Insect cell culture for industrial production of recombinant proteins

Insect cells used in conjunction with the baculovirus expression vector system (BEVS) are gaining ground rapidly as a platform for recombinant protein production. Insect cells present several comparative advantages to mammalian cells, such as ease of culture, higher tolerance to osmolality and by-product concentration and higher expression levels when infected with a recombinant baculovirus. Here we review some of the recent developments in protein expression by insect cells and their potential application in large-scale culture. Our current knowledge of insect cell metabolism is summarised and emphasis is placed on elements useful in the rational design of serum-free media. The culture of insect cells in the absence of serum is reaching maturity, and promising serum substitutes (hydrolysates, new growth and production-enhancing factors) are being evaluated. Proteolysis is a problem of the BEVS system due to its lytic nature, and can, therefore, be a critical issue in insect cell bioprocessing. Several cell- or baculovirus proteases are involved in degradation events during protein production by insect cells. Methods for proteolysis control, the optimal inhibitors and culture and storage conditions which affect proteolysis are discussed. Finally, engineering issues related to high-density culture (new bioreactor types, gas exchange, feeding strategies) are addressed in view of their relevance to large-scale culture.     

Ring-based versus disc-based separation of spatial scales: a case study on the impact of arable land proportions on invertebrates in freshwater streams

The impact of different land-use types on species is traditionally estimated by correlating landscape proportions recorded in buffer areas around focal points with species data observed at these sites. If a high proportion of a specific land-use type exists within a small radius, it will be accumulated in larger buffers and may confound the interpretation at larger scales. We sampled freshwater invertebrates in ten streams using cages with artificial substrate and compared the effects of arable land proportions calculated in disc-shaped buffers of increasing radius versus areas calculated from non-overlapping rings of increasing radius. We hypothesize that (1) the accumulative disc-based approach leads to confounding effects across increasing buffer size and that (2) the use of ring-based methods facilitates the identification of relevant scales for conservation measures. The abundance of crustaceans showed a positive relationship with arable land proportions, but Plecoptera abundance and the taxonomic richness of Ephemeroptera and Plecoptera decreased with increasing arable land proportions in the surrounding landscape. Our results further support the presence of confounding effects in disc-based analyses, as correlations between arable land proportions and Crustacea, or Plecoptera, respectively, were affected by the accumulation of small-scale area proportions. The distance at which arable land proportions significantly affected benthic fauna in freshwater streams was consistently shorter if calculated from rings rather than from discs. Although an a priori definition of ring width introduces new challenges, a combined use of disc- and ring-based techniques for the estimation of land-use effects may substantially improve the realization of conservation and protection measures in terrestrial and aquatic systems.     

Analysis of regenerating hepatic cell replication in vivo by differential chromatid staining

The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.     

Sodium phenylbutyrate controls neuroinflammatory and antioxidant activities and protects dopaminergic neurons in mouse models of Parkinson's disease

Neuroinflammation and oxidative stress underlie the pathogenesis of various neurodegenerative disorders. Here we demonstrate that sodium phenylbutyrate (NaPB), an FDA-approved therapy for reducing plasma ammonia and glutamine in urea cycle disorders, can suppress both proinflammatory molecules and reactive oxygen species (ROS) in activated glial cells. Interestingly, NaPB also decreased the level of cholesterol but involved only intermediates, not the end product of cholesterol biosynthesis pathway for these functions. While inhibitors of both geranylgeranyl transferase (GGTI) and farnesyl transferase (FTI) inhibited the activation of NF-κB, inhibitor of GGTI, but not FTI, suppressed the production of ROS. Accordingly, a dominant-negative mutant of p21(rac), but not p21(ras), attenuated the production of ROS from activated microglia. Inhibition of both p21(ras) and p21(rac) activation by NaPB in microglial cells suggests that NaPB exerts anti-inflammatory and antioxidative effects via inhibition of these small G proteins. Consistently, we found activation of both p21(ras) and p21(rac)in vivo in the substantia nigra of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Oral administration of NaPB reduced nigral activation of p21(ras) and p21(rac), protected nigral reduced glutathione, attenuated nigral activation of NF-κB, inhibited nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Consistently, FTI and GGTI also protected nigrostriata in MPTP-intoxicated mice. Furthermore, NaPB also halted the disease progression in a chronic MPTP mouse model. These results identify novel mode of action of NaPB and suggest that NaPB may be of therapeutic benefit for neurodegenerative disorders.     

Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater

The glycosyltransferase SnogD from Streptomyces?nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6?? resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in?vivo and in?vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction. Database The atomic coordinates and structure factors have been deposited with the RCSB Protein Data Bank under accession numbers 4AMB, 4AMG and 4AN4 Structured digital abstract ? snogD?and?snogD?bind?by?x-ray crystallography?(View Interaction:?1,?2).     

Shared Protein Complex Subunits Contribute to Explaining Disrupted Co-occurrence

The gene composition of present-day genomes has been shaped by a complicated evolutionary history, resulting in diverse distributions of genes across genomes. The pattern of presence and absence of a gene in different genomes is called its phylogenetic profile. It has been shown that proteins whose encoding genes have highly similar profiles tend to be functionally related: As these genes were gained and lost together, their encoded proteins can probably only perform their full function if both are present. However, a large proportion of genes encoding interacting proteins do not have matching profiles. In this study, we analysed one possible reason for this, namely that phylogenetic profiles can be affected by multi-functional proteins such as shared subunits of two or more protein complexes. We found that by considering triplets of proteins, of which one protein is multi-functional, a large fraction of disturbed co-occurrence patterns can be explained.     

Hypomorphic mutations in the gene encoding a key Fanconi anemia protein, FANCD2, sustain a significant group of FA-D2 patients with severe phenotype

FANCD2 is an evolutionarily conserved Fanconi anemia (FA) gene that plays a key role in DNA double-strand-type damage responses. Using complementation assays and immunoblotting, a consortium of American and European groups assigned 29 patients with FA from 23 families and 4 additional unrelated patients to complementation group FA-D2. This amounts to 3%-6% of FA-affected patients registered in various data sets. Malformations are frequent in FA-D2 patients, and hematological manifestations appear earlier and progress more rapidly when compared with all other patients combined (FA-non-D2) in the International Fanconi Anemia Registry. FANCD2 is flanked by two pseudogenes. Mutation analysis revealed the expected total of 66 mutated alleles, 34 of which result in aberrant splicing patterns. Many mutations are recurrent and have ethnic associations and shared allelic haplotypes. There were no biallelic null mutations; residual FANCD2 protein of both isotypes was observed in all available patient cell lines. These analyses suggest that, unlike the knockout mouse model, total absence of FANCD2 does not exist in FA-D2 patients, because of constraints on viable combinations of FANCD2 mutations. Although hypomorphic mutations arie involved, clinically, these patients have a relatively severe form of FA.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.