In vitro and in silico analysis of signal peptides from the human blood fluke, Schistosoma mansoni

Proteins secreted by and anchored on the surfaces of parasites are in intimate contact with host tissues. The transcriptome of infective cercariae of the blood fluke, Schistosoma mansoni, was screened using signal sequence trap to isolate cDNAs encoding predicted proteins with an N-terminal signal peptide. Twenty cDNA fragments were identified, most of which contained predicted signal peptides or transmembrane regions, including a novel putative seven-transmembrane receptor and a membrane-associated mitogen-activated protein kinase. The developmental expression pattern within different life-cycle stages ranged from ubiquitous to a transcript that was highly upregulated in the cercaria. A bioinformatics-based comparison of 100 signal peptides from each of schistosomes, humans, a parasitic nematode and Escherichia coli showed that differences in the sequence composition of signal peptides, notably the residues flanking the predicted cleavage site, might account for the negative bias exhibited in the processing of schistosome signal peptides in mammalian cells. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Laboratory culture studies of Trichodesmium isolated from the great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6 years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR similar to 40-50 mumol quanta m(-2) s(-1)). N-2 fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2-0.3 day(-1) and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR similar to 5-120 mumol quanta m(-2) s(-1)) with the maximum growth occurring at - 40-50 mumol quanta m(-2) s(-1). These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37 psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45 nM. No active growth was observed with the 4.5 nM Fe addition.

Visualizing levels of osteoblast differentiation by a two-color promoter-GFP strategy: Type I collagen-GFPcyan and osteocalcin-GFPtpz

A 3.9 kb DNA fragment of human osteocalcin promoter and 3.6 kb DNA fragment of the rat collagen type1a1 promoter linked with visually distinguishable GFP isomers, topaz and cyan, were used for multiplex analysis of osteoblast lineage progression. Three patterns of dual transgene, expression can be appreciated in primary bone cell cultures derived from the transgenic mice and by histology of their corresponding bones. Our data support the interpretation that strong pOBCol3.6GFPcyan alone is found in newly formed osteoblasts, while strong pOBCol3.6GFPcyan and hOC-GFPtpz are present in osteoblasts actively making a new matrix. Osteoblasts expressing strong hOC-GFPtpz and weak pOBCol3.6GF-Pcyan are also present and may or may not be producing mineralized matrix. This multiplex approach reveals the heterogeneity within the mature osteoblast population that cannot be appreciated by current histological methods. It should be useful to identify and isolate populations of cells within an osteoblast lineage as they progress through stages of differentiation.

Isoform specific changes in PPAR alpha and beta in colon and breast cancer with differentiation

To investigate the role of peroxisome proliferator-activated receptors (PPARs) chi and beta in the differentiation of colon cancer cells, we differentiated HT-29 cells using sodium butyrate (NaB) and culturing post-confluence and assessed differentiation using the marker intestinal alkaline phosphatase. While PPAR chi levels only changed with culturing post confluence, PPAR beta levels increased independent of the method of differentiation. To explore further the differences induced by NaB. we assessed changes in both PPAR isoforms in MCF-7 breast cancer cells cultured in the presence of NaB over 48 h. Again a very different expression pattern was observed with PPAR-1 increasing after 4 h and remaining elevated, while PPAR beta increased transiently. Our studies suggest that the expression of PPARs is dependent upon both the method of differentiation and on time. Moreover, these studies show that changes in levels are not required for the differentiation of colon cancer cell lines, whereas changes in PPAR beta are more closely associated with differentiation. (c) 2005 Elsevier Inc. All rights reserved.

Role of growth hormone receptor signaling in osteogenesis from murine bone marrow progenitor cells

Growth hormone (GH) regulates many of the factors responsible for controlling the development of bone marrow progenitor cells (BMPCs). The aim of this study was to elucidate the role of GH in osteogenic differentiation of BMPCs using GH receptor null mice (GHRKO). BMPCs from GHRKO and their wild-type (WT) littermates were quantified by flow cytometry and their osteogenic differentiation in vitro was determined by cell morphology, real-time RT-PCR, and biochemical analyses. We found that freshly harvested GHRKO marrow contains 3% CD34 (hernatopoietic lineage), 43.5% CD45 (monocyte/macrophage lineage), and 2.5% CD106 positive (CFU-F/BMPC) cells compared to 11.2%, 45%, and 3.4% positive cells for (WT) marrow cells, respectively. When cultured for 14 days under conditions suitable for CFU-F expansion, GHRKO marrow cells lost CD34 positivity, and were markedly reduced for CD45, but 3- to 4-fold higher for CD106. While WT marrow cells also lost CD34 expression, they maintained CD45 and increased CD106 levels by 16-fold. When BMPCs from GHRKO mice were cultured under osteogenic conditions, they failed to elongate, in contrast to WT cells. Furthermore, GHRKO cultures expressed less alkaline phosphatase, contained less mineralized calcium, and displayed lower osteocalcin expression than WT cells. However, GHRKO cells displayed similar or higher expression of cbfa-1, collagen 1, and osteopontin mRNA compared to WT. In conclusion, we show that GH has an effect on the proportions of hematopoietic and mesenchymal progenitor cells in the bone marrow, and that GH is essential for both the induction and later progression of osteogenesis. (c) 2005 Elsevier Inc. All rights reserved.

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells

In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG(4) monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 mu g 10(6) cells(-1) mL(-1) at a PEI nitrogen: DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from similar to 13 to similar to 39 mg L-1. Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended activated hypothermic synthesis.

In vitro heterogeneity of osteogenic cell populations at various equine skeletal sites

Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted. The numbers of nodules produced from osteogenic cells harvested from different skeletal sites were compared with the use of a mixed linear model. On average, cortical bone sites yielded significantly greater numbers of nodules than did cancellous bone sites. Between cortical bone sites, there was no significant difference in nodule numbers. Among cancellous sites, the radial cancellous bone yielded significantly more nodules than did the tibial cancellous bone. Among appendicular skeletal sites, tibial metaphyseal bone yielded significantly fewer nodules than did all other long bone sites. This study detected evidence of heterogeneity of equine osteogenic cell populations at various skeletal sites. Further characterization of the dissimilarities is warranted to determine the potential role heterogeneity plays in differential rates of fracture healing between skeletal sites.

Long-term loading inhibits ERK1/2 phosphorylation and increases FGFR3 expression in MC3T3-E1 osteoblast cells

Bone tissue homeostasis relies upon the ability of cells to detect and interpret extracellular signals that direct changes in tissue architecture. This study utilized a four-point bending model to create both fluid shear and strain forces (loading) during the time-dependent progression of MC3T3-E1 preosteoblasts along the osteogenic lineage. Loading was shown to increase cell number, alkaline phosphatase (ALP) activity, collagen synthesis, and the mRNA expression levels of Runx2, osteocalcin (OC), osteopontin, and cyclo-oxygenase-2. However, mineralization in these cultures was inhibited, despite an increase in calcium accumulation, suggesting that loading may inhibit mineralization in order to increase matrix deposition. Loading also increased fibroblast growth factor receptor-3 (FGFR3) expression coincident with an inhibition of FGFR1, FGFR4, FGF1, and extracellular signal-related kinase (ERK)1/2 phosphorylation. To examine whether these loading-induced changes in cell phenotype and FGFR expression could be attributed to the inhibition of ERK1/2 phosphorylation, cells were grown for 25 days in the presence of the MEK1/2 inhibitor, U0126. Significant increases in the expression of FGFR3, ALP, and OC were observed, as well as the inhibition of FGFR1, FGFR4, and FGF1. However, U0126 also increased matrix mineralization, demonstrating that inhibition of ERK1/2 phosphorylation cannot fully account for the changes observed in response to loading. in conclusion, this study demonstrates that preosteoblasts are mechanoresponsive, and that long-term loading, whilst increasing proliferation and differentiation of preosteoblasts, inhibits matrix mineralization. In addition, the increase in FGFR3 expression suggests that it may have a role in osteoblast differentiation.

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus. (c) 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Effect of Mn deficiency and legume inoculation on rhizosphere pH in highly alkaline soils

Although plant growth is often limited at high pH, little is known about root-induced changes in the rhizospheres of plants growing in alkaline soils. The effect of Mn deficiency in Rhodes grass (Chloris gayana cv. Pioneer) and of legume inoculation in lucerne (Medicago sativa L. cv. Hunter River), on the rhizosphere pH of plants grown in highly alkaline bauxite residue was investigated. Rhizosphere pH was measured quantitatively, with a micro pH electrode, and qualitatively, with an agar/pH indicator solution. Manganese deficiency in Rhodes grass increased root-induced acidification of the rhizosphere in a soil profile in which N was supplied entirely as NO3-. Rhizosphere pH in the Mn deficient plants was up to 1.22 pH units lower than that of the bulk soil, while only 0.90 to 0.62 pH units lower in plants supplied with adequate Mn. When soil N was supplied entirely as NO3-, rhizosphere acidification was more efficient in inoculated lucerne (1.75 pH unit decrease) than in non-inoculated lucerne (1.16 pH unit decrease). This difference in capacity to lower rhizosphere pH is attributable to the ability of the inoculated lucerne to fix atmospheric N2 rather than relying on the soil N (NO3 ) reserves as the non-inoculated plants. Rhizosphere acidification in both Rhodes grass and lucerne was greatest in the meristematic root zone and least in the maturation root zone.

Mg induced Ca deficiency under alkaline conditions

Little is known about Mg induced Ca deficiency in alkaline conditions, and the relationship between Mg induced Ca deficiency and Na induced Ca deficiency. Dilute nutrient solutions (dominated by Mg) were used to investigate the effect of Ca activity ratio (CAR) on the growth of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald). At pH 9.0, root growth was reduced below a critical CAR of 0.050 (corresponding to 90 % relative root length). Root growth was found to be limited more in Mg solutions than had been previously observed for Na solutions. Using a CAR equation modified with plasma membrane binding constants (to incorporate the differing antagonistic effects of Mg and Na), new critical CAR values were calculated for both Na (0.56) and Mg (0.44) dominated solutions. This modified CAR equation permits the calculation of CAR irrespective of the dominant salt present.

Characterization of serum antibodies to Porphyromonas gingivalis in individuals with and without periodontitis

Although Porphyromonas gingivalis is a defined pathogen in periodontal disease, many subjects control the infection without experiencing loss of attachment. Differences in host susceptibility to the disease may be reflected in the pattern of humoral antibodies against specific P. gingivalis antigens. The aim of this study was to determine the presence of antibodies against immunodominant P. gingivalis antigens as well as the isotype and subclass of anti-P. gingivalis antibodies against outer membrane antigens in four groups of patients: P. gingivalis-positive, 1) with and 2) without periodontitis, and P. gingivalis-negative, 3) with and 4) without periodontitis. Antigens of molecular weight 92, 63, and 32 kDa and lipopolysaccharide were found to be immunodominant. Group 1 subjects showed a significantly higher response to the 92 and 63 kDa antigens compared with other groups. The response to lipopolysaccharide was significantly higher in group 1, and lower in group 4 than in groups 2, 3. Immunoglobulin G(1) (IgG(1)), IgG(2) and IgM antibodies against P. gingivalis outer membrane were present in all subjects, while only some subjects were seropositive for IgG(3), IgG(4) and IgA. There were no differences in concentrations for IgG(1), IgG(3) and IgM. The IgG(2) concentration in group 4 was significantly higher than in groups 1 and 2, while the IgG(4) concentration in group 4 was significantly lower than in other groups. The frequency of seropositivity for IgG(4) and IgA was lowest in group 4, while IgG; seropositivity was almost exclusively seen in healthy patients iii groups 2, 4. These findings suggest that the presence of IgG(3) may reflect non-susceptibility to the disease, while lack of IgG(4) may be indicative of periodontal health and lack of infection.

Dragmacidins: New protein phosphatase inhibitors from a southern Australian deep-water marine sponge, Spongosorites sp.

A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

Analysis of immunosuppressive proteins in serum of liver-transplanted rats by using an anti-LSF-1 affinity column

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the nonrejector combination of DA donors into PVG; recipients. In the present study, the purification of proteins from rat OLT serum taken 60 days after transplantation Mras performed by affinity chromatography using the anti-LSF-1 polyclonal antibody (pAb). The assessment of column eluates using anti-LSF-1 and OLT serum was studied using rat heart and liver transplantation models. Rejection was not suppressed by the administration of OLT serum in heart or liver allografts. However, heart allografts treated with peak eluates (450 mu g single shot im, dissolved in Intralipos) taken from the affinity OLT serum survived significantly longer than untreated rats (median = 36.5 days; n = 7 vs 6.5 days; n = 5, respectively, P = 0.011). The same treatment with anti-LSF-1 column eluates also prolonged liver allografts significantly (>200 days) than those in either the untreated group (median = 11 days; n = 7) or those which received only Intralipos (median = 10.5 days; n = 5, P = 0.019). Subsequent analysis of the N-terminal sequences of some of the proteins which were eluted from the affinity column revealed that the homology of a 30-kDa protein was identical to hemoglobin alpha-chain, a 59-kDa protein to granulocyte inhibitory factor, a 70-kDa and a 90-kDa to albumin and its precursor, respectively. Although the specific immunosuppressive component has not been isolated, our results suggested that the anti-LSF-1 column can extract immunosuppressive moiety of LSF-1 from OLT serum. (C) 1998 Academic Press.