PurposeIdentification of human insulin analogs' impurity with a mass shift +14Da in comparison to a parent protein.MethodsThe protein sequence variant was detected and identified with the application of peptide mapping, liquid chromatography, tandem mass spectrometric analysis, nuclear magnetic resonance spectroscopy (NMR) and Edman sequencing.ResultsThe misincorporated lysine (Lys) at asparagine (Asn) position A21 was detected in recombinant human insulin and its analogs.ConclusionsAlthough there are three asparagine residues in the insulin derivative, the misincorporation of lysine occurred only at position A21. The process involves G/U or A/U wobble base pairing.

The NMR derived translational diffusion coefficients were performed on unlabeled and uniformly labeled 13C,15N human insulin in water, both in neat, with zinc ions only, and in pharmaceutical formulation, containing only m-cresol as phenolic ligand, glycerol and zinc ions. The results show the dominant role of the pH parameter and the concentration on aggregation. The diffusion coefficient Dav was used for monitoring the overall average state of oligomeric ensemble in solution. The analysis of the experimental data of diffusion measurements, using the direct exponential curve resolution algorithm (DECRA) allows suggesting the two main components of the oligomeric ensemble. The 3D HSQC-iDOSY, (diffusion ordered HSQC) experiments performed on 13C, 15N-fully labeled insulin at the two pH values, 4 and 7.5, allow for the first time a more detailed experimental observation of individual components in the ensemble. The discussion involves earlier static and dynamic laser light scattering experiments and recent NMR derived translational diffusion results. The results bring new informations concerning the preparation of pharmaceutical formulation and in particular a role of Zn2+ ions. They also will enable better understanding and unifying the results of studies on insulin misfolding effects performed in solution by diverse physicochemical methods at different pH and concentration.

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations.

A monomer structure of a novel human insulin analog A22S-B3K-B31R (SK3R) has been characterized by NMR in water/acetonitrile solution and compared with the structure of human insulin (HIS) established in the same medium. The composition of the oligomer ensemble for neat insulins in water was qualitatively assessed by monitoring, derived from NMR experiment, translational diffusion coefficient Dix10-10m2s-1, whose value is a population averaged of individual coefficients for species in oligomeric ensemble. Nanospray ESI/MS experiment was used to establish the masses of oligomers in pharmaceutical formulation of the SK3R insulin. The pharmacodynamic data were established and compared to insulin glargine characterized by the same profile of action in diabetics. The oligomerization process of insulin during development of pharmaceutical formulation with routinely used excipients has been studied using translation diffusion coefficient Dix10-10m2s-1 established in water solution. These properties were compared with those of human insulin (HIS) which is a standard reference for novel recombinant insulins.

The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

Human insulin analogues with modified amino acid sequence may exert stronger proliferative effect and carcinogenic potential than insulin itself. The objective of the study was to assess mitogenic activity and activation of proliferation in tumorigenic and non-tumorigenic cell lines upon stimulation with a new-developed short-acting B28Lys-B29Pro human insulin (KP) in comparison to EU Pharmacopeia human insulin standard (HIS) and insulin AspB10 with strong mitogenic, cancerogenic activity.

Short acting insulin analogues are the essential drugs of an intensive antidiabetic therapy. The analogues administered before meals together with the basal long acting insulins allow to achieve pharmacokinetic profile that most closely mimics physiological secretion of endogenous insulin.

BACKGROUND:Numerous bacterial human growth hormone (hGH) expression methods under conventional fermentation and induction conditions have been described. Despite significant progress made in this area over the past several years, production of recombinant hGH by using cellular expression systems still requires further optimization. Fusion of the ubiquitin (Ub) tag to the hGH protein allowed to increase of the overall efficiency of the biosynthesis and improve the protein stability. Ub is a protein composed of 76 amino acid residues with a molecular mass of 8.6 kDa, expressed in all eukaryotes. This protein is an element of the universal protein modification system, which does not occur in bacteria, and is a useful carrier for heterologous proteins obtained through expression in Escherichia coli. Purification of Ub-fusion proteins is easier than that of unconjugated recombinant proteins, and Ub can be removed by deubiquitinating proteases (DUBs or UBPs).RESULTS AND CONCLUSION:In the present study the UBPD2C protease, a stable UBP1 analog, was produced as a recombinant protein in E. coli and used for production of recombinant human growth hormone (rhGH). hGH was expressed as a fusion protein with Ub as a tag. Our findings show that the UBPD2C protease is very effective in removing the Ub moiety from recombinant Ub-fused hGH. The described approach enables obtaining a considerable yield of rhGH in a purity required for pharmaceutical products.

A specific cucumber seed peptide (CSP) was successfully produced by liquid state fermentation with B. subtilis HYT. Then the CSP was divided into four kind molecular weight components (CSP, untreated; CSP1, MW > 10 kDa; CSP2, 6 kDa < MW < 10 kDa; CSP3, MW < 6 kDa) by ultrafiltration. Among them, the CSP3 displayed the most excellent ability to chelate with calcium (CSP3-Ca). SEM, EDS, ultra-violet spectroscopy, FTIR and Fluorescence spectroscopy were applied to distinguish the characterizations between CSP3 and CSP3-Ca chelate. The results suggested that the apparent characteristics and internal structure of CSP3 were both changed. There were abundant mental-binding sites presented in CSP3, and the COOH, OH, NH2 as well CONH might participate in the coordination with Ca2+ to form the CSP3-Ca chelate. Meanwhile, the finding showed that cucumber seeds peptide could chelate with calcium and play the role of novel carries for calcium supplement.

Analyses and visualizations by the ISSCOR method of influenza virus hemagglutinin genes of different A-subtypes revealed some rather striking temporal relationships between groups of individual gene subsets. Based on these findings we consider application of the ISSCOR-PCA method for analyses of large sets of homologous genes to be a worthwhile addition to a toolbox of genomics--allowing for a rapid diagnostics of trends, and ultimately even aiding an early warning of newly emerging epidemiological threats.

The influence of cladribine (2-chloro-2'-deoxyadenosine, CdA) on in vitro response of human acute lymphoblastic leukemia MOLT-4 cells, human histiocytic lymphoma U-937 cells, and human promyelocytic leukemia HL-60 cells, was determined using the MTT spectrophotometric and Beckman Coulter methods. Cell viability, cell volume and count were compared 24h and 48h after cladribine application at four concentrations--50 nM, 100 nM, 250 nM, and 500 nM. Different patterns of temporary changes in the viability, volume and count of pathological hematopoietic cells exposed to the action of CdA were found. The effects of CdA on MOLT-4, U-937, and HL-60 cells were dependent on the agent tested and its concentration, the time intervals after agent application, and the cell line used. The various in vitro cytotoxic activities of CdA against the three human pathological hematopoietic cell lines were shown.

Radiotherapy is an established treatment for malignant localised disease. Pancreatic cancer however seems relatively insensitive to this form of therapy.Pancreatic cancer cell lines MiaPaca-2 and Panc-1 were pre-treated with 3000 IU/ml IFNα or 100 IU/ml IFNβ followed by 0, 2, 4, or 6 Gray (Gy) irradiation. Colony forming assay was used to assess the effects on cellgrowth. To measure the surviving fraction at the clinically relevant dose of 2 Gy (SF2), cells were pre-treated with 1000–10.000 IU/ml IFNα or 50–500 IU/ml IFNβ followed by 2 Gy irradiation.The plating efficiency was 49% for MiaPaca-2 and 22% for Panc-1. MiaPaca-2 was more radiosensitive than Panc-1 (surviving fraction of 0.28 versus 0.50 at 4 Gray). The SF2 of MiaPaca-2 was 0.77 while the SF2 of Panc-1 was 0.70. The SF2 significantly decreased after pretreatment with IFNα 1000 IU/ml (p < 0.001) and IFNβ 100 IU/ml (p < 0.001) in MiaPaca-2 and with IFNα 5000 IU/ml (p < 0.001) and IFNβ 100 IU/ml (p < 0.01) in Panc-1. The sensitising enhancement ratio (SER) for IFNα 3000 IU/ml was 2.15 in MiaPaca-2 and 1.90 in Panc-1. For IFNβ 100 IU/ml the SER was 1.72 for in MiaPaca-2 and 1.51 in Panc-1.Type I interferons have radiosensitising effects in pancreatic cancer cell lines. This radiosensitising property might lead to an improved response to treatment in pancreatic cancer. Interferon β is the most promising drug due to its effect in clinically obtainable doses.

Hypoglycemic activity is one of the most important features of insulin derivatives and can be examined both in vitro and in vivo. The aim of the study was to evaluate activity of a new long-acting insulin analogue using 3 different test systems.

The senescence-accelerated mouse (SAM) is used as an animal model of senescence acceleration and age-associated disorders. SAM is derived from unexpected crosses between the AKR/J and unknown mouse strains. There are nine senescence-prone (SAMP) strains and three senescence-resistant (SAMR) strains. Although SAMP strains exhibit strain-specific and age-related pathological changes, the genes responsible for the pathologic changes in SAMP strains have not been comprehensively identified. In the present study, we evaluated sweet taste perception using the two-bottle test. We compared genotypes of the taste related gene, Tas1r3, using SAM strains and the parental AKR/J strain. The two-bottle test revealed that SAMR1 (R1), SAMP6 (P6), SAMP8 (P8), and SAMP10 (P10) mice were saccharin-preferring strains, whereas AKR/J did not prefer saccharin. All genotypes of the R1, P6, P8, and P10 strains at the polymorphic sites in Tas1r3, which is known to influence saccharin preference, were identical to those of C57BL6/J, a well-known saccharin-preferring strain, and were completely different from those of the parental AKR/J strain. These genetic alterations in SAM strains appear to arise from an unknown strain that is thought to have been crossed with AKR/J initially.