Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.     

Single intra-arterial injection of mesenchymal stromal cells for treatment of steroid-refractory acute graft-versus-host disease: a pilot study

Background aimsCell therapy with mesenchymal stromal cells (MSC) has been reported recently as a promising treatment for severe acute graft-versus-host disease (GvHD).MethodsWe designed a pilot study to treat severe hepatic or gut GvHD using MSC derived from only the donor and cultured without bovine serum. Because the number of cultured MSC is smaller using this method, we planned to treat patients by intra-arterial regional administration directly to the target organs.ResultsThree patients were enrolled, and the MSC could be expanded using donor serum. There were no obvious side-effects immediately after arterial injection. The maximum response was partial in one of three patients and did not continue for more than 2 months. Idiopathic pneumonia syndrome developed in two of the three patients.ConclusionsA single local arterial MSC injection was unable to save these patients' lives and so might not be more effective than multiple systemic intravenous MSC injection. Further clinical research and additional strategies are required to develop appropriate methods for using MSC to achieve extended remission of GvHD.     

Development of wrapped liposomes: Novel liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids

Novel wrapped liposomes comprised of polyanion drug and cationic lipid complexes wrapped with neutral lipids were prepared using an efficient, innovative procedure. In this study, dextran fluorescein anionic (DFA) was used as an example of a polyanionic compound. During the process, neutral lipids accumulated around the complexes and eventually covered the complexes. The resulting liposomes were 120-140 nm in diameter and the encapsulation efficiency was up to 90%. In fetal bovine serum, DFA/cationic lipid complexes degraded rapidly but the wrapped liposomes were considerably more stable. Following intravenous administration to rats, DFA/cationic lipid complexes were rapidly eliminated whereas the wrapped liposomes exhibited a much longer blood half-life. These data suggest that DFA is located on the surface of the complexes, but DFA is present inside the wrapped liposomes. The drug-delivery properties of the wrapped liposomes established in the present study suggests that formulations based on this technology could offer important advantages for the administration of many types of drug including antisense oligonucleotides, plasmids and siRNAs which may therefore lead to improved therapeutic effectiveness of this range of drugs. The method of preparation of the wrapped liposomes is so simple that it should be straightforward to adapt to a manufacturing scale.     

A novel method for construction of gene fragment library to searching epitopes

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.     

Visualization of the post-Golgi trafficking of multiphoton photoactivated transferrin receptors

Newly synthesized membrane proteins are sorted in the trans-Golgi network (TGN) on the basis of sorting signals carried in their cytoplasmic domains and delivered to their final destinations in the secretory and endocytic pathways. Although previous studies have suggested the involvement of early endosomes in the biosynthetic pathway of transmembrane proteins, the precise trafficking routes followed by the newly synthesized plasma membrane proteins, such as transferrin receptors (TfRs), after exit from the TGN remain unclear. In this report, first, we demonstrated the advantages of photoactivating PA-GFP, a variant of the Aequorea victoria green fluorescent protein (GFP), with multiphoton laser light rather than single-photon laser light, in terms of photoactivation efficiency and spatial resolution. We then applied the multiphoton photoactivation technique to selectively photoactivate the TfR tagged with PA-GFP (PA-GFP-TfR) at the TGN, and monitored the movement of the photoactivated PA-GFP-TfR in live cells. We observed that the PA-GFP-TfR photoactivated at the TGN are transported to the Tfn(+)EEA1(+) endosomal compartments after exiting the TGN. These data support the notion that early endosomes can serve as a sorting station for not only internalized plasma membrane proteins in the endocytic pathway but also newly synthesized membrane proteins in the post-Golgi secretory pathway.     

Novel carbohydrate-binding activity of pancreatic trypsins to N-linked glycans of glycoproteins

How glycosylation affects the reactivity of proteins to trypsin is not well understood. Bovine and porcine pancreatic trypsins were discovered to bind to alpha-Man, Neu5Acalpha2,6Galbeta1,4Glc, and alpha-galactose sequences by binding studies with biotinylated sugar-polymers. Quantitative kinetic studies supported that phenylmethylsulfonyl fluoride (PMSF)-treated trypsin binds to glycolipid analogues possessing alpha-Man or alpha-NeuAc but not to those possessing beta-galactose or beta-GlcNAc residue. Enzyme-linked immunosorbent assay (ELISA) showed that trypsin binds to six kinds of biotinylated glycoproteins possessing high mannose-type and complex-type N-glycans but not to bovine submaxillary mucin, which possesses only O-glycans. Further, the binding of trypsin to glycoproteins was differentially changed by treatments with sequential exoglycosidases, endoglycosidase H, or N-glycosidase F. Quantitative kinetic studies indicated that PMSF-treated trypsin binds with bovine thyroglobulin with the affinity constant of 10(10) m(-1), which was the highest among the glycoproteins examined, and that alpha-galactosidase treatment decreased it to 10(5) m(-1). PMSF-treated trypsin bound to other glycoproteins, including ovomucoid, a trypsin inhibitor, with the affinity constants of 10(8)-10(5) mol(-1) and were markedly changed by glycosidase treatments in manners consistent with the sugar-binding specificities suggested by ELISA. Thus, the binding site for glycans was shown to be distinct from the catalytic site, allowing trypsin to function as an uncompetitive activator in the hydrolysis of a synthetic peptide substrate. Correspondingly the carbohydrate-binding activities of trypsin were unaffected by treatment with PMSF or soybean trypsin inhibitor. The results indicate the presence of an allosteric regulatory site on trypsin that sugar-specifically interacts with glycoproteins in addition to the proteolytic catalytic site.     

Regulation of IkappaB kinase (IKK) complex by IKKgamma-dependent phosphorylation of the T-loop and C terminus of IKKbeta

The mechanistic relationship of phosphorylation of the C terminus of IKKbeta with phosphorylation of its T-loop kinase domain within the IKK complex remained unclear. We investigated the regulatory role of the serine cluster residing immediately adjacent to the HLH domain and of the serines in the NEMO/IKKgamma-binding domain (NBD/gammaBD) in the C-terminal portion of IKKbeta in MEFs deficient in IKKbeta and IKKalpha and in yeast reconstitution system. We show that phosphorylation events at the C terminus of IKKbeta can be divided into autophosphorylation of the serine cluster adjacent to the HLH domain and phosphorylation of the NBD/gammaBD. Autophosphorylation of the serine cluster occurs immediately after IKK activation and requires IKKgamma. In MEFs, this autophosphorylation does not have the down-regulatory function on the IKK complex that was previously described (1). On the other hand, phosphorylation of the NBD/gammaBD regulates IKKgamma-dependent phosphorylation of the T-loop activation domain in IKKbeta and, hence, IKK complex activation. Our study suggests that, within the IKK complex, modulation of the NBD/gammaBD by IKKgamma is upstream to the T-loop phosphorylation.     

Genetic variation of the MHC DQB locus in the finless porpoise (Neophocaena phocaenoides)

The Major Histocompatibility Complex (MHC) is a large multigene coding for glycoproteins that play a key role in the initiation of immune responses in vertebrates. The exon 2 region of the MHC DQB locus was analyzed using 160 finless porpoises from 5 populations in Japanese waters. The 5 populations were based on a previous mitochondrial DNA control region analysis, which showed distinct geographical separation. Eight DQB alleles were detected, and the geographical distribution of the alleles indicated that most of them are shared among the populations. Heterozygosity of the DQB alleles in each population ranged from 0.55 to 0.78, and for all 5 populations was 0.78. Low MHC variability is not a common feature in marine mammals, but the finless porpoise populations inhabiting coastal waters had a relatively high MHC heterozygosity. Balancing selection in the MHC DQB alleles of the finless porpoise was indicated by the higher rate of nonsynonymous than synonymous substitutions for PBR; however, an excess of hetrozygotes compared to expectation was not observed. This suggests that the MHC DQB locus in the finless porpoise may have been under balancing selection for a long evolutionary time period, and is influenced by genetic drift beyond the effect of balancing selection for short time periods in small local populations.