Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal-regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, a cycle where multisite phosphorylations are performed by different kinases, but dephosphorylation reactions are catalyzed by the same phosphatase, can also exhibit bistability and hysteresis. Hence, bistability induced by multisite covalent modification may be a widespread mechanism of the control of protein activity.     

Identification of a sporozoite-specific member of the Toxoplasma SAG superfamily via genetic complementation

Toxoplasma gondii sporozoites possess an array of stage-specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site-specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.