Transient production of recombinant proteins by chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG(4) encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 mug mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn(297) was not significantly affected by nocodazole during transient production by this method. (C) 2004 Wiley Periodicals, Inc.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. Mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor. (c) 2006 Elsevier Inc. All rights reserved.

Irradiation-induced oral candidiasis in an experimental murine model

The aim of this experiment was to establish a mouse model of irradiation-induced oral candidiasis and to explore the cellular populations and mechanisms by which the infection is cleared from the oral mucosa. BALB/c mice received irradiation to the head and neck equivalent to 800 Rad using a Cobalt 60 gamma source. Both irradiated and non-irradiated mice were infected orally with 1 X 10(8) Candida albicans yeasts. Compared with untreated controls, irradiated animals developed a more severe infection of longer duration, with hyphae penetrating the oral mucosa. Monoclonal antibody depletion of CD4(+) but not CD8(+) T cells from the systemic circulation prolonged the infection in irradiated mice, but not in controls. Supernatants of submandibular and superficial cervical lymph node cultures from irradiated animals demonstrated significantly higher titers of interleukin-12, but similar levels of interferon-gamma compared with controls. Screening for cytokine production by an RNase protection assay detected only macrophage migration inhibition factor in irradiated and non-irradiated oral tissues from day 8 onwards. The results of this study demonstrate a requirement for CD4(+) T cells in the recovery from oral candidiasis induced by head and neck irradiation in mice, and are consistent with a role for Th-1-type cytokines in host resistance.

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect

Aims: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. Methods and Results: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. Conclusions: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. Significance and Impact of the Study: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Clarithromycin and prednisolone inhibit cytokine production in chronic rhinosinusitis

Objectives. Long-term, low-dose macrolide therapy is effective in the treatment of chronic rhinosinusitis. The mechanism of its anti-inflammatory effect and how this differs from corticosteroids remains unclear. The effect of clarithromycin and prednisolone on interleukin-5, interleukin-8, and granulocyte-macrophage colony-stimulating factor production by cultured chronic sinusitis nasal mucosa was examined in the study. Study Design and Methods. Nasal mucosa was obtained from 11 patients with chronic sinusitis. This tissue was cultured for 24 hours in the presence of clarithromycin or prednisolone at a variety of concentrations. Cytokine levels were determined by enzyme-linked immunoassay. Results. Clarithromycin and prednisolone each produced significant reductions in interleukin-5, interleukin-8, and granulocyte-macrophage colony-stimulating factor production. There was no significant difference between the effects of clarithromycin and prednisolone. Conclusion: Macrolide antibiotics are capable of inhibiting pro-inflammatory cytokine production in vitro and are as potent as prednisolone. This mechanism is likely to be at least partly responsible for the clinical efficacy of macrolide antibiotics in chronic rhinosinusitis. Key Words. Macrolide, prednisolone, sinusitis, enzyme-linked immunosorbent assay, cytokine.

L-arginine-dependent nitric oxide production of a murine macrophage-like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg-LPS). RAW264.7 cells were incubated with i) various concentrations of Pg-LPS or Salmonella typhosa LPS (St-LPS), ii) Pg-LPS with or without L-arginine and/or N-G-monomethyl-L-arginine (NMMA), an arginine analog or iii) Pg-LPS and interferon-gamma (IFN-gamma) with or without anti-IFN-gamma antibodies or interleukin-10 (IL-10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg-LPS, but was observed after stimulation with St-LPS. Exogenous L-arginine restored the ability of Pg-LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg-LPS with exogenous L-arginine was abolished by NMMA. IFN-gamma induced independent NO production by Pg-LPS-stimulated macrophages and this stimulatory effect of IFN-gamma could be completely suppressed by anti-IFN-gamma antibodies and IL-10. These results suggest that Pg-LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an L-arginine-dependent mechanism which is itself independent of the action of IFN-gamma.

Insertion of an E-coli lacZ gene in Acetobacter xylinus for the production of cellulose in whey

A mini-Tn10:lacZ: kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in beta-galactosidase activity when grown in lactose medium. beta-Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and beta-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l(-1) after 4 days incubation. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Antibiotic therapy for Klebsiella pneumoniae bacteremia: Implications of production of extended-spectrum beta-lactamases

The prevalence of extended-spectrum beta-lactamase (ESBL) production by Klebsiella pneumonia approaches 50% in some countries, with particularly high rates in eastern Europe and Latin America. No randomized trials have ever been performed on treatment of bacteremia due to ESBL-producing organisms; existing data comes only from retrospective, single-institution studies. In a prospective study of 455 consecutive episodes of Klebsiella pneumoniae bacteremia in 12 hospitals in 7 countries, 85 episodes were due to an ESBL-producing organism. Failure to use an antibiotic active against ESBL-producing K. pneumoniae was associated with extremely high mortality. Use of a carbapenem ( primarily imipenem) was associated with a significantly lower 14-day mortality than was use of other antibiotics active in vitro. Multivariate analysis including other predictors of mortality showed that use of a carbapenem during the 5-day period after onset of bacteremia due to an ESBL-producing organism was independently associated with lower mortality. Antibiotic choice is particularly important in seriously ill patients with infections due to ESBL-producing K. pneumoniae.

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87-0.97 g/g starch associated with 1.5-2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.

Simultaneous saccharification and fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of simultaneous saccharification and fermentation (SSF) for lactic acid production by fungal species of Rhizopus arrhizus 36017 and Rhizopus oryzae 2062 was studied with respect to growth pH, temperature and substrate. Both R. arrhizus 36017 and R. oryzae 2062 had a capacity to carry out a single stage SSF process for lactic acid production from potato starch wastewater. The kinetic characteristics, termed as starch hydrolysis, accumulation of reducing sugars, lactic acid production and fungal biomass formation, were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.85-0.92 g/g associated with 1.5-3.5 g/l fungal biomass produced in 36-48 h fermentation. R. arrhizus 36017 had a higher capacity to produce lactic acid, while R. oryzae 2062 produced more fungal biomass under similar conditions. (c) 2005 Elsevier B.V. All rights reserved.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.

Isotopes, wool, and rangeland monitoring: Let the sheep do the sampling

Stable carbon isotope analyses of wool staples provided insight into the vegetation consumed by sheep at a temporal resolution not previously studied. Contemporary Australian and historic South African samples dating back to 1916 were analyzed for their stable carbon isotope ratio, a proxy for the proportion of C-3 and C-4 plant species consumed by animals. Sheep sample vegetation continuously throughout a year, and as their wool grows it integrates and stores information about their diet. In subtropical and tropical rangelands the majority of grass species are C-4. Since sheep prefer to graze, and their wool is an isotopic record of their diet, we now have the potential to develop a high resolution index to the availability of grass from a sheep's perspective. Isotopic analyses of wool suggest a new direction for monitoring grazing and for the reconstruction of past vegetation changes, which will make a significant contribution to traditional rangeland ecology and management. It is recommended that isotopic and other analyses of wool be further developed for use in rangeland monitoring programs to provide valuable feedback for land managers.

A phase-segregated model for plant cell culture: The effect of cell volume fraction

Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases, Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus, Large errors in the simulated results were observed if the phase-segregation was not considered.