A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Characterizing the Data in Online Companion-dog Obituaries to Assess Their Usefulness as a Source of Information about Human–Animal Bonds

Online pet obituary sites host hundreds of obituaries regarding the passing of companion animals. Often composed by the owner or primary caretaker of the animal, they are a potential source of data about human–animal bonds where there were strong positive human emotions surrounding the animal at point of death. The aim of the present study was to characterize on-line pet obituaries and to evaluate their usefulness as a source of information on the human–animal bond. One hundred and thirty full obituaries of dogs were studied. Where the role of the writer could be identified, the majority of obituary writers identified themselves as a female parental figure to the dog (34.6%); however, obituaries were also written by male parental figures (7.7%) and children (5.4%). Most obituaries (60%) fell within the 100-400 words length range. Obituaries were seen to express several key concepts. For instance, dogs were described as “child-like,” “part of the family,” showing “sympathy” and/or “gratitude” to the owner, and having a “sense of humor.” For their part, writers expressed “guilt” over the dog’s death, discussed a concept of the “afterlife” and noted an “instant connection” between themselves and the dog. A high proportion of the obituaries discussed the afterlife (51%) and indicated that the dog was considered part of the family (49%). There were some significant associations between concept usage within obituaries. Dogs that were described as “child-like” were more often perceived to be in an “afterlife” and to have had an “instant connection” with obituary writers (x² = 38.08, p < 0.001). We conclude that online pet obituaries can be a valuable source of information on human feelings surrounding a companion animal death.     

Cells activated for wound repair have the potential to direct collective invasion of an epithelium

Mechanisms regulating how groups of cells are signaled to move collectively from their original site and invade surrounding matrix are poorly understood. Here we develop a clinically relevant ex vivo injury invasion model to determine whether cells involved in directing wound healing have invasive function and whether they can act as leader cells to direct movement of a wounded epithelium through a three-dimensional (3D) extracellular matrix (ECM) environment. Similar to cancer invasion, we found that the injured cells invade into the ECM as cords, involving heterotypical cell–cell interactions. Mesenchymal cells with properties of activated repair cells that typically locate to a wound edge are present in leader positions at the front of ZO-1–rich invading cords of cells, where they extend vimentin intermediate filament–enriched protrusions into the 3D ECM. Injury-induced invasion depends on both vimentin cytoskeletal function and MMP-2/9 matrix remodeling, because inhibiting either of these suppressed invasion. Potential push and pull forces at the tips of the invading cords were revealed by time-lapse imaging, which showed cells actively extending and retracting protrusions into the ECM. This 3D injury invasion model can be used to investigate mechanisms of leader cell–directed invasion and understand how mechanisms of wound healing are hijacked to cause disease.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

Invariant chain modulates HLA class II protein recycling and peptide presentation in nonprofessional antigen presenting cells

The expression of MHC class II molecules and the invariant chain (Ii) chaperone, is coordinately regulated in professional antigen presenting cells (APC). Ii facilitates class II subunit folding as well as transit and retention in mature endosomal compartments rich in antigenic peptides in these APC. Yet, in nonprofessional APC such as tumors, fibroblasts and endocrine tissues, the expression of class II subunits and Ii may be uncoupled. Studies of nonprofessional APC indicate class II molecules access antigenic peptides by distinct, but poorly defined pathways in the absence of Ii. Here, investigations demonstrate that nonprofessional APC such as human fibroblasts lacking Ii internalize antigenic peptides prior to the binding of these ligands to recycling class II molecules. By contrast, fibroblast lines expressing Ii favor exogenous peptides binding directly to cell surface class II molecules without a need for ligand internalization. Endocytosis of class II molecules was enhanced in cells lacking Ii compared with Ii-expressing APC. These results suggest enhanced reliance on the endocytic recycling pathway for functional class II presentation in nonprofessional APC.     

Slow endocytosis of the LDL receptor-related protein 1B: implications for a novel cytoplasmic tail conformation

The LDL receptor-related protein 1B (LRP1B) is a putative tumor suppressor homologous to LRP1. Both LRP1 and LRP1B contain cytoplasmic tails with several potential endocytosis motifs. Although the positions of these endocytic motifs are similar in both receptors, LRP1B is internalized at a 15-fold slower rate than LRP1. To determine whether the slow endocytosis of LRP1B is due to the utilization of an endocytosis motif other than the YATL motif used by LRP1, we tested minireceptors with mutations in each of the five potential motifs in the LRP1B tail. Only mutation of both NPXY motifs together abolished LRP1B endocytosis, suggesting that LRP1B can use either of these motifs for internalization. LRP1B contains a unique insertion of 33 amino acids not present in LRP1 that could lead to altered recognition of trafficking motifs. Surprisingly, deletion of this insertion had no effect on the endocytosis rate of LRP1B. However, replacing either half of the LRP1B tail with the corresponding LRP1 sequence markedly accelerated LRP1B endocytosis. From these data, we propose that both halves of the LRP1B cytoplasmic tail contribute to a unique global conformation, which results in less efficient recognition by endocytic adaptors and a slow endocytosis rate.     

A comparative proteomic analysis of skin secretions of the tammar wallaby (Macropus eugenii) and the wombat (Vombatus ursinus)

The secretome of the pouch skin of the model marsupial the tammar wallaby, Macropus eugenii has been investigated using techniques of two-dimensional gel electrophoresis, in-gel trypsin digestion followed by nanoliquid chromatography coupled tandem mass spectrometry (LC-MS/MS). Differences in the patterns of secreted proteins were observed in the female pouch at three stages of maturity — reproductively immature; reproductively mature and active and mature, postreproductively active. Skin from the underarm area of mature females had a markedly different secreted protein profile. The greatest diversity of proteins was seen in the mature reproductive pouch and from an opportunistic sample collected from the pouch another mature female marsupial, the common wombat, Vombatus ursinus. A total of 20 proteins were confidently identified from the pouch skin secretions of the tammar wallaby and wombats, whilst 20 proteins were tentatively identified. In all skin secretomes, globins were the most abundant proteins whilst the antimicrobial, dermcidin was detected in the wombat sample. Some proteins such as keratin and actin could be sourced to sloughed and degraded skin cells. A number of proteins were present at such low concentrations that confident identification was not possible. This was compounded by the lack of a comprehensive database of marsupial proteins which constrains the reliability of automated identification protocols.     

Drosophila klaroid encodes a SUN domain protein required for Klarsicht localization to the nuclear envelope and nuclear migration in the eye

KASH (Klarsicht/Anc-1/Syne homology) domain proteins are cytoskeleton-associated proteins localized uniquely to the outer nuclear membrane. Klarsicht is a KASH protein required for nuclear migration in differentiating cells of the Drosophila eye. The C-terminal KASH domain of Klarsicht resides in the perinuclear space, and the cytoplasmic moiety connects to the microtubule organizing center. In C. elegans and vertebrate cells, SUN (Sad1/UNC-84) domain proteins reside in the inner nuclear membrane and tether KASH proteins to the outer nuclear membrane. Is there a Drosophila SUN protein that performs a similar function, and if so, is it like Klarsicht, obviously essential for nuclear positioning only in the eye? Here, we identify Drosophila Klaroid, a SUN protein that tethers Klarsicht. klaroid loss-of-function mutants are indistinguishable phenotypically from klarsicht mutants. Remarkably, neither gene is essential for Drosophila viability or fertility, and even in klaroid klorsicht double mutants, the only obvious external morphological defect is rough eyes. In addition, we find that klaroid and klarsicht are required for nuclear migration in differentiating neurons and in non-neural cells. Finally, while perinuclear Klaroid is ubiquitous in the eye, Klarsicht expression is limited to differentiating cells and may be part of the trigger for apical nuclear migration.