Quantitative proteomic profiling of shake flask versus bioreactor growth reveals distinct responses of Agrobacterium tumefaciens for preparation in molecular pharming.

The preparation of Agrobacterium tumefaciens cultures with strains encoding proteins intended for therapeutic or industrial purposes is an important activity prior to treatment of plants for transient expression of valuable protein products. The rising demand for biologic products such as these underscores the expansion of molecular pharming and warrants the need to produce transformed plants at an industrial scale. This requires large quantities of A. tumefaciens culture, which is challenging using traditional growth methods (e.g., shake flask). To overcome this limitation, we investigate the use of bioreactors as an alternative to shake flasks to meet production demands. Here, we observe differences in bacterial growth among the tested parameters and define conditions for consistent bacterial culturing between shake flask and bioreactor. Quantitative proteomic profiling of cultures from each growth condition defines unique growth-specific responses in bacterial protein abundance and highlights the functional roles of these proteins, which may influence bacterial processes important for effective agroinfiltration and transformation. Overall, our study establishes and optimizes comparable growth conditions for shake flask versus bioreactors and provides novel insights into fundamental biological processes of A. tumefaciens influenced by such growth conditions.

Exposure of Agrobacterium tumefaciens to agroinfiltration medium demonstrates cellular remodelling and may promote enhanced adaptability for molecular pharming.

Agroinfiltration is used to treat plants with modified strains of Agrobacterium tumefaciens for the purpose of transient in planta expression of genes transferred from the bacterium. These genes encode valuable recombinant proteins for therapeutic or industrial applications. Treatment of large quantities of plants for industrial-scale protein production exposes bacteria (harboring genes of interest) to agroinfiltration medium that is devoid of nutrients and carbon sources for prolonged periods of time (possibly upwards of 24 h). Such conditions may negatively influence bacterial viability, infectivity of plant cells, and target protein production. Here, we explored the role of timing in bacterial culture preparation for agroinfiltration using mass spectrometry-based proteomics to define changes in cellular processes. We observed distinct profiles associated with bacterial treatment conditions and exposure timing, including significant changes in proteins involved in pathogenesis, motility, and nutrient acquisition systems as the bacteria adapt to the new environment. These data suggest a progression towards increased cellular remodelling over time. In addition, we described changes in growth- and environment-specific processes over time, underscoring the interconnectivity of pathogenesis and chemotaxis-associated proteins with transport and metabolism. Overall, our results have important implications for the production of transiently expressed target protein products, as prolonged exposure to agroinfiltration medium suggests remodelling of the bacterial proteins towards enhanced infection of plant cells.

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle.

AIMS: To produce a method of distinguishing between type 1 and 2 skeletal muscle fibres that would be more economical and reproducible than the standard ATPase method and be applicable to both fixed and frozen tissue. Because the ATPase method has been accepted as the basis for fibre identification for the past 50 years, the new method should not give significantly different results. METHODS: Isoforms of myosin correlate with isoforms of myofibrillar ATPase and an immunohistochemical (IHC) double labelling protocol was devised using monoclonal antibodies to fast and slow myosin. This required one tissue section rather than four. The results of the two methods were compared by means of morphometric analysis of skeletal muscle biopsies from 20 normal healthy volunteers. RESULTS: There were no significant differences (p = 0.57) in the percentages of type 1 (46% using the IHC method v 48% using ATPase) or type 2 fibres (54% v 52%, respectively). The 2a and 2b subtypes were distinguished easily. Analysis of variance revealed that cross sectional area (mu m(2)), diameter (mu m), form factor, and density of fibre staining (a measure of substrate-enzyme or protein) were all similar. The method worked equally well on fixed material. CONCLUSION: An IHC method based on the fast and slow isoforms of myosin shows no significant differences in fibre type analysis from the standard ATPase method although it provides important advantages because it is applicable to fixed (including archival) material, is economical and reproducible, and yields a permanent preparation.

Peptidases affecting recombinant protein production by Streptomyces lividans.

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.

Development of fermentation process for rDNA products.

Fermentation process development for recombinant DNA-derived products is becoming increasingly important in the current commercial and regulatory framework. This article provides an overview of the current approach to process development, and the contribution of developmental data to final process validation is highlighted. Cited literature is restricted to between 1995 and 2001.

Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.

A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.

The immunohistochemical localization of S100 in the diagnosis of papillary carcinoma of the thyroid.

In general, the diagnosis of papillary carcinoma of the thyroid is readily achieved based on a defined aggregate of histopathologic features. A papillary architecture is an important but not pivotal component of the diagnosis. The recognition of classic nuclear features is the essential diagnostic element. However, both the architectural and cytological hallmarks may be encountered in other conditions and produce problems in histopathologic interpretation. A papillary architecture may be encountered in hyperplastic areas of follicular neoplasms, multinodular goiter, and Graves' disease. Moreover, there may be scattered cells within several thyroid lesions that display some of the nuclear characteristics of papillary carcinoma. The distinction of these lesions from papillary carcinoma has important implications for clinical management. Thus, the availability of supportive diagnostic evidence would be helpful. In the authors' experience, the strong expression of S100 is of value in identifying papillary neoplasia and distinguishing it from examples of papillary hyperplasia. It is of supportive but not conclusive use in distinguishing follicular adenoma from the follicular variant of papillary carcinoma. The authors stress that the overwhelming factor in the distinction remains the identification of the nuclear characteristics of a papillary carcinoma. However, the authors have encountered several cases wherein the latter are either focal or absent for reasons addressed previously and have found immunohistochemistry a valuable adjunct to diagnosis. In examining papillary foci within Graves' disease, caution must be exercised; S100 expression is a phenomenon of the hyperplastic, hyperfunctional state.

Differential release of prostaglandins by organ cultures of human fetal trachea and lung.

Human fetal lung at 16-19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE2. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4-6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF1 alpha, PGF2 alpha, and PGE2, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF2 alpha is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG2 alpha,PGF2 alpha,PGE2, and 6-keto-PGF1 alpha. Human fetal lung in vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE2 relative to PGE2 suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturation in vivo by limiting PGE2 availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and with in utero ontogeny.

Prostaglandin production and metabolism in self-differentiating human fetal lung organ culture.

PGE2 and PGF2 alpha are released into the media of human fetal lung organ cultures in decreasing amounts with time. This decline in PGs is not due to culture failure or loss of synthetic capacity, which can be stimulated by fetal bovine serum, nor is it due to increased catabolism of PGE2 to 13,14-dihydro-15-keto-PGE2 (PGEM) or of PGF2 alpha to 13,14-dihydro-15-keto-PGF2 alpha (PGFM). Immunohistochemically reactive PGs are not retained within lung cells. Antisera against methyl-moximated derivatives of PGEM or PGFM and preceded by derivatization on tissue sections of PGs by methyl-moximation not only demonstrate the localization of PGEM and PGFM in epithelial cells and blood vessels, but also show an overall decline in immunoreactivity with time. In addition electron microscopy of uncultured fetal lung removed directly after termination reveals various degrees of mitochondrial damage and in some cases plasma membrane blebs which resolve during the period in culture and as fetal lung self-differentiates. It is proposed that oxidative and mechanical stresses, occurring during termination of pregnancy or tissue preparation, result in cell damage and increased lung prostaglandin production, which, although decreasing during culture as cells recover, is sufficient to trigger terminal self-differentiation.

Development of human fetal lung in organ culture compared with in utero ontogeny.

In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes. Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial immunoreactivity to cytokeratins CK 7, 8, and 18 decreases with differentiation both in utero and in organ culture, whereas reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0 +/- 7.3 wk. In organ culture, capillary blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost synchronous in human lung in utero but clearly dissociate in organ culture.

Prostaglandins PGE2 and PGF2 alpha in human fetal lung: immunohistochemistry and release from organ culture.

Immunohistochemical studies in human fetal lung have shown that epithelial and endothelial cells are both strongly and equally reactive for PGE2. In contrast, epithelial PGF2 alpha reactivity varied between fetuses, in some as intense as endothelial staining and in others very much less. As lung organ cultures differentiated, the intensity of PGE2 staining declined in airways and blood vessels, although it was still weakly positive at 10 days. In contrast, epithelial cells rapidly became negative for PGF2 alpha, whereas PGF2 alpha positivity was retained in blood vessels, albeit less obviously. PGF2 alpha and PGE2 were released into the media of organ cultures in decreasing amounts as cultures progressed. Amounts of released PGF2 alpha were greater by 2- to 10-fold than PGE2. Our findings suggest that the endogenous production of prostaglandins by human fetal lung in organ culture has a key role in the self-differentiation process that occurs in the absence of sera or added growth factors or hormones.

Self-differentiation of human fetal lung organ culture: the role of prostaglandins PGE2 and PGF2 alpha.

Addition of PGE2, but not PGF2 alpha, to fetal lung organ cultures accelerates the process of self-differentiation with increased dilatation of terminal airsacs and differentiation of the epithelial lining. Indomethacin reduces the endogenous production by organ cultures of PGE2, PGF2 alpha, 13,14-dihydro-15-keto-PGE2, and 13,14-dihydro-15-keto-PGF2 alpha and retards the process of self-differentiation. Prolonged exposure of cultures to indomethacin results in cell necrosis. Indomethacin inhibition of self-differentiation can be reversed and accelerated by the addition of PGE2. Addition of PGF2 alpha in the presence of indomethacin prevents indomethacin-associated cell necrosis but does not accelerate dilatation or differentiation beyond that of cultures in sera-free media without additions. We propose that the endogenous production of PGE2 is a key process in the mechanism of self-differentiation of human fetal lung in organ culture.

The alpha and pi isoenzymes of glutathione S-transferase in human fetal lung: in utero ontogeny compared with differentiation in lung organ culture.

Polyclonal antisera to the alpha and pi isoenzymes of glutathione S-transferase have been used in immunohistochemical studies of developing human lung. In utero expression of the pi set was down-regulated in distal airway cells and the first appearance of pi-negative cells coincided with phenotypic differentiation. In contrast, in the early phase of fetal lung organ culture pi isoenzyme was detected in all differentiated epithelial cells and only as culture progressed did focal negativity develop. The alpha set showed no developmental changes in utero or in organ culture.

Immunohistochemical analysis of human MHC class II antigens in B-cell non-Hodgkin's lymphomas.

Twenty cases of B-cell non-Hodgkin's lymphoma (NHL) were evaluated by immunohistochemical staining of frozen sections with a panel of anti MHC class II monoclonal antibodies (MCA). These studies showed marked differences in the expression of class II antigens both between different cases and within the population of cells of individual cases. In all of the cases studied the majority of the tumour cells reacted with MCAs directed against determinants common to the products of SB and DR loci. However, MCAs specific for DC determinants failed to react with 3/20 cases and in several other cases stained only a minority of cells. Absent or reduced expression of DC antigens was most marked in lymphocytic lymphoma; however, in centroblastic-centrocytic and centroblastic lymphomas, DC antigens could be detected on the majority of cells.