24 research outputs found
Proteolytic rafts for improving intraparenchymal migration of minimally invasively administered hydrogel-embedded stem cells
Isolation and characterization of acetylated derivative of recombinant insulin lispro produced in Escherichia coli
PURPOSE: Insulin lispro is a rapid-acting insulin analogue produced by recombinant DNA technology. As a biosynthetic drug, the protein undergoes strict monitoring aiming for detection and characterization of impurities. The goal of this study was to isolate and identify a derivative of insulin lispro formed during biosynthesis. METHODS: For this purpose, ion exchange chromatography in combination with endoproteinase Glu-C digestion, MALDI-TOF/TOF mass spectrometry and Edman sequencing were employed. RESULTS: Ion exchange chromatography analysis of related proteins in development batches of recombinant insulin lispro revealed the existence of unknown derivative in excess of the assumed limit. Its molecular mass was 42 Da higher than the theoretical mass of Lys(B31) insulin lispro—one of the expected process-related intermediates. Endoproteinase Glu-C cleavage enabled indication of the modified peptide. Tandem mass spectrometry (MS/MS) allowed to explore the location and type of the modification. The 42 amu shift was present in the mass of y-type ions, while b-type ions were in agreement with theoretical values. It suggested that the modification is present on B31 lysine. Further inquiry revealed the presence of two diagnostic ions for lysine acetylation at m/z 143.1 and 126.1. In addition, the peptide was isolated and sequenced by Edman degradation. Standards of phenylthiohydantoin derivatives of N-ε-acetyl-L-lysine and N-ε-trimethyl-L-lysine, not available commercially, were synthesized in the laboratory. The retention time of the modified residue confirmed its identity as N-ε-acetyl-L-lysine. CONCLUSIONS: The derivative of insulin lispro formed during biosynthesis of the drug was identified to be N-ε-acetyl-L-lysine (B31) insulin lispro
Explosive Fibrillation Kinetics of Two-Chain Insulin Fragment Released upon Partial Digestion with Pepsin
Adipose-derived stromal cells seeded on dermal regeneration template improve post-burn wound reconstruction
A peptide factor secreted by Staphylococcus pseudintermedius exhibits properties of both bacteriocins and virulence factors
Staphylococcus pseudintermedius is a common commensal bacterium colonizing the skin and mucosal surfaces of household animals. However, it has recently emerged as a dangerous opportunistic pathogen, comparable to S. aureus for humans. The epidemiological situation is further complicated by the increasing number of methicillin-resistant S. pseudintermedius infections and evidence of gene transmission driving antibiotic resistance between staphylococci colonizing human and zoonotic hosts. In the present study, we describe a unique peptide, BacSp222, that possesses features characteristic of both bacteriocins and virulence factors. BacSp222 is secreted in high quantities by S. pseudintermedius strain 222 isolated from dog skin lesions. This linear, fifty-amino-acid highly cationic peptide is plasmid-encoded and does not exhibit significant sequence similarities to any other known peptides or proteins. BacSp222 kills gram-positive bacteria (at doses ranging from 0.1 to several micromol/l) but also demonstrates significant cytotoxic activities towards eukaryotic cells at slightly higher concentrations. Moreover, at nanomolar concentrations, the peptide also possesses modulatory properties, efficiently enhancing interferon gamma-induced nitric oxide release in murine macrophage-like cell lines. BacSp222 appears to be one of the first examples of multifunctional peptides that breaks the convention of splitting bacteriocins and virulence factors into two unrelated groups
Isolation and Characterization of Acetylated Derivative of Recombinant Insulin Lispro Produced in Escherichia coli
Elaboration of the semi-preparative method of obtaining of reduced insulin chains. Aplication of chemically modified insulin B-chain in cell culture.
Giant supernumerary parathyroid adenoma as a cause of persistent primary hyperparathyroid in a patient with multiglandular parathyroid disease
Cell therapy in surgical treatment of fistulas. Preliminary results
Risk of recurrence after surgical treatment of a recurrent fistula is up to 50%. It has be known that more aggressive surgical treatment is associated with a high risk of anal sphincter damage and leads to incontinence. Several studies have been designed to elaborate minimally invasive treatment of rectovaginal and anal fistulas. The properties of Adipose-derived Stem Cells (ASC) significantly enhance a natural healing potency. Here, we present our experience with combined surgical and cell therapy in the treatment of fistulas. Materials and Methods: Four patients were enrolled in our study after unsuccessful treatments in the past – patients 1-3 with rectovaginal fistulas including two women after graciloplasty, and patient 4 - a male with complex perianal fistula. Adipose tissue was obtained from subcutaneous tissue. ASCs were isolated, cultured up to 10+/-2 mln cells and injected into the walls of fistulas. Follow-up physical examination and anoscopy were performed at 1, 4, 8, and 12 weeks, 6 and 12 months after implantation. Results: Up to 8 weeks after ASC implantation, symptoms of fistulas’ tracts disappeared. At 8 weeks, in patients 1-3, communication between vaginal and rectal openings was closed and at 12-16 w. intestinal continuity was restored in patient 3 and 4. After a 6-month follow-up, the fistula tract of patient 4 was closed. Up to 12 m. after ASC implantation no recurrences or adverse events were observed. Conclusion: ASCs combined with surgical pre-treated fistula tracts were used in four patients. All of them were healed. This encouraging result needs further trials to evaluate the clinical efficiency and the cost-effectiveness ratio