Some kinetic properties of a cysteine proteinase (cruzipain) from Trypanosoma cruzi

A cysteine proteinase, purified to homogeneity from epimastigotes of Trypanosoma cruzi, was strongly inhibited by L-trans-epoxysuccinylleucylamido(4-guanidino)butane (E-64). The second-order rate constant was 20,800 M-1.s-1, and the reagent could be used for active site titration. The enzyme hydrolysed chromogenic peptides at the carboxyl Arg or Lys; it required at least one more amino acid, preferably Arg, Phe, Val or Leu, between the terminal Arg or Lys and the amino-blocking group. Enzyme activity on azocasein at pH 5.0 was increased by urea, maximal activity being attained at 2 M, and was still as active at 5 M urea as in its absence. Guanidine hydrochloride and KSCN also activated at low concentrations, but caused a strong inhibition above 2 M and 1 M, respectively. When azocasein was tested as a substrate at pH 7.0, there was no activation, and when synthetic substrates were used all chaotropic agents tested were inhibitory. The results suggest that the enzyme, for which we propose the trivial name 'cruzipain', differs in some aspects from all other cysteine proteinases described so far, although it shares several of the properties of mammalian cathepsin L.     

H-2 histocompatibility antigens of subcellular membranes of mouse liver

Purified fractions of plasma membrane, Golgi apparatus, rough endoplasmic reticulum vesicles, nuclear envelope, and mitochondria were isolated from mouse liver and the distribution of H-2 histocompatibility antigens determined by indirect radioimmunoassay before and after membrane disruptive treatments. Fractions enriched in plasma membrane (surface membrane) revealed H-2 antigens in highest concentration; disruptive treatments were not necessary to reveal H-2 antigens with surface membranes. In contrast, internal membranes did not possess H-2 antigens which were accessible to antibody. Golgi apparatus fractions or some component of these fractions (e.g. secretory vesicles) possessed the antigens but in a latent form where accessibility was provided by simple rupture of the membrane vesicles. With endoplasmic reticulum, detergent solubilization of the membranes was required before H-2 antigen could be detected. Nuclear envelope preparations contained little or no demonstrable H-2 activity. These results were confirmed by several techniques including immunoprecipitation of labelled solubilized membrane components with anti-H-2 serum and subsequent analysis in SDS-PAGE.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.     

Reaction of the anastral mitotic apparatus of endosperm cells of the plant Leucojum aestivum with antibodies to tubulin from porcine brain as revealed by immunofluorescence microscopy.

Structures binding an antibody against tubulin from porcine brain were localized in the giant anastral mitotic apparatus of endosperm cells of the monocotyledonous plant, Leucojum aestivum, by indirect immunofluorescence microscopy. Both continuous and chromosomal spindle fibers were strongly stained. Postive fluorescence was also noted in polar cap regions and, in prometaphase stages, to some extent at the fragmented nuclear envelope. Intermingling and branching of subfiber elements was frequently noted.     

IL-28A,IL-28B,and IL-29: Promising cytokines with type I interferon-like properties

IL-28A, IL-28B and IL-29 (also designated type III interferons) constitute a new subfamily within the IL-10–interferon family. They are produced by virtually any nucleated cell type, particularly dendritic cells, following viral infection or activation with bacterial components, and mediate their effects via the IL-28R1/IL-10R2 receptor complex. Although IL-28/IL-29 are closer to the IL-10-related cytokines in terms of gene structure, protein structure, and receptor usage, they display type I interferon-like anti-viral and cytostatic activities. Unlike type I interferons, the target cell populations of IL-28/IL-29 are restricted and mainly include epithelial cells and hepatocytes. These properties suggest that IL-28/IL-29 are potential therapeutic alternatives to type I interferons in terms of viral infections and tumors. This review describes the current knowledge about these cytokines.     

Scale‐down simulators for mammalian cell culture as tools to access the impact of inhomogeneities occurring in large‐scale bioreactors

During the scale‐up of a bioprocess, not all characteristics of the process can be kept constant throughout the different scales. This typically results in increased mixing times with increasing reactor volumes. The poor mixing leads in turn to the formation of concentration gradients throughout the reactor and exposes cells to varying external conditions based on their location in the bioreactor. This can affect process performance and complicate process scale‐up. Scale‐down simulators, which aim at replicating the large‐scale environment, expose the cells to changing environmental conditions. This has the potential to reveal adaptation mechanisms, which cells are using to adjust to rapidly fluctuating environmental conditions and can identify possible root causes for difficulties maintaining similar process performance at different scales. This understanding is of utmost importance in process validation. Additionally, these simulators also have the potential to be used for selecting cells, which are most robust when encountering changing extracellular conditions. The aim of this review is to summarize recent work in this interesting and promising area with the focus on mammalian bioprocesses, since microbial processes have been extensively reviewed.     

Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.     

Increasing picocyanobacteria success in shelf waters contributes to long‐term food web degradation

Continental margins are disproportionally important for global primary production, fisheries and CO₂ uptake. However, across the Northeast Atlantic shelves, there has been an ongoing summertime decline of key biota—large diatoms, dinoflagellates and copepods—that traditionally fuel higher tropic levels such as fish, sea birds and marine mammals. Here, we combine multiple time series with in situ process studies to link these declines to summer nutrient stress and increasing proportions of picophytoplankton that can comprise up to 90% of the combined pico‐ and nanophytoplankton biomass in coastal areas. Among the pico‐fraction, it is the cyanobacterium Synechococcus that flourishes when iron and nitrogen resupply to surface waters are diminished. Our field data show how traits beyond small size give Synechococcus a competitive edge over pico‐ and nanoeukaryotes. Key is their ability to grow at low irradiances near the nutricline, which is aided by their superior light‐harvesting system and high affinity to iron. However, minute size and lack of essential biomolecules (e.g. omega‐3 polyunsaturated fatty acids and sterols) render Synechococcus poor primary producers to sustain shelf sea food webs efficiently. The combination of earlier spring blooms and lower summer food quantity and quality creates an increasing period of suboptimal feeding conditions for zooplankton at a time of year when their metabolic demand is highest. We suggest that this nutrition‐related mismatch has contributed to the widespread, ~50% decline in summer copepod abundance we observe over the last 60 years. With Synechococcus clades being prominent from the tropics to the Arctic and their abundances increasing worldwide, our study informs projections of future food web dynamics in coastal and shelf areas where droughts and stratification lead to increasing nutrient starvation of surface waters.