Search

The induced pluripotent stem cell (iPSC) technology enables derivation of patient-specific pluripotent stem cells from adult somatic cells without using an embryonic cell source. Redifferentiation of iPSCs from diabetic patients into pancreatic islets will allow patient-specific disease modeling and autologous cell replacement therapy for failing islets. To date, diabetes-specific iPSCs have been generated from patients with type 1 diabetes using integrating retroviral vectors. However, vector integration into the host genome could compromise the biosafety and differentiation propensities of derived iPSCs. Although various integration-free reprogramming systems have been described, their utility to reprogram somatic cells from patients remains largely undetermined. Here, we used nonintegrating Sendai viral vectors to reprogram cells from patients with type 1 and type 2 diabetes (T2D). Sendai vector infection led to reproducible generation of genomic modification-free iPSCs (SV-iPSCs) from patients with diabetes, including an 85-year-old individual with T2D. SV-iPSCs lost the Sendai viral genome and antigens within 8–12 passages while maintaining pluripotency. Genome-wide transcriptome analysis of SV-iPSCs revealed induction of endogenous pluripotency genes and downregulation of genes involved in the oxidative stress response and the INK4/ARF pathways, including p16INK4a, p15INK4b, and p21CIP1. SV-iPSCs and iPSCs made with integrating lentiviral vectors demonstrated remarkable similarities in global gene expression profiles. Thus, the Sendai vector system facilitates reliable reprogramming of patient cells into transgene-free iPSCs, providing a pluripotent platform for personalized diagnostic and therapeutic approaches for diabetes and diabetes-associated complications.

Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.

By Northern blot analyses with DNA probes carrying 6-demethylchlortetracycline (6-DCT) biosynthetic genes from Streptomyces aureofaciens NRRL3203, a highly expressed gene (tcrC) was detected in a high titer producing mutant derived from the parental strain NRRL3203 by NTG mutagenesis. The analysis of the nucleotide sequence of the 2.8-kb BamHI fragment containing tcrC gene showed that the predicted tcrC gene product is a protein consisting of 512 amino acids. The deduced amino acid sequence had a high level identity with that of the self-defense gene (tet347) of Streptomyces rimosus, known to mediate oxytetracycline efflux. The tcrC gene-inactivated strains generated from strain NRRL3203 by gene replacement had a 90% decrease in the level of resistance to tetracycline and the antibiotic productivity when compared with the parental strain.

A 72-kDa major cell-surface protein (72K-CSP) was purified from the wash fluid of Porphyromonas gingivalis OMZ409. Using the synthetic oligonucleotide probes corresponding to the determined amino-terminal amino acid sequence of 72K-CSP, recombinant plasmid clones carrying approx. 3.4-kb KpnI-XhoI fragments in XL1-Blue libraries of P. gingivalis OMZ409 and 381 were obtained. The premature form proteins of 558 and 563 amino acids led by putative signal sequences were thought to be processed to form the mature proteins of a predicted size of 55,655 Da for strain OMZ409 and of 55,654 for strain 381. Both proteins had unusual proline-rich regions in their carboxyl-terminal regions. No homologous sequences could be found in protein databases. Examination of antigen-specific antibody responses in the serum of patients with adult periodontitis by ELISA revealed that 72K-CSP had a different immunoreactivity from that of P. gingivalis 381 fimbriae.

To clarify the mechanism of the γ-L-glutamylpeptide formation in the L-glutamic acid fermentation with Corynebacterium glutamicum, γ-glutamylpeptide synthetic activity of the intact cells and the cell extracts of the bacteria was studied. γ-L-Glu-L-Glu and other various γ-glutamylpeptides were formed by these crude preparations under high substrate amino acid concentrations without direct or indirect biological energy supplying systems. It was re-vealed that these enzyme preparations possessed strong hydrolytic activity to γ-glutamyl-peptides, and that notable amounts of these peptides could be formed by the reverse reaction of the hydrolysis. These reactions were found to be catalyzed by an enzyme. To give the evidence, the equilibrium constants of the hydrolysis of γ-L-glutamylpeptides, the substrate specificity and the distribution of the enzyme among various strains of the bacteria were studied. The partial purification of the enzyme gave sufficient proof to the idea. The mecha-nism was thought to contribute to the γ-L-GIu-L-Glu formation in the fermentation.

Five γ-glutamylpeptides were isolated from the l-glutamic acid fermentation broths using Corynebacterium glutamicum, and were identified as γ-l-glutamyl-l-gIutamic acid, γ-l-glutamyl-l-glutamine, γ-l-glutamyl-l-valine, γ-l-glutamyl-l-leucine, and γ-l-glutamyl-γ-l-glutamyl-l-glutamic acid, respectively. γ-l-Glutamyl-l-glutamine and the tripeptide are new compounds found in fermentation. It was suggested that these peptides were synthesized by the bacteria. The studies on the crystallization of l-glutamic acid revealed that γ-l-glutamyl-l-glutamic acid was the essential compound that affected the crystallization. More than 0.5 % existence of the peptide lowered the crystallization rate, and more than 1.0% existence caused the change of morphological tendency of the crystallization, α-form to β-form. The other peptides did not show any actions to the crystallization by themselves, but when they coexisted with γ-l-glutamyl-l-glutamic acid, they enhanced the action of the peptide.

The enzyme of Corynebacterium glutamicum that catalyzes the formation of various γ-L(D)-glutamylpeptides by the reversal of hydrolysis was found to be γ-glutamyltranspeptidase. The enzyme catalyzed γ-l(d)-glutamyl transfer reaction from various γ-L(D)-glutamyl- peptides including glutathione. The susceptibility of D-isomers was 17% of that of l-isomers. Many kinds of α-l-amino acids and peptides such as γ-l-GIu-l-GIu could be γ-glutamyl acceptors. These findings offered full explanation on the formation of γ-l-glutamylpeptides in the l-glutamic acid fermentation. In the supposed mechanism, γ-l-GIu-l-GIu is formed mainly by the reversal of hydrolysis from l-glutamic acid being richly produced by the bacteria. The other four minor peptides are formed by the γ-glutamyltranspeptidatkm from γ-l-GIu-l-Glu to some α-l-amino acids and to γ-l-GIu-l-GIu itself in the broths. The enzyme was found to be widely distributed among bacteria, especially in some strains of Corynebacterium, Bacillus, Erwinea, Serratia, Pseu...