Identification of P18, a surface protein produced by the fish pathogen Flavobacterium psychrophilum

AIMS: This study was focused on the identification of associated outer membrane proteins which may play a role in the specific interactions between Flavobacterium psychrophilum (the aetiological agent of cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide) and the fish tissues. METHODS AND RESULTS: The surface protein interactions with the outer membrane being mainly ionic, different methods were used for the detachment of proteins from the cell surface of Fl. psychrophilum involving detergent-free buffers or solutions known to perturb the ionic interactions. Such treatments led to the isolation of a surface protein, named P18 in accordance with its relative molecular mass. The expression of P18 was not related to the growth conditions (liquid or solid medium, temperature and aeration) or the strains of Fl. psychrophilum tested here. CONCLUSIONS: Preliminary characterization indicated that P18 is a surface antigen which is not sugar-modified and might be a subunit of a surface layer (i.e. S-layer), one of the most common surface structures on bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Data reported here should be used as the basis for further works involving the purification and characterization of P18 to identify the specific roles of such a surface protein, especially the interaction between this protein and the host surface.     

Fatty acid protection from palmitic acid-induced apoptosis is lost following PI3-kinase inhibition

We have previously shown that saturated fatty acids induce DNA damage and cause apoptotic cell death in insulin-producing beta-cells. Here we examine further the effects of single or combined dietary fatty acids on RINm5F survival or cell death signalling. Palmitate and stearate, but not linoleate, oleate or palmitoylmethyl ester, induced growth inhibition and increased apoptosis in RINm5F cells following 24 h exposure. Co-incubation with inhibitors of ceramide synthesis, myriocin or fumonisin B(1), did not improve viability of palmitic acid treated RINm5F cells. The inhibitor of inducible nitric oxide synthase, 1400 W, similarly had no protective effect. However, linoleic acid protected against palmitic acid-induced apoptotic and necrotic cell death. The specific pharmacological inhibitors of phosphatidylinositol 3-kinase, LY294002 and wortmannin, abolished the protective effect of linoleic acid on apoptosis but not on necrosis. These data show that the growth inhibitory and apoptosis-inducing effect of the saturated fatty acid palmitate on RINm5F cells is prevented by co-incubation with the polyunsaturated fatty acid linoleate but not inhibitors of ceramide or nitric oxide generation. A key role for phosphatidylinositol 3-kinase in mediating the linoleic-acid reduction in apoptosis is suggested.     

Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice

BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.     

Aryl Acylamidase Activity on Acetylcholinesterase Is High During Early Chicken Brain Development

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to exhibit aryl acylamidase activities (here called AAA(AChe) and AAA(BChe), respectively), which have been suggested to be involved in developmental and pathological processes. We here have investigated the developmental profiles of both AAA(AChe) and AAA(BChe) activities along with their AChE and BChE activities from embryonic days E3 to hatching (E21) in Triton-extracted homogenates from chicken embryonic brains. AAA(AChe) follows continuously an increase that is typical for AChE expression itself, whereas AAA(BChe) was relatively high before E10 to then become negligible toward hatching. Sucrose gradient centrifugation of both homogenized and immunopurified samples from E6-E18 brains showed that all globular forms (G1, G2, G4) of AChE present AAA(AChe) activity. Interestingly, the ratio of AAA(AChe) to AChE is highest at E6, and here again higher on G1/G2- over the G4-form. Noticeably, the sensitivity of AAA(AChe) toward the specific AChE inhibitor BW284c51 at all stages is higher than that of AChE itself. These data of high ratios of AAA associated at young stages with cholinesterases strongly indicate a role of AAA in early brain development.     

Comparison of bulk enucleation methods for porcine oocytes

Cloning of mammalian oocytes requires that the recipient oocyte is enucleated to remove all genetic material associated with the chromosomes. The procedure currently used in most species requires careful micromanipulation of oocytes treated with cytochalasin B to prevent structural damage. Although functional, this procedure requires time and limits the number of oocytes available for cloning, and our ability to understand the mechanisms of nuclear reprogramming. Therefore, this study aimed at evaluating different procedures to enucleate large pools of oocytes in a time-efficient manner. Two different approaches were tested. The first approach involved centrifugation of zona-free oocytes through a percoll gradient to separate the portion containing the chromatin from the cytoplasmic portion. The second used etoposide to prevent chromatin segregation at first metaphase and resulting in the expulsion of all chromosomes in the polar body. Using the chemical approach an average enucleation rate of 39.4 +/- 7.5% was obtained, while the centrifugation approach resulted in an average enucleation rate of 66.9 +/- 6. In terms of time efficiency, the control manipulation method takes 0.11 min and the centrifugation took an average of 0.52 min per oocyte. The MPF activity at the end of procedure was estimated through the measurement of H1 activity and as expected, the etoposide-cycloheximide treated oocytes had lower H1 activity which was restored by further incubation in the maturation medium for 5 hr while the centrifugation gave a nonsignificant intermediary result. In conclusion, the results presented suggest that both the chemical and the mechanical methods are usable alternatives to micromanipulation of oocytes to generate a large number of chromosome free cytoplasm for biochemical analysis. Mol. Reprod. Dev. 67: 70-76, 2004.     

Primordial germ cells: what does it take to be alive?

Specification of primordial germ cells (PGCs) in the proximal epiblast enables about 45 founder PGCs clustered at the base of the allantoic bud to enter the embryo by active cell movement. Specification of the PGC lineage depends on paracrine signals derived from the somatic cell neighbors in the extraembryonic ectoderm. Secretory bone morphogenetic proteins (BMP) 4, BMP8b, and BMP2 and components of the Smad signaling pathway participate in the specification of PGCs. Cells in the extraembryonic ectoderm induce expression of the gene fragilis in the epiblast in the presence of BMP4, targeting competence of PGCs. The fragilis gene encodes a family of transmembrane proteins presumably involved in homotypic cell adhesion. As PGCs migrate throughout the hindgut, they express nanos3 protein. In the absence of nanos3 gene expression, no germ cells are detected in ovary and testis. During migration and upon arrival at the genital ridges, the population of PGCs is regulated by a balanced proliferation/programmed cell death or apoptosis. Paracrine and autocrine mechanisms, involving transforming growth factor-beta1 and fibroblast growth factors exert stimulatory or inhibitory effects on PGCs proliferation, modulated in part by the membrane-bound form of stem cell factor. Apoptosis requires the participation of the pro-apoptotic family member Bax, whose activity is balanced by the anti-apoptotic family member Bcl21/Bcl-x. In addition, a loss of cell-cell contacts in vitro results in the apoptotic elimination of PGCs. It needs to be determined whether apoptosis is triggered by a failure of PGC to establish and maintain appropriate cell-cell contacts with somatic cells or whether undefined survival factors released by adjacent somatic cells cannot reach physiological levels to satisfy needs of the expanding population of PGCs.     

Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1–283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS–PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.     

Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-beta (TGF-beta) in various renal diseases. To elucidate the role of CTGF in renal tubular epithelial-myofibroblast transdifferentiation, we examined the expression of alpha-smooth muscle actin (alpha-SMA), vimentin, tenascin-C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelial cell line (HKC), and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF-beta. It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 microg/L), the expression of alpha-SMA and tenascin-C mRNA increased significantly (p<0.01), while collagen IV gene expression decreased significantly (p<0.01), all in a dose-dependent manner. The percentage of alpha-SMA-positive cells was significantly larger in the rhCTGF-stimulated groups than that in negative control (38.9%, 65.5% vs. 2.4%, respectively, p<0.01) as confirmed by flow cytometry. Both cytoplasmic and secretory tenascin-C expression was upregulated by the stimulation of rhCTGF (p<0.01). Under this condition, collagen IV secreted into the culture media was lowered markedly (p<0.01). On RT-PCR analysis, TGF-beta1 upregulated CTGF gene expression, preceding that of alpha-SMA. The alpha-SMA mRNA expression induced by TGF-beta1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p<0.01). With prolonged incubation time, CTGF antisense ODN also inhibited intracellular alpha-SMA protein synthesis, as demonstrated by indirect immuno-fluorescence. So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis.