Enabling Low Cost Biopharmaceuticals: A Systematic Approach to Delete Proteases from a Well-Known Protein Production Host Trichoderma reesei

The filamentous fungus Trichoderma reesei has tremendous capability to secrete proteins. Therefore, it would be an excellent host for producing high levels of therapeutic proteins at low cost. Developing a filamentous fungus to produce sensitive therapeutic proteins requires that protease secretion is drastically reduced. We have identified 13 major secreted proteases that are related to degradation of therapeutic antibodies, interferon alpha 2b, and insulin like growth factor. The major proteases observed were aspartic, glutamic, subtilisin-like, and trypsin-like proteases. The seven most problematic proteases were sequentially removed from a strain to develop it for producing therapeutic proteins. After this the protease activity in the supernatant was dramatically reduced down to 4% of the original level based upon a casein substrate. When antibody was incubated in the six protease deletion strain supernatant, the heavy chain remained fully intact and no degradation products were observed. Interferon alpha 2b and insulin like growth factor were less stable in the same supernatant, but full length proteins remained when incubated overnight, in contrast to the original strain. As additional benefits, the multiple protease deletions have led to faster strain growth and higher levels of total protein in the culture supernatant.     

Volatile emissions and phenolic compound concentrations along a vertical profile of <Emphasis Type="Italic">Populus nigra</Emphasis> leaves exposed to realistic ozone concentrations

Plants are exposed to increasing levels of tropospheric ozone concentrations. This pollutant penetrates in leaves through stomata and quickly reacts inside leaves, thus making plants valuable ozone sinks, but at the same time triggers oxidation processes which lead to leaf injuries. To counteract these negative effects, plants produce an array of antioxidants which react with ozone and reactive molecules which ozone generates in the leaf tissues. In this study, we measured the effect of an ozone concentration which is likely to be attained in many areas of the world in the near future (80 ppb) on leaves of the vertical profile of the widespread agroforestry species Populus nigra. Changes in (1) physiological parameters (photosynthesis and stomatal conductance), (2) ozone uptake, (3) emission of volatile organic compounds (VOCs, i.e. isoprene, methanol and other oxygenated compounds), (4) concentration of antioxidant surface compounds, and (5) concentration of phenolic compounds were assessed. The aim was to assess whether the defensive pathways leading to isoprenoids and phenolics formation were induced when a moderate and chronic increment of ozone is not able to damage photosynthesis. No visual injuries and minor changes in physiology and ozone uptake were observed. The emission of isoprene and oxygenated six-carbon (C6) volatiles were inhibited by ozone, whereas methanol emission was increased, especially in developing leaves. We interpret these results as suggesting an ontogenetic shift in ozone-treated leaves, leading to a slower development and a faster senescence. Most surface and phenolic compounds showed a declining trend in concentration from the youngest to the fully expanded leaves. Ozone reduced the concentrations of chlorogenic acid derivatives at the leaf surface, whereas in total leaf extracts a metabolic shift towards few phenolics with higher antioxidant capacity was observed.     

A trophic pathway from biogenic methane supports fish biomass in a temperate lake ecosystem

Although some primary consumers such as chironomid larvae are known to exploit methane‐derived carbon via microbial consortia within aquatic food webs, few studies have traced the onward transfer of such carbon to their predators. The ruffe Gymnocephalus cernuus is a widespread benthivorous fish which feeds predominantly on chironomid larvae and is well adapted for foraging at lower depths than other percids. Therefore, any transfer of methanogenic carbon to higher trophic levels might be particularly evident in ruffe. We sampled ruffe and chironomid larvae from the littoral, sub‐littoral and profundal areas of Jyväsjärvi, Finland, a lake which has previously been shown to contain chironomid larvae exhibiting the very low stable carbon isotope ratios indicative of methane exploitation. A combination of fish gut content examination and stable isotope analysis was used to determine trophic linkages between fish and their putative prey. Irrespective of the depth from which the ruffe were caught, their diet was dominated by chironomids and pupae although the proportions of taxa changed. Zooplankton made a negligible contribution to ruffe diet. A progressive decrease in δ¹³C and δ¹⁵N values with increasing water column depth was observed for both chironomid larvae and ruffe, but not for other species of benthivorous fish. Furthermore, ruffe feeding at greater depths were significantly larger than those feeding in the littoral, suggesting an ontogenetic shift in habitat use, rather than diet, as chironomids remained the predominant prey item. The outputs from isotope mixing models suggested that the incorporation of methane‐derived carbon to larval chironomid biomass through feeding on methanotrophic bacteria increased at greater depth, varying from 0% in the littoral to 28% in the profundal. Using these outputs and the proportions of littoral, sub‐littoral or profundal chironomids contributing to ruffe biomass, we estimated that 17% of ruffe biomass in this lake was ultimately derived from chemoautotrophic sources. Methanogenic carbon thus supports considerable production of higher trophic levels in lakes.     

Real‐time monitoring of herbivore induced volatile emissions in the field

When plants are damaged by herbivorous insects they emit a blend of volatile organic compounds (VOCs) which include a range or terpenoids and green leaf volatiles (GLVs) formed via different metabolic pathways. The precise timing of these emissions upon the onset of herbivore feeding has not been fully elucidated, and the information that is available has been mainly obtained through laboratory based studies. We investigated emissions of VOCs from Populus tremula L. ×P. tremuloides Michx. during the first 20 h of feeding by Epirrita autumnata (autumnal moth) larvae in a field site. The study was conducted using Proton Transfer Reaction‐Mass Spectrometry (PTR‐MS) to measure emissions online, with samples collected for subsequent analysis by complementary gas chromatography‐mass spectrometry for purposes of compound identification. GLV emission peaks occurred sporadically from the outset, indicating herbivore activity, while terpene emissions were induced within 16 h. We present data detailing the patterns of monoterpene (MT), GLV and sesquiterpene (SQT) emissions during the early stages of herbivore feeding showing diurnal MT and SQT emission that is correlated more with temperature than light. Peculiarities in the timing of SQT emissions prompted us to conduct a thorough characterization of the equipment used to collect VOCs and thus corroborate the accuracy of results. A laboratory based analysis of the throughput of known GLV, MT and SQT standards at different temperatures was made with PTR‐MS. Enclosure temperatures of 12, 20 and 25°C had little influence on the response time for dynamic measurements of a GLV or MT. However, there was a clear effect on SQT measurements. Elucidation of emission patterns in real‐time is dependent upon the dynamics of cuvettes at different temperatures.     

Amyloid-beta 1-42 induced endocytosis and clusterin/apoJ protein accumulation in cultured human astrocytes

Recent studies indicate that astrocytes may be the primary target of secreted amyloid-beta 1-42 peptides, with the neurotoxicity representing a secondary response to astrocytic stress. Our purpose was to clarify the astrocytic stress response induced by amyloid-beta peptides in human and rat astrocytes. Human amyloid-beta 1-42 peptides and fibrils induced the appearance of cytoplasmic vacuoles in normal human astrocytes (NHA) and CCFsttg1 astrocytoma cells. Vacuoles appeared 9-12h after the amyloid-beta exposure and remained present for several days. Rat primary neonatal astrocytes showed similar but less prominent vacuolar response. Human amyloid-beta peptides 1-16, 1-28, 10-20, 17-21 and 25-35 did not cause vacuole formation. Electron microscopic observations revealed large endocytic vacuoles containing fibrillar amyloid material. Stress marker analysis did not show any increase in protein levels of HSP70, HSP90, GRP78 and GRP94. However, the protein level of clusterin/apoJ, a secreted chaperone, was strongly increased both in NHA and CCFsttg1 astrocytes. Endocytic response associated with the accumulation of clusterin/apoJ protein suggests that clusterin/apoJ has a role in the clearance of amyloid-beta peptides.     

Structure of three tandem filamin domains reveals auto-inhibition of ligand binding

Human filamins are large actin-crosslinking proteins composed of an N-terminal actin-binding domain followed by 24 Ig-like domains (IgFLNs), which interact with numerous transmembrane receptors and cytosolic signaling proteins. Here we report the 2.5 A resolution structure of a three-domain fragment of human filamin A (IgFLNa19-21). The structure reveals an unexpected domain arrangement, with IgFLNa20 partially unfolded bringing IgFLNa21 into close proximity to IgFLNa19. Notably the N-terminus of IgFLNa20 forms a beta-strand that associates with the CD face of IgFLNa21 and occupies the binding site for integrin adhesion receptors. Disruption of this IgFLNa20-IgFLNa21 interaction enhances filamin binding to integrin beta-tails. Structural and functional analysis of other IgFLN domains suggests that auto-inhibition by adjacent IgFLN domains may be a general mechanism controlling filamin-ligand interactions. This can explain the increased integrin binding of filamin splice variants and provides a mechanism by which ligand binding might impact filamin structure.     

Conjugation of oligosaccharides by reductive amination to amine modified chondroitin oligomer and γ-cyclodextrin

Carbohydrates present on cell surfaces participate in numerous biological recognition phenomena including cell–cell interactions, cancer metastasis and pathogen invasion. Therefore, synthetic carbohydrates have a potential to act as pharmaceutical substances for treatment of various pathological phenomena by inhibiting specifically the interaction between cell surface carbohydrates and their protein receptors (lectins). However, the inherently low affinity of carbohydrate-protein interactions has often been an obstacle for successful generation of carbohydrate based pharmaceuticals. Multivalent glycoconjugates, i.e. structures carrying several copies of the active carbohydrate sequence in a carrier molecule, have been constructed to overcome this problem. Here we present two novel types of multivalent carbohydrate conjugates based on chondroitin oligomer and cyclodextrin carriers. These carriers were modified to express primary amino groups, and oligosaccharides were then bound to carrier molecules by reductive amination. Multivalent conjugates were produced using the human milk type oligosaccharides LNDFH I (Lewis-b hexasaccharide), LNnT, and GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc.