Osmoregulation, immunolocalization of Na+/K+-ATPase, and ultrastructure of branchial epithelia in the developing brown shrimp, Crangon crangon (Decapoda, Caridea)

Aspects of osmoregulation including salinity tolerance, osmoregulatory capacity, location of transporting epithelia, and the expression of the enzyme Na+/K+-ATPase were investigated in the developing brown shrimp, Crangon crangon (L.), from the North Sea. Early developmental stages and large juveniles were exposed to a wide range of salinities for measurement of hemolymph osmolality and survival rates. In media ranging from 17.0 per thousand to 32.2 per thousand, salinity tolerance was generally high (survival rates: 70%-100%) in all developmental stages, but it decreased in media <10.2 per thousand. Zoeal stages and decapodids slightly hyperregulated at 17.0 per thousand and osmoconformed in media > or =25.5 per thousand. At 10.2 per thousand, these stages showed high mortality, and only juveniles survived at 5.3 per thousand. Juveniles hyperregulated at 10.2 per thousand and 17.0 per thousand, osmoconformed at 25.5 per thousand, and hyporegulated in media > or =32.2 per thousand. Large juveniles hyperregulated also at 5.3 per thousand. Expression of the Na+/K+-ATPase and ion-transporting cells was located through immunofluorescence microscopy and transmission electron microscopy. In zoeae I and VI, a strong immunoreactivity was observed in cells of the inner epithelia of the branchiostegites and in epithelial cells lining the pleurae. Their ultrastructure showed typical features of ion-transporting cells. In decapodids and juveniles, ionocytes and expression of Na+/K+-ATPase remained located in the branchiostegite epithelium, but they disappeared from the pleurae and appeared in the epipodites. In large juveniles, the cells of the gill shaft showed positive immunolabeling and ultrastructural features of ionocytes. In summary, the adult pattern of osmoregulation in C. crangon is accomplished after metamorphosis from a moderately hyperosmoconforming decapodid to an effectively hyper-/hyporegulating juvenile stage. Salinity tolerance and osmoregulatory capacity are closely correlated with the development of ion-transporting cells and the expression of Na+/K+-ATPase.     

Francisella tularensis proteome: low levels of ASB-14 facilitate the visualization of membrane proteins in total protein extracts

Proteomic analysis of bacterial pathogens isolated from in vivo sources, such as infected tissues, provides many challenges not the least of which is the limited quantity of sample available for analysis. It is, therefore, highly desirable to develop a one-step cellular lysis and protein solubilization method that minimizes protein losses and allows the maximum possible coverage of the proteome. Here, we have used standard sample buffer constituents including urea, thiourea and DTT, but varied the detergent composition of the buffers in order to achieve the best quality of gels and the greatest spot resolution. We found that the most efficient solubilizing solution in this case consisted of 7 M urea, 2 M thiourea, 1% DTT, 0.5% amidosulfobetaine-14 (ASB-14) and 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Inclusion of low levels of ASB-14 in solutions allowed visualization of a subset of 24 new protein spots in the Live Vaccine Strain (LVS) of Francisella tularensis and 21 spots in a virulent A-strain of the pathogen. Further investigation showed that 15 of the 24 enriched LVS spots were membrane or membrane-associated proteins suggesting that the optimized lysis and solubilization solution aids in the detection of more hydrophobic proteins. This methodology is now being applied to the analysis of Francisella obtained from in vivo sources.     

Dual expression system suitable for high-throughput fluorescence-based screening and production of soluble proteins

Many studies that aim to characterize the proteome structurally or functionally require the production of pure protein in a high-throughput format. We have developed a fast and flexible integrated system for cloning, protein expression in Escherichia coli, solubility screening and purification that can be completely automated in a 96-well microplate format. We used recombination cloning in custom-designed vectors including (i) a (His)(6) tag-encoding sequence, (ii) a variable solubilizing partner gene, (iii) the DNA sequence corresponding to the TEV protease cleavage site, (iv) the gene (or DNA fragment) of interest, (v) a suppressible amber stop codon, and (vi) an S.tag peptide-encoding sequence. First, conditions of bacterial culture in microplates (250 microL) were optimized to obtain expression and solubility patterns identical to those obtained in a 1-L flask (100-mL culture). Such conditions enabled the screening of various parameters in addition to the fusion partners (E. coli strains, temperature, inducer...). Second, expression of fusion proteins in amber suppressor strains allowed quantification of soluble and insoluble proteins by fluorescence through the detection of the S.tag. This technique is faster and more sensitive than other commonly used methods (dot blots, Western blots, SDS-PAGE). The presence of the amber suppressor tRNA was shown to affect neither the expression pattern nor the solubility of the target proteins. Third, production of the most interesting soluble fusion proteins, as detected by our screening method, could be performed in nonsuppressor strains. After cleavage with the TEV protease, the target proteins were obtained in a native form with a unique additional N-terminal glycine.     

Simplified ultraviolet liquid chromatographic method for determination of sertindole, dehydrosertindole and norsertindole, in human plasma

An ultra-violet high-performance liquid chromatographic method was developed for the determination of sertindole, an atypical antipsychotic drug and its main metabolites dehydrosertindole and norsertindole, in human plasma. With a small sample volume, after a single-step liquid-liquid extraction, the compounds were separated on a reversed-phase XTerra RP(18) column, eluted with 45% of acetonitrile and 55% of ammonium acetate buffer (0.05 M, adjusted pH 8) and detected at 256 nm within 11 min. This method shows a good linearity for plasma concentration between 5-100 ng/ml and 100-1000 ng/ml, a good precision (inter and intra day CV < 11%) and a good inter-assay accuracy (bias < 11%). The limit of quantification concentration was 5 ng/ml. The absolute recovery of sertindole was higher than 99%. This rapid and sensitive method could be used for therapeutic drug monitoring as well as for overdose management.     

Stromal cell-derived factor-1 (SDF-1) expression in embryonic mouse cerebral cortex starts in the intermediate zone close to the pallial-subpallial boundary and extends progressively towards the cortical hem

We describe the onset and the expansion of stromal cell-derived factor 1 (SDF-1) expression in the intermediate zone of embryonic mouse cerebral cortex between embryonic days (E)11.5 and 18.5, and on postnatal day 1. Using in situ hybridisation with a digoxigenin-labeled probe, SDF-1 mRNA was detectable by E 12.5 in a small area of the intermediate zone just dorsal to the pallial-subpallial boundary. During the following days, SDF-1 expression extended towards the dorso-lateral pallium, and then the hippocampus and cortical hem. The position of the SDF-1 positive cells within the intermediate zone was closely correlated with the stream of tangentially migrating cells carrying the polysialylated form of neural cell adhesion molecule (PSA-NCAM). However, whereas these cells form a ventro-dorsal stream passing from the subpallium into the pallium, SDF-1 was not detectable on the ventral side of the pallial-subpallial boundary at any of the developmental stages studied. By E 16.5, the intensity of SDF-1 hybridisation signal in the intermediate zone decreased, to become undetectable by E 18.5.     

Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions

ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.     

Generation of peptide-MHC class I complexes through UV-mediated ligand exchange

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.     

TNFAIP8: A new effector for Galpha(i) coupling to reduce cell death and induce cell transformation

Galpha(i)‐coupled receptors comprise a diverse family of receptors that induce transformation by largely unknown mechanisms. We previously found that the Galpha(i)‐coupled dopamine‐D2short (D2S) receptor transforms Balb‐D2S cells via Gαi3. To identify new Gαi effectors, a yeast two‐hybrid screen was done using constitutively active Gαi3‐Q204L as bait, and tumor necrosis factor‐alpha (TNFα)‐induced protein 8 (TNFAIP8, SCC‐S2/NDED/GG2‐1) was identified. In contrast, TNFAIP8‐related TIPE1 and TIPE2 showed a very weak interaction with Gαi3. In yeast mating, in vitro pull‐down, co‐immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays, TNFAIP8 preferentially interacted with activated Gαi proteins, consistent with direct Gαi‐TNFAIP8 coupling. Over‐expression or depletion of TNFAIP8 using antisense constructs in Balb‐D2S cells did not affect D2S‐induced signaling to Gαi‐dependent inhibition of cAMP. In contrast, antisense depletion of TNFAIP8 completely inhibited spontaneous and D2S‐induced foci formation, consistent with a role for TNFAIP8 in Gαi‐dependent transformation. To address possible mechanisms, the effect of D2S signaling via TNFAIP8 on TNFα action was examined. D2S receptor activation inhibited TNFα‐induced cell death in Balb‐D2S cells, but not in cells depleted of TNFAIP8. However, depletion of TNFAIP8 did not prevent D2S‐induced inhibition of TNFα‐mediated caspase activation, suggesting that D2S/TNFAIP8‐induced protection from TNFα‐induced cell death is caspase‐independent. The data suggest that Gαi‐TNFAIP8‐mediated rescue of pre‐oncogenic cells enhances progression to oncogenic transformation, providing a selective target to inhibit cellular transformation. J. Cell. Physiol. 225: 865–874, 2010. © 2010 Wiley‐Liss, Inc.     

KiSS-1: a likely candidate for the photoperiodic control of reproduction in seasonal breeders

In seasonal species, photoperiod exerts tight regulation of reproduction to ensure that birth occurs at the most favorable time of yr. A distinct photoneuroendocrine circuit composed of the retina, suprachiasmatic nucleus (SCN) of the hypothalamus, and pineal gland transduces daylength into a rhythmic secretion of melatonin. The duration of the night-time rise of this hormone conveys daylength information to the organism. Melatonin is known to mediate the control of seasonal reproduction, but how it modulates sexual activity is far from understood. Recent data indicate that the product of the KiSS-1 gene is a potent stimulator of the hypothalamic-pituitary-gonadal axis and may play, together with its receptor GPR54, a central role in the neuroendocrine regulation of gonadotropin secretion. This article briefly reviews these findings and presents arguments that KiSS-1 could take part in the seasonal control of reproduction.     

Apoptotic death concurrent with CD3 stimulation in primary human CD8+ T lymphocytes: a role for endogenous granzyme B

We exposed primary CD8(+) T cells to soluble CD3 mAb plus IL-2 and limited numbers of monocytes (3%). These cells were activated but concurrently subjected to ongoing apoptosis ( approximately 25% were apoptotic from day 2 of culture). However, their costimulated CD4(+) counterparts were much less prone to apoptosis. The apoptotic signaling pathway bypassed Fas and TNFRs, and required the activity of cathepsin C, a protease which performs the proteolytic maturation of granzyme (Gr) A and GrB proenzymes within the cytolytic granules. Silencing the GrB gene by RNA interference in activated CD8(+) T cells prevented the activation of procaspase-3 and Bid, and indicated that GrB was the upstream death mediator. A GrB-specific mAb immunoprecipitated a approximately 70-kDa molecular complex from cytolytic extracts of activated CD8(+) (but not resting) T cells, that was specifically recognized by a nucleocytoplasmic protease inhibitor 9 (PI-9) specific mAb. This complex was also detected after reciprocal immunoprecipitation of PI-9. It coexisted in the cytosol with the 32-kDa form of GrB. As neither were detected in the cytosol of CD4(+) bystander T cells (which poorly synthesized GrB), and as silencing the perforin (Pf) gene had no effect in our system, endogenous GrB was likely implicated. Immunoprecipitation experiments failed to reveal Pf in the cytosol of CD8(+) T cells, and only a tiny efflux of granular GrA was detected by ELISA. We propose that some GrB is released from cytolytic granules to the cytosol of CD8(+) T lymphocytes upon CD3/TCR stimulation and escapes PI-9, thereby mediating apoptotic cell death.