Design and physical characteristics of a multistage,continuous tower fermentor

A multistage tower laboratory fermentor has been constructed consisting of eight compartments separated by sieve plates. Flow of substrate and air is concurrent from the bottom to the top of the column. It, was hoped that this system could be used to reproduce, simultaneously on a continuous basis, eight distinct phases of a batch growth curve. It was believed that the extent of batch curve simulation would depend upon the character of hydraulic mean residence time of broth in the column and in the individual compartments. The expected relationship did not occur. Rather it was found that growth in the column involved residence time characteristics not only for the fluid but also for the microorganisms, and for the growth limiting substrate. Depending upon the column operation, these could be distinct and different. The purpose of this investigation was to study the residence time distribution (RTD) of the continous (fluid) and dispersed (microorganisms) phases for model systems as well as for a yeast fermentation. Various degrees of flow nonideality, i.e., fluid blackflow and dispersed phase sedimentation, were noticed. The former seems to be due to interaction of the concurrent gas and liquid flow; it is particularly dependent upon void area of the sieve plate holes. Sedimentation is probably a function of plate design as well as cell size and density. It wa concluded that for a particular plate design the gas hold-up wass controlled by superficial air velocity and was the main parameter governing the differences between dispersed and continous phase(Rt1). This conclusion was supported by a computeraided styudy utilizing a mathematical model of fluid flow to fit the growth kinetics and cell distribution observed experimentally throughout the fermentor. Some advantages of foam control in the tower fermentor by surface active compounds are mentioned. Also, suggestions are made for carrying out fermentations that have two liquid phases, such as a hydrocarbon fermentation. The possibility of closely approximating plug-flow conditions in the multistage tower fermentor, a necessary condition for batch growth simulation, is discussed from a practical point of view.     

Genetic studies on the acidification capacity of sunflower roots induced under iron stress

Under stress of iron deficiency roots of sunflower (Helianthus annuus L.) increase proton efflux which acidifies the root medium, increase the ferric reducing capacity and the exudation of phenolic compounds. Differences have been found previously among sunflower inbred lines in the capacity of their roots to lower pH and it was also found that this character is under genetic control.This work presents the results of an inheritance study made by crossing two genotypes, one (CMS HA 89) without acidification capacity and another (RHA 271) with it. Plants were grown individually in 75 mL vessels with an aerated solution low in iron. After 4 days, solutions were changed to one without iron and the pH of the medium was measured during the following days. Results from F₁, F₂, and backcross generations can be explained with two pairs of alleles controlling the character, being the relation between alleles of complete dominance at both gene pairs, but either gene, when dominant is epistatic to the other.     

Internal protein sequence analysis: enzymatic digestion for less than 10 micrograms of protein bound to polyvinylidene difluoride or nitrocellulose membranes.

A procedure for the generation and isolation of internal peptide fragments for less than 10 micrograms of protein bound to either polyvinylidene difluoride (PVDF) or nitrocellulose membranes after electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) is presented. This technique has produced internal sequence data for 120 peptides, with an average initial yield of 20 pmol. Membrane-bound proteins were enzymatically digested with either trypsin or endoproteinase Lys-C in the presence of 1% hydrogenated Triton X-100/10% acetonitrile/100 mM Tris-HCl, pH 8.0, for 24 h at 37 degrees C. The eluted peptides were then directly isolated by microbore HPLC for subsequent sequence analysis. One percent hydrogenated Triton X-100 did not inhibit enzymatic activity, distort HPLC resolution of peptides, or contain uv-absorbing contaminants that could interfere with peptide identification. Reproducible peptide maps and consistent recoveries are presented for standard proteins (3.5-8.0 micrograms) bound to either membrane, with higher recoveries for PVDF-bound proteins. Ninety percent of the proteins analyzed by this technique have produced results; representative peptide maps and sequence data are presented. This technique has a wide range of applications, particularly for proteins with blocked amino termini or those that can only be purified by SDS-PAGE or 2D isoelectric focusing SDS-PAGE.     

Acyl carrier protein (ACP) import into chloroplasts does not require the phosphopantetheine: evidence for a chloroplast holo-ACP synthase.

Import of the acyl carrier protein (ACP) precursor into the chloroplast resulted in two products of about 14 kilodalton (kD) and 18 kD when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Time course experiments indicate that the latter is a modification derivative of the 14-kD peptide after the removal of the transit peptide. Substitution of serine 38 by alanine, eliminating the phosphopantetheine prosthetic group attachment site of ACP, produced a precursor mutant that gave rise to only the 14-kD peptide during import, showing that the modified form depends on the presence of serine 38. Furthermore, these results demonstrate that the prosthetic group is not essential for ACP translocation across the envelope or proteolytic processing. Analysis of the products of import by nondenaturing, conformationally sensitive gels showed reversal of the relative mobility of the 14-kD peptide and the modified form, raising the possibility that the modification is the addition of the phosphopantetheine. Proteolytic processing and the modification reaction were reconstituted in an organelle-free assay. The addition of coenzyme A to the organelle-free assay completely converted the 14-kD peptide to the modified form at 10 micromolar, and this only occurred with the wild-type substrate. Reciprocally, treatment of the products of a modification reaction with Escherichia coli phosphodiesterase converted the modified ACP from back to the 14-kD peptide. These results strongly support the conclusion that there is a holo-ACP synthase in the soluble compartment of the chloroplast capable of transferring the phosphopantetheine of coenzyme A to ACP.     

Ultrastructural analysis of mineralized matrix from human osteoblastic cells: Effect of tumor necrosis factor-alpha

The study, addressed to understand whether or not human platelets possess a unique thiol-oxidase whose activity could be modulated by signalling pathway initiated upon the activation of Receptor-C_k revealed the existence of disulphide-dependent oxidation within these cells and this phenomenon was regulated by Receptor-C_k-dependent generation of second messengers especially phosphatidic acid (PA); cAMP and cGMP. Purification of this activity revealed the existence of 47 kDa protein having thiol-oxidase activity. Keeping in view these results we propose that the existence of this novel 47 kDa Thiol-oxidase within human platelets may provide a crucial switch for the regulation of Receptor-C_k-dependent mevalonate pathway in human platelets.     

Patterns of carbon and nitrogen uptake during blooms of Emiliania huxleyi in two Norwegian fjords

Blooms of the coccolithophorid Emiliania huxleyi were monitoredin two land-locked fjords, Fauskangerpollen and Nordåsvannet(Western Norway), in May 1993. The chemical composition of particulatematter, size-fractionated photosynthesis, calcification, nitrogenuptake rates and the patterns of macromolecular synthesis wereexamined during the peak and decline of E.huxleyi blooms. Thetemporal evolution of the bloom in Fauskangerpollen was definedby a gradual decrease in cell abundance and cell-specific calcificationrates, and by increasing numbers of empty coccospheres and theratio detached coccoliths/living cells. A large proportion ofthe nitrogen required for microplankton growth was suppliedby aminonium and dissolved organic compounds such as urea and,as a consequence, the f-ratios were low (0.2). In general, nitrogenuptake patterns were consistent with ambient concentrationsof nitrogenous species. The photosynthetic carbon metabolismof E.huxleyi dominated phytoplankton assemblages was characterizedby high carbon allocation into lipids and relatively low carbonincorporation into protein as compared with diatom-dominatedassemblages. Consequently, the organic C/nitrogen uptake ratiodetermined stoichiometrically was significantly higher (up to10.8) when coccolithophorids were dominant than in diatom-basedor mixed-phyto-plankton assemblages. These carbon incorporationpatterns were reflected in differences in the chemical compositionof particulate matter.     

Involvement of the proteasome in the programmed cell death of NGF-deprived sympathetic neurons.

Sympathetic neurons undergo programmed cell death (PCD) upon deprivation of nerve growth factor (NGF). PCD of neurons is blocked by inhibitors of the interleukin-1beta converting enzyme (ICE)/Ced-3-like cysteine protease, indicating involvement of this class of proteases in the cell death programme. Here we demonstrate that the proteolytic activities of the proteasome are also essential in PCD of neurons. Nanomolar concentrations of several proteasome inhibitors, including the highly selective inhibitor lactacystin, not only prolonged survival of NGF-deprived neurons but also prevented processing of poly(ADP-ribose) polymerase which is known to be cleaved by an ICE/Ced-3 family member during PCD. These results demonstrate that the proteasome is a key regulator of neuronal PCD and that, within this process, it is involved upstream of proteases of the ICE/Ced-3 family. This order of events was confirmed in macrophages where lactacystin inhibited the proteolytic activation of precursor ICE and the subsequent generation of active interleukin-1beta.     

The Physiological Relevance of Na+-Coupled K+-Transport

Plant roots utilize at least two distinct pathways with high and low affinities to accumulate K+. The system for high-affinity K+ uptake, which takes place against the electrochemical K+ gradient, requires direct energization. Energization of K+ uptake via Na+ coupling has been observed in algae and was recently proposed as a mechanism for K+ uptake in wheat (Triticum aestivum L.). To investigate whether Na+ coupling has general physiological relevance in energizing K+ transport, we screened a number of species, including Arabidopsis thaliana L. Heynh. ecotype Columbia, wheat, and barley (Hordeum vulgare L.), for the presence of Na+-coupled K+ uptake. Rb+-flux analysis and electrophysiological K+-transport assays were performed in the presence and absence of Na+ and provided evidence for a coupling between K+ and Na+ transport in several aquatic species. However, all investigated terrestrial species were able to sustain growth and K+ uptake in the absence of Na+. Furthermore, the addition of Na+ was either without effect or inhibited K+ absorption. The latter characteristic was independent of growth conditions with respect to Na+ status and pH. Our results suggest that in terrestrial species Na+-coupled K+ transport has no or limited physiological relevance, whereas in certain aquatic angiosperms and algae this type of secondary transport energization plays a significant role.