Expression, purification, and molecular analysis of the Necator americanus glutathione S-transferase 1 (Na-GST-1): a production process developed for a lead candidate recombinant hookworm vaccine antigen

The enzyme Necator americanus glutathione S-transferase 1 (Na-GST-1) belongs to a unique Nu class of GSTs and is a lead candidate antigen in a bivalent human hookworm vaccine. Here we describe the expression of Na-GST-1 in the yeast Pichia pastoris at the 20 L manufacturing scale and its purification process performed by three chromatographic steps, comprised of a Q Sepharose XL anion exchange column, followed by a Butyl Sepharose HP hydrophobic affinity column and a Superdex 75 size-exclusion column. Approximately 1.5 g of recombinant protein was recovered at an overall process yield of 51%, with a purity grade of 98% and the absence of detectable host cell protein. By mass spectrometry the recombinant protein exhibits a mass of 23,676Da, which closely matches the predicted molecular mass of the protein. The expression and purification methods described here are suitable for further scale-up product development and for its use to design formulation processes suitable to generate a vaccine for clinical testing.     

PDGFRβ triggered by bFGF promotes the proliferation and migration of endothelial progenitor cells via p-ERK signalling

We have examined the effects of bFGF (basic fibroblast growth factor) on p-ERK (phosphorylated extracellular signal-regulated kinase) through PDGFRβ (platelet-derived growth factor receptor β) in the proliferation and migration of EPCs (endothelial progenitor cells). EPC migration was detected using the Transwell system. The expression of PDGFRβ mRNA and protein, total ERK and p-ERK proteins was respectively assessed by real-time PCR and Western blottings. bFGF promote the proliferation and migration of EPCs, the effects of bFGF being implemented by activating ERK signalling through the expression of PDGFRβ, whereas an anti-bFGF antibody and inhibitor of PDGF (platelet-derived growth factor) receptor kinase (AG1296) could respectively decrease the expression of PDGFRβ mRNA and protein and p-ERK protein. Total ERK protein did not change under the same experimental conditions, and an inhibitor of p-ERK (PD98059) inhibited the proliferation and migration of EPCs. The findings strongly suggest that a PDGFRβ/p-ERK signalling pathway triggered by bFGF plays an important role in the proliferation and migration of EPCs.     

New insights into the important roles of tumor cell-intrinsic PD-1

PD-1 (Programmed cell death protein-1) is mainly expressed in various immune cells, while its ligands PD-L1/PD-L2 (Programmed death ligand-1/Programmed death ligand-2) are mostly expressed in tumor cells. Generally, the binding of PD-L1/PD-L2 and PD-1 could lead to the tumor immune evasion. However, some recent studies showed that PD-1 could also be expressed in tumor cells and could activate mTOR (Mammalian Target of Rapamycin) or Hippo signaling pathway, therefore facilitating tumor proliferation independent of the immune system. While there was evidence that tumor cell-intrinsic PD-1 inhibited the activation of AKT and ERK1/2 pathways, thereby inhibiting tumor cell growth. Based on TCGA and CCLE database, we found that PD-1 was expressed in a variety of tumors and was associated with patient''s prognosis. Besides, we found that PD-1 may be involved in many carcinogenic signaling pathway on the basis of PD-1 gene enrichment analysis of cancer tissues and cancer cells. Our understanding of the tumor cell-intrinsic PD-1 function is still limited. This review is aimed at elaborating the potential effects of tumor cell-intrinsic PD-1 on carcinogenesis, providing a novel insight into the effects of anti-PD-1/PD-L1 immunotherapy, and helping to open a major epoch of combination therapy.     

Cloning and analysis of peptidoglycan recognition protein‐LC and immune deficiency from the diamondback moth,Plutella xylostella

Peptidoglycan (PGN) exists in both Gram‐negative and Gram‐positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP‐LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP‐LC and IMD (PxPGRP‐LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP‐LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP‐LC and IMD homologs. PxPGRP‐LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP‐LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full‐length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP‐LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella.     

Meta‐Analysis of the Association of the Trp64Arg Polymorphism in the β3 Adrenergic Receptor with Insulin Resistance

Objective: To clarify the possible association between the Trp64Arg polymorphism and insulin resistance (IR). Research Methods and Procedures: Articles evaluating the effect of the Trp64Arg polymorphism on IR were identified on the MEDLINE and PubMed databases from 1995 to February, 2004. After extraction of relevant data, main and subgroup meta‐analyses were performed to assess the differences in IR indices between Trp/Trp and Trp/Arg genotypes. Results: Forty eligible papers containing 56 subgroups were included in this meta‐analysis. Among a total of 12, 805 subjects, 21.9% had Trp64Arg mutation: 20.8%, heterozygotes and 1.1%, homozygotes. Significant associations were found between this mutation and some indices of IR. The weighted mean difference in fasting insulin, 120‐minute insulin level after oral glucose tolerance test, and homeostasis model assessment between Arg64 and Trp64 was 0.23 [95% confidence interval (CI), 0.05 to 0.42] pM, 0.89 (95% CI, 0.30 to 1.48) pM, and 0.55 (95% CI, 0.14 to 0.96), respectively. Subgroup analysis further indicated that this significant association existed only in the Asian population (p < 0.01) and in the obese (p = 0.02) and diabetes subgroups (p = 0.03). Discussion: Numerous studies have been conducted to examine the relationship between the β3‐adrenergic receptor Trp64Arg polymorphism and components of IR syndrome. However, the results have been inconsistent and have led to controversy about whether this polymorphism is associated with these clinical features. The current meta‐analysis demonstrated the moderate effects of the Trp64Arg polymorphism on IR in the Asian population and in obese and diabetic subgroups.     

Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue

Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 A increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.     

An intron loss of Dfak gene in species of the Drosophila melanogaster subgroup and phylogenetic analysis

Drosophila focal adhesion kinase (Dfak) gene is a single-copy nuclear gene. Previous study revealed that Drosophila melanogaster and Drosophila simulans had lost an intron precisely within the tyrosine kinase (TyK) domain of this gene. However, this did not happen in several other Drosophila species, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila jambulina, Drosophila prostipennis, Drosophila takahashii, and Drosophila pseudoobscura. In the current study, homologous sequences of Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were amplified by polymerase chain reaction, and further sequencing analysis indicated that these species were missing a TyK domain intron, indicating they were closely related. The relationship of the D. melanogaster species group was reconstructed using TyK domain nucleotide sequences. The resulting phylogenetic tree revealed that these 8 species were the most related species in the melanogaster group. These results strongly support previously proposed classifications based on morphological and molecular data.