Bioreactors for surface-immobilized cells

Surface immobilization of plant cells avoids the problem of hydrodynamic or shear stress, which tends to be characteristic of suspended cells cultured in typical, mechanically agitated bioreactor systems. Surface immobilization also promotes the natural tendency for plant cells to aggregate, which may improve the synthesis and accumulation of secondary metabolites. In addition, exchange of medium is made simple in surface-immobilized systems, and extracellular secondary products are easily recovered on a continuous basis. However, problems related to regulation of the thickness of the immobilized cell layer, maintenance of the biomass in a productive condition, and vacuolar retention of secondary products have yet to be resolved satisfactorily. This review focusses on two surface-immobilization technologies, differing primarily in the nature and the configuration of the inert support. Prototypes of these designs have been applied to a variety of plant cell systems at bioreactor volumes up to 20 litres. Results obtained with several alternative technologies are also summarized.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - SIPCB surface-immobilized plant cell bioreactor National Research Council of Canada publication no. 38460     

Calcitonin gene-related peptide and its receptor in the thymus

Calcitonin gene-related peptide (CGRP), a 37-amino acid residue neuropeptide, was immunostained in rat thymus at two sites: a subpopulation of thymic epithelial cells, namely subcapsular/perivascular cells, were heavily stained besides some nerve fibers surrounding arteries and arterioles. The administration of nanomolar concentrations of rat -CGRP dose-dependently raised intracellular cyclic adenosine monophosphate (cAMP) levels in isolated rat thymocytes (half-maximum stimulation 1 nM) but not in cultured rat thymic epithelial cells. Peptides structurally related to CGRP (i.e., rat calcitonin or amylin) had no effect. CGRP(8–37), an N-terminally truncated form, acted as an antagonist. Peripheral blood lymphocytes did not respond to CGRP, suggesting that receptors are present only on a subpopulation of thymocytes but not on mature T cells. This was substantiated by visualization of CGRP receptors on single cells by use of CGRP-gold and -biotin conjugates of established biological activity: only a small proportion of isolated thymocytes was surface labeled. In situ, the CGRP conjugates labeled receptors on large thymocytes residing in the outer cortical region of rat thymus pseudolobules. Thus, immunoreactive CGRP is found in subcapsular/perivascular thymic epithelial cells and acts via specific CGRP receptors on thymocytes by raising their intracellular cAMP level. It is suggested that CGRP is a paracrine thymic mediator that might influence the differentiation, maturation, and proliferation of thymocytes.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.     

Alkaloid production in Catharanthus roseus (L.) G. Don.

Analysis of 76 cell clones derived from one leaf of a periwinkle plant (Catharanthus roseus (L.) G. Don) showed the occurrence of Corynanthe-, Strychnos-, and Aspidosperma-type alkaloids. The majority of clones (62%) displayed compounds of all three types. Variation of the alkaloid spectra of the cell clones was low when compared to that found previously with serially subcultured callus and cell suspensions derived from different plants.NRCC # 19100     

The kinetics of the alkaline dissociation of myosin.

The rates of two processes in alkaline (pH 10.5–11.5) myosin solutions at 0 °C have been investigated: production of ionized tyrosine residues and production of light subunits. The progressive absorbance change is shown to result from a first-order irrevocable exposure to solvent and subsequent ionization of 40% of the tyrosine residues. Extrapolation to zero time gives the spectrophotometric ionization curve for native myosin; the pK of the abnormal tyrosines exceeds 12. Similarly, extrapolation to infinite time gives the curve for denatured myosin; the pK of the normal tyrosines (and of all tyrosines after denaturation) is 11.0–11.6. From the pH dependence of the rate, it is found that activation requires ionization of six residues and that their pK is much greater than 11.3. The rate of production of subunits was determined by fractionating the reaction mixture and determining the weight of light subunits produced. The process is also first order. Within experimental error, the rate constants for these two processes are equal. We conclude that they have the same rate-determining step. The data are consistent with either of two simple possible mechanisms. These are a rapid conformation change, followed by rate-determining subunit dissociation, followed by a rapid, irrevocable conformation change; or, a rapid conformation change, followed by a rate-determining, irrevocable conformation change, followed by rapid subunit dissociation.     

Metal thiolate coordination in the E7 proteins of human papilloma virus 16 and cottontail rabbit papilloma virus as expressed in Escherichia coli.

The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.