Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

The yeast Saccharomyces cerevisiae has been successfully established as a commercially viable system for the production of recombinant proteins. Manipulation of chaperone gene expression has been utilized extensively to increase recombinant protein production from S. cerevisiae, focusing predominantly on the products of the protein disulfide isomerase gene PDI1 and the hsp70 gene KAR2. Here we show that the expression of the genes SIL1, LHS1, JEM1, and SCJ1, all of which are involved in regulating the ATPase cycle of Kar2p, is increased in a proprietary yeast strain, developed by several rounds of random mutagenesis and screening for increased production of recombinant human albumin (rHA). To establish whether this expression contributes to the enhanced-production phenotype, these genes were overexpressed both individually and in combination. The resultant strains showed significantly increased shake-flask production levels of rHA, granulocyte-macrophage colony-stimulating factor, and recombinant human transferrin.     

Leishmania adaptor protein-1 subunits are required for normal lysosome traffic, flagellum biogenesis, lipid homeostasis, and adaptation to temperatures encountered in the mammalian host

The adaptor protein-1 (AP-1) complex is involved in membrane transport between the Golgi apparatus and endosomes. In the protozoan parasite Leishmania mexicana mexicana, the AP-1 μ1 and σ1 subunits are not required for growth at 27°C but are essential for infectivity in the mammalian host. In this study, we have investigated the function of these AP-1 subunits in order to understand the molecular basis for this loss of virulence. The μ1 and σ1 subunits were localized to late Golgi and endosome membranes of the major parasite stages. Parasite mutants lacking either AP-1 subunit lacked obvious defects in Golgi structure, endocytosis, or exocytic transport. However, these mutants displayed reduced rates of endosome-to-lysosome transport and accumulated fragmented, sterol-rich lysosomes. Defects in flagellum biogenesis were also evident in nondividing promastigote stages, and this phenotype was exacerbated by inhibitors of sterol and sphingolipid biosynthesis. Furthermore, both AP-1 mutants were hypersensitive to elevated temperature and perturbations in membrane lipid composition. The pleiotropic requirements for AP-1 in membrane trafficking and temperature stress responses explain the loss of virulence of these mutants in the mammalian host.     

The golgin GCC88 is required for efficient retrograde transport of cargo from the early endosomes to the trans-Golgi network

Retrograde transport pathways from early/recycling endosomes to the trans-Golgi network (TGN) are poorly defined. We have investigated the role of TGN golgins in retrograde trafficking. Of the four TGN golgins, p230/golgin-245, golgin-97, GCC185, and GCC88, we show that GCC88 defines a retrograde transport pathway from early endosomes to the TGN. Depletion of GCC88 in HeLa cells by interference RNA resulted in a block in plasma membrane-TGN recycling of two cargo proteins, TGN38 and a CD8 mannose-6-phosphate receptor cytoplasmic tail fusion protein. In GCC88-depleted cells, cargo recycling was blocked in the early endosome. Depletion of GCC88 dramatically altered the TGN localization of the t-SNARE syntaxin 6, a syntaxin required for endosome to TGN transport. Furthermore, the transport block in GCC88-depleted cells was rescued by syntaxin 6 overexpression. Internalized Shiga toxin was efficiently transported from endosomes to the Golgi of GCC88-depleted cells, indicating that Shiga toxin and TGN38 are internalized by distinct retrograde transport pathways. These findings have identified an essential role for GCC88 in the localization of TGN fusion machinery for transport from early endosomes to the TGN, and they have allowed the identification of a retrograde pathway which differentially selects TGN38 and mannose-6-phosphate receptor from Shiga toxin.     

Tyrosine 311 is phosphorylated by c-Abl and promotes the apoptotic effect of PKCdelta in glioma cells

In this study we characterized the phosphorylation of tyrosine 311 and its role in the apoptotic function of PKCdelta in glioma cells. We found that c-Abl phosphorylated PKCdelta on tyrosine 311 in response to H2O2 and that this phosphorylation contributed to the apoptotic effect of H2O2. In contrast, Src, Lyn, and Yes were not involved in the phosphorylation of tyrosine 311 by H2O2. A phosphomimetic PKCdelta mutant, in which tyrosine 311 was mutated to glutamic acid (PKCdeltaY311E), induced a large degree of cell apoptosis. Overexpression of the PKCdeltaY311E mutant induced the phosphorylation of p38 and inhibition of p38 abolished the apoptotic effect of the PKCdelta mutant. These results suggest an important role of tyrosine 311 in the apoptotic function of PKCdelta and implicate c-Abl as the kinase that phosphorylates this tyrosine.     

Patterns that define the four domains conserved in known and novel isoforms of the protein import receptor Tom20

Tom20 is the master receptor for protein import into mitochondria. Analysis of motifs present in Tom20 sequences from fungi and animals found several highly conserved regions, including features of the transmembrane segment, the ligand-binding domain and functionally important flexible segments at the N terminus and the C terminus of the protein. Hidden Markov model searches of genome sequence data revealed novel isoforms of Tom20 in vertebrate and invertebrate animals. A three-dimensional comparative model of the novel type I Tom20, based on the structurally characterized type II isoform, shows important differences in the amino acid residues lining the ligand-binding groove, where the type I protein from animals is more similar to the fungal form of Tom20. Given that the two receptor types from mouse interact with the same set of precursor protein substrates, comparative analysis of the substrate-binding site provides unique insight into the mechanism of substrate recognition. No Tom20-related protein was found in genome sequence data from plants or protozoans, suggesting the receptor Tom20 evolved after the split of animals and fungi from the main lineage of eukaryotes.     

Solid‐phase synthesis and screening of a library of C‐terminal arginine peptide aldehydes against Murray Valley encephalitis virus protease

Murray Valley encephalitis virus is a member of the flavivirus group, a large family of single‐stranded RNA viruses, which cause serious disease in all regions of the world. Unfortunately, no suitable antivirals are available, and there are commercial vaccines for only three flaviviruses. The solid‐phase synthesis of a library of 400 C‐terminal arginine peptide aldehydes and their screening against Murray Valley encephalitis virus protease are demonstrated. The library was utilised to elucidate several tripeptide sequences that can be used as inhibitors in further SAR studies. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The trans-Golgi network golgin, GCC185, is required for endosome-to-Golgi transport and maintenance of Golgi structure

Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.     

Comparative floral structure and systematics of the clade of Lophopyxidaceae and Putranjivaceae (Malpighiales)

In molecular phylogenetic studies, Lophopyxidaceae and Putranjivaceae are well supported as sisters in the large rosid order Malpighiales. As the floral structure of both families is poorly known and the two families have never been compared, the present comparative study was carried out, as part of a larger project on the comparative floral structure of Malpighiales, using microtome section series and scanning electron microscopy (SEM) studies. Similar to other angiosperm clades, it appears that the structure of the ovules is a strong marker for suprafamilial relationships in Malpighiales. Both families have two collateral pendant antitropous ovules per carpel associated with obturators (as in some Euphorbiaceae s.l., to which Putranjivaceae belonged in earlier classifications). However, in contrast with Euphorbiaceae s.l., the ovules are not crassinucellar, but either incompletely tenuinucellar or only weakly crassinucellar with a long and conspicuously slender nucellus and an endothelium, and do not have a nucellar beak, but a normal micropyle, features they share with families other than Euphorbiaceae s.l. among Malpighiales. Other shared features of the two families include the following. The outer sepals tend to be smaller than the inner ones and the sepals do not protect the gynoecium in older buds. Sepals of some taxa have a single vascular trace. A short zone of synsepaly tends to be present. Stamens tend to be antesepalous in haplostemonous flowers. A short gynophore is present. The synascidiate zone extends up to above the placenta, but is restricted to the ovary in taxa with more than one carpel. The micropyle is formed by the inner integument. The ventral carpel slits extend down into the synascidiate zone as postgenitally fused furrows. The carpels have a broad dorsal band of vascular bundles in the style. The overall floral structure of the two families corroborates their sister position well and does not support the earlier association of Putranjivaceae with Euphorbiaceae s.l. or of Lophopyxidaceae with Geraniales–Sapindales–Celastrales, which rely on shared superficial floral similarities. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 404–448.     

Clear obstacles and hidden challenges: understanding recruiter perspectives in six pragmatic randomised controlled trials

Background

Recruitment of sufficient participants in an efficient manner is still widely acknowledged to be a major challenge to the mounting and completion of randomised controlled trials (RCTs). Few recruitment interventions have involved staff undertaking recruitment. This study aimed i) to understand the recruitment process from the perspective of recruiters actively recruiting RCT participants in six pragmatic RCTs, and ii) to identify opportunities for interventions to improve recruitment.

Methods

Interviews were undertaken with 72 individuals (32 doctors or RCT Chief investigators (CIs); 40 nurses/other health professionals) who were actively recruiting participants in six RCTs to explore their experiences of recruitment. The RCTs varied in scale, duration, and clinical contexts. Interviews were fully transcribed and analysed using qualitative content and thematic analytic methods derived from grounded theory. For this analysis, data were systematically extracted from each RCT and synthesised across all six RCTs to produce a detailed and nuanced understanding of the recruitment process from the perspectives of the recruiters.

Results

Recruiters readily identified organisational difficulties, fewer than expected eligible patients, and patients’ treatment preferences as the key barriers to recruitment. As they described their experiences of recruitment, several previously hidden issues related to their roles as researchers and clinicians emerged, imbued with discomfort and emotion. The synthesis across the RCTs showed that doctors were uncomfortable about aspects of patient eligibility and the effectiveness of interventions, whereas nurses were anxious about approaching potential RCT participants and conflicts between the research and their clinical responsibilities. Recruiters seemed unaware that their views contributed to recruitment difficulties. Their views were not known to RCT CIs. Training and support needs were identified for both groups of staff.

Conclusions

The synthesis showed that recruitment to these RCTs was a complex and fragile process. Clear obstacles were identified but hidden challenges related to recruiters’ roles undermined recruitment, unbeknown to RCT CIs. Qualitative research can elicit and identify the hidden challenges. Training and support are then needed for recruiters to become more comfortable with the design and principles of RCTs, so that they can engage more openly with potentially eligible participants and create a more resilient recruitment process.