Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21 R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function

The hydrolysis of urea by the widely distributed enzyme urease is special in that it is one of the few biologically occurring reactions that can generate carbonate ions without an associated production of protons. When this hydrolysis occurs in a calcium-rich environment, calcite (calcium carbonate) precipitates from solution forming a solid-crystalline material. The binding strength of the precipitated crystals is highly dependent on the rate of carbonate formation and under suitable conditions it is possible to control the reaction to generate hard binding calcite cement (or Biocement). The objective of this thesis was to develop an industrially suitable cost-effective microbial process for the production of urease active cells and investigate the potential for urease active cells to act as a catalyst for the production of Biocement.

The biocementation capability of two suitable strains was compared. Sporosarcina pasteurii (formally Bacillus pasteurii) produced significantly higher levels of urease activity compared to Proteus vulgaris, however the level of urease activity was variable with respect to biomass suggesting that the enzyme was not constitutive as indicated by the literature, but subject to regulation. The environmental and physiological conditions for maximum urease activity in S. pasteurii were investigated and it was found that the potential urease capacity of the organism was very high (29 mM urea.min-1.OD-1) and sufficient for biocementation without additional processing (e.g. concentration, cell lysis). The regulation mechanism for S. pasteurii urease was not fully elucidated in this study, however it was shown that low specific urease activity was not due to depletion of urea nor due to the high concentrations of the main reaction product, ammonium. pH conditions were shown to have a regulatory effect on urease but it was evident that another co-regulating mechanism existed. Despite not fully exploiting the urease capability of S. pasteurii, sufficient urease activity to allow direct application of the enzyme without additional processing could still be achieved and the organism was considered suitable for biocementation.

Urease was the most expensive component of the cementation process and cost-efficient production was desired, thus an economic growth procedure was developed for large-scale cultivation of S. pasteurii. The organism is a moderate alkaliphile (growth optimum pH 9.25) and it was shown that sufficient activity for biocementation could be cultivated in non-sterile conditions with a minimum of upstream and downstream processing. The cultivation medium was economised and expensive components were replace with a food-grade protein source and acetate, which lowered production costs by 95%. A high level of urease activity (21 mM urea hydrolysed.min-1) was produced in the new medium at a low cost ($0.20 (AUD) per L).

The performance of urease in whole S. pasteurii cells was evaluated under biocementation conditions (i.e. presence of high concentrations of urea, Ca2+, NH4 +/NH3, NO3 - and Cl- ions). It was established that the rate of urea hydrolysis was not constant during cementation, but largely controlled by the external concentrations of urea and calcium, which constantly changed during cementation due to precipitation of solid calcium carbonate from the system. A simple model was generated that predicted the change in urea hydrolysis rate over the course of cementation. It was shown that whole cell S. pasteurii urease was tolerant to concentrations of up to 3 M urea and 2 M calcium, and the rate of urea hydrolysis was unaffected up to by 3 M ammonium. This allowed the controlled precipitation of up to 1.5 M CaCO3 within one treatment, and indicated that the enzyme was very stable inspite of extreme chemical conditions.

A cost-efficient cementation procedure for the production of high cementation strength was developed. Several biocementation trials were conducted into order to optimise the imparted cementation strength by determining the effect of urea hydrolysis rate on the development of strength. It was shown that high cementation strength was produced at low urea hydrolysis rates and that the development of cementation strength was not linear over the course of the reaction but mostly occurred in the first few hours of the reaction. In addition, the whole cell bacterial enzyme had capacity to be immobilised in the cementation material and re-used to subsequent applications, offering a significant cost-saving to the process.

An industry-sponsored trial was undertaken to investigate the effectiveness of Biocement for increasing in-situ strength and stiffness of two different sandy soils; (a) Koolschijn sand and (b) 90% Koolschijn sand mixed with 10% peat (Holland Veen). After biocementation treatment, Koolschijn sand indicated a shear strength of 1.8 MPa and a stiffness of 250 MPa, which represents an 8-fold and 3-fold respective improvement in strength compared to unconsolidated sand. Significantly lower strength improvements were observed in sand mixed with peat.

In combination, trials of producing bacteria under economically acceptable conditions and cementation trials support the possibility of on-site production and in-situ application of large field applications.

/pdf/microbial-caco3-precipitation-for-the-production-of-kctukme0qr.pdf

Microbial CaCO3 Precipitation: For the Production of Biocement

Random Amplified Polymorphic DNA (RAPD) analysis was applied to three species of the tilapia genus Oreochromis and four subspecies of O. niloticus. Thirteen random 10-mer primers were used to assay polymorphisms within and between populations. Different RAPD fragment patterns were observed for different species, although not always for different subspecies. Evidence is presented that RAPD markers might be useful for systematic investigation at the level of species and subspecies.

/pdf/application-of-the-rapd-technique-in-tilapia-fish-species-4f89dylt9v.pdf

Application of the RAPD technique in tilapia fish: species and subspecies identification.

The intracellular levels and spatial localizations of metabolites and peptides reflect the state of a cell and its relationship to its surrounding environment. Moreover, the amounts and dynamics of metabolites and peptides are indicative of normal or pathological cellular conditions. Here we highlight established and evolving strategies for characterizing the metabolome and peptidome of single cells. Focused studies of the chemical composition of individual cells and functionally defined groups of cells promise to provide a greater understanding of cell fate, function and homeostatic balance. Single-cell bioanalytical microanalysis has also become increasingly valuable for examining cellular heterogeneity, particularly in the fields of neuroscience, stem cell biology and developmental biology.

/pdf/profiling-metabolites-and-peptides-in-single-cells-5fkdswj27s.pdf

Profiling metabolites and peptides in single cells

Preface Cell Culture Technology: An Overview Recombinant DNA Technology and Cell Line Development Medium Development Cell Metabolism Protein Glycosylation Cell Culture Bioreactors Aeration, Mixing and Hydrodynamics in Bioreactors Instrumentation and Process Control Cell Culture Kinetics and Modeling Fed-Batch Cultivation Of Mammalian Cells For Production Of Recombinant Proteins Optimization of High Density Perfusion Bioreactors Cell Separation And Product Capture Downstream Processing Formulation, Finishing, and Packaging Validation Of Cell Culture Based Processes & Qualification Of Associated Equipment And Facility Facility Design Transient Expression of Proteins Large-Scale Propagation of Insect Cells Advances In Adult Stem Cell Culture Ex Vivo Culture Of Hematopoietic And Mesenchymal Stem Cells For Tissue Engineering And Cell-Based Therapies

https://www.progenericos.org.br/upload/Sadettin_Ozturk_Aug2009_UFRJ.pdf

Cell culture technology for pharmaceutical and cell-based therapies

Classical rabies vaccine strains (genotype 1) protect inefficiently against rabies-related lyssaviruses (other genotypes) and in particular against the Mokola virus (genotype 3). DNA vaccine modem approach was used to produce Mokola valency. Purified Plasmid vector was injected into each anterior tibialis muscle of BALB/c mice. It appeared that the Mokola DNA vaccine has induced a sustained level of VNAb, a T helper cell response and confered protection against Mokola virus challenge. Therefore, it seems that the DNA vaccine approach is a very promising technology, which needs further investigations for a possible application.

Animal Cell Technology

Immobilization of animal cells in porous carrier culture

Can cell culture medium costs be reduced? Strategies and possibilities

Perfusion permits culture homogeneity rather than a feed-and-fast regime, and has permitted the development of bioreactors which have no gas headspace and significant scale-up of cell density. A wide range of culture systems has been reviewed with the emphasis on attached cells. The prime objective of using perfusion techniques is to achieve a controlled and homogeneous environment within the bioreactor. The use of perfusion to keep small fragments of tissue viable in microscope chambers developed in complexity between 1912 and 1970, culminating in Rose's dual-rotary circumfusion system. The cytogenerator also encouraged the development of perfusion systems for substrate or anchorage-dependent cells. The use of perfusion techniques carries this risk because of continuous entry and exit of materials from the reactor, often through vulnerable tubing in pumps, etc. Both the bioreactor and the associated equipment should be critically evaluated with this in mind.

Perfusion systems for cell cultivation.

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.

/pdf/adaptation-of-bhk-cells-producing-a-recombinant-protein-to-togpglntun.pdf

Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium.

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