Total Synthesis of desB30 Insulin Analogues by Biomimetic Folding of Single‐Chain Precursors

Insulin is a peptide hormone consisting of 51 amino acids in two chains with three disulfide bridges. Human insulin and various analogues are used for the treatment of diabetes and are produced recombinantly at ton scale. Herein, we report the chemical synthesis of insulin by the step‐wise, Fmoc‐based, solid‐phase synthesis of single‐chain precursors with solubilising extensions, which under redox conditions, spontaneously fold with the correct pairing of the three disulfide bridges. The folded, single‐chain, insulin precursors can be transformed into bioactive two‐chain desB30 insulin by the simultaneous removal of the solubilising extension (4–5 residues) and the chain‐bridging C‐peptide (3–5 residues) by employing Achromobacter lyticus protease—a process well‐known from the yeast‐based recombinant production of insulin. The overall yields of synthetic insulins were as much as 6 %, and the synthetic process was straightforward and not labour intensive.     

Crystallization of Enantiomerically Pure Proteins from Quasi‐Racemic Mixtures: Structure Determination by X‐Ray Diffraction of Isotope‐Labeled Ester Insulin and Human Insulin

As a part of a program aimed towards the study of the dynamics of human insulin‐protein dimer formation using two‐dimensional infrared spectroscopy, we used total chemical synthesis to prepare stable isotope labeled [(1‐¹³C=¹⁸O)Phe^B24)] human insulin, via [(1‐¹³C=¹⁸O)Phe^B24)] ester insulin as a key intermediate product that facilitates folding of the synthetic protein molecule (see preceding article). Here, we describe the crystal structure of the synthetic isotope‐labeled ester insulin intermediate and the product synthetic human insulin. Additionally, we present our observations on hexamer formation with these two proteins in the absence of phenol derivatives and/or Zn metal ions. We also describe and discuss the fractional crystallization of quasi‐racemic protein mixtures containing each of these two synthetic proteins.     

Synthesis of water‐insoluble functional copolymers containing amide,amine, and carboxylic acid groups and their metal‐ion‐uptake properties

The crosslinked poly[N‐(3‐dimethylamino)propylmethacrylamide] [P(NDAPA)] and poly[N‐(3‐dimethylamino)propylmethacrylamide‐co‐acrylic acid] [P(NDAPA‐co‐AA)] were synthesized by radical polymerization. The resins were completely insoluble in water. The metal‐ion‐uptake properties were studied by a batch equilibrium procedure for the following metal ions: silver(I), copper(II), cadmium(II), zinc(II), lead(II), mercury(II), chromium(III), and aluminum(III). The P(NDAPA‐co‐AA) resin showed a lower metal‐ion affinity than P(NDAPA), except for Hg(II), which was retained at 71% at pH 2. At pH 5, the resin showed a higher affinity for Pb(II) (80%) and Cu(II) (60%), but its affinity was very low for Zn(II) and Cr(III). The polymer ligand–metal‐ion equilibrium was achieved during the first 20 min. By changing the pH, we found it possible to remove between 60 and 70% of Cd(II) and Zn(II) ions with (1M, 4M) HClO₄ and (1M, 4M) HNO₃. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102:5232–5239, 2006     

胰岛素样生长因子I(IGF-I)基因的克隆及分泌表达

目的　构建原核分泌型表达载体并高效表达胰岛素样生长因子I(IGF I)。方法　构建出 2种分泌型表达载体 ,命名为pCSA和pCST ,并将胰岛素样生长因子I(IGF I)基因插入pCSA和pCST中 ,转化E .coliW3110 ,经筛选获得工程菌pCSA/IGF I/W3110和pCST/IGF 1/W3110 ,并进行表达。结果　经SDS PAGE检测 ,在相对分子质量 76 0 0处有明显表达带 ,Westernblot表明重组蛋白具有IGF I的抗原性。结论　原核分泌型IGF I的高效表达 ,为进一步研究人IGF I的功能和生物学特性打下基础     

Flaxseed Oil Supplementation Improve Gene Expression Levels of PPAR-γ, LP(a), IL-1 and TNF-α in Type 2 Diabetic Patients with Coronary Heart Disease

This study was carried out to determine the effects of flaxseed oil administration on gene expression levels related to insulin, lipid and inflammation in overweight diabetic patients with coronary heart disease (CHD). This randomized double‐blind, placebo‐controlled trial was conducted among 60 diabetic patients with CHD. Subjects were randomly allocated into two groups to intake either 1000 mg n‐3 fatty acid from flaxseed oil containing 400 mg α‐Linolenic acid [ALA (18:3n‐3)] (n = 30) or placebo (n = 30) twice a day for 12 weeks. Gene expression related to insulin, lipid and inflammation were quantified in peripheral blood mononuclear cells (PBMC) of diabetic patients with CHD with RT‐PCR method. Results of RT‐PCR demonstrated that after the 12‐week intervention, compared with the placebo, flaxseed oil supplementation could up‐regulate gene expression of peroxisome proliferator‐activated receptor gamma (PPAR‐γ) (P = 0.02) in PBMC of diabetic patients with CHD. In addition, compared with the placebo, taking flaxseed oil supplements down‐regulated gene expression levels of lipoprotein(a) [LP(a)] (P = 0.001), interleukin‐1 (IL‐1) (P = 0.001) and tumor necrosis factor alpha (TNF‐α) (P = 0.02) in PBMC of diabetic patients with CHD. We did not observe any significant effect of flaxseed oil supplementation on gene expression levels of low‐density lipoprotein receptor (LDLR), IL‐8 and transforming growth factor beta (TGF‐β) in PBMC of diabetic patients with CHD. Overall, flaxseed oil supplementation for 12 weeks in diabetic patients with CHD significantly improved gene expression levels of PPAR‐γ, LP(a), IL‐1 and TNF‐α, but did not influence LDLR, IL‐8 and TGF‐β.     

Virtual Screening of Specific Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors from the National Cancer Institute (NCI) Molecular Database

Insulin-like growth factor 1 receptor (IGF1R) is an attractive drug target for cancer therapy and research on IGF1R inhibitors has had success in clinical trials. A particular challenge in the development of specific IGF1R inhibitors is interference from insulin receptor (IR), which has a nearly identical sequence. A few potent inhibitors that are selective for IGF1R have been discovered experimentally with the aid of computational methods. However, studies on the rapid identification of IGF1R-selective inhibitors using virtual screening and confidence-level inspections of ligands that show different interactions with IGF1R and IR in docking analysis are rare. In this study, we established virtual screening and binding-mode prediction workflows based on benchmark results of IGF1R and several kinase receptors with IGF1R-like structures. We used comprehensive analysis of the known complexes of IGF1R and IR with their binding ligands to screen specific IGF1R inhibitors. Using these workflows, 17 of 139,735 compounds in the NCI (National Cancer Institute) database were identified as potential specific inhibitors of IGF1R. Calculations of the potential of mean force (PMF) with GROMACS were further conducted for three of the identified compounds to assess their binding affinity differences towards IGF1R and IR.     

Polyelectrolyte‐assisted removal of metal ions with ultrafiltration

The water‐soluble polymers poly(styrene sulfonic acid‐co‐maleic acid) and poly(acrylic acid‐co‐maleic acid) were investigated with respect to their metal‐ion‐binding ability with ultrafiltration. The studied metal ions included Ag(I), Cu(II), Ni(II), Co(II), Ca(II), Mg(II), Pb(II), Cd(II), Zn(II), Al(III), and Cr(III) ions. The retention properties of the polyelectrolytes for the metal ions depended strongly on the ligand type. As for the carboxylate ligands, with increasing concentration and pH, the metal‐binding affinity increased. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 95: 1091–1099, 2005     

Insulin release dynamics from poly(diethylaminoethyl methacrylate) hydrogel systems

Novel glucose‐sensitive systems for the release of insulin from poly(diethylaminoethyl methacrylate) (PDEAEM) microparticles and nanoparticles decorated with glucose oxidase and catalase enzymes have been developed. The effect of polymer composition and loading conditions on the insulin loading efficiency and release was studied. The optimal conditions for loading insulin into PDEAEM microparticles were found to be at a loading pH of 5.6, particle to insulin mass ratio of 7:1, a concentration of 1.0 mg/mL insulin, and a collapsing pH of approximately 9.5. Microparticles exhibited a responsive (pH) or intelligent (glucose) release of insulin from a stimulus. Microparticles that had a nominal crosslinking ratio of 10% released a third of the insulin payload after a single stimulus, compared to nearly 70% for microparticles with a 3% crosslinking ratio. PDEAEM microparticles of 150 μm diameter showed promise as components of a system of automated, intelligent delivery method for insulin to type I diabetics. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3578–3585, 2013     

Resveratrol Affects Insulin Signaling in Type 2 Diabetic Goto-Kakizaki Rats

Resveratrol is a biologically active diphenolic compound exerting multiple beneficial effects in the organism, including anti-diabetic properties. This action is, however, not fully elucidated. In the present study, we examined effects of resveratrol on some parameters related to insulin signaling, and also on diabetes-associated dysregulation in Goto-Kakizaki (GK) rats with congenital type 2 diabetes. Resveratrol was given at the dose of 20 mg/kg b.w. for 10 weeks. It was shown that the expression and phosphorylation levels of insulin receptor in the skeletal muscle of GK rats were significantly decreased, compared with control animals. However, these changes were totally prevented by resveratrol. Liver expression of the insulin receptor was also reduced, but in this case, resveratrol was ineffective. Resveratrol was also demonstrated to significantly influence parameters of insulin binding (dissociation constant and binding capacity) in the skeletal muscle and liver. Moreover, it was shown that the expression levels of proteins related to intracellular glucose transport (GLUT4 and TUG) in adipose tissue of GK rats were significantly decreased. However, treatment with resveratrol completely abolished these changes. Resveratrol was found to induce normalization of TUG expression in the skeletal muscle. Blood levels of insulin and GIP were elevated, whereas proinsulin and GLP-1 diminished in GK rats. However, concentrations of these hormones were not affected by resveratrol. These results indicate that resveratrol partially ameliorates diabetes-associated dysregulation in GK rats. The most relevant finding covers the normalization of the insulin receptor expression in the skeletal muscle and also GLUT4 and TUG in adipose tissue.     

Deep Resequencing of 9 Candidate Genes Identifies a Role for ARAP1 and IGF2BP2 in Modulating Insulin Secretion Adjusted for Insulin Resistance in Obese Southern Europeans

Type 2 diabetes is characterized by impairment in insulin secretion, with an established genetic contribution. We aimed to evaluate common and low-frequency (1–5%) variants in nine genes strongly associated with insulin secretion by targeted sequencing in subjects selected from the extremes of insulin release measured by the disposition index. Collapsing data by gene and/or function, the association between disposition index and nonsense variants were significant, also after adjustment for confounding factors (OR = 0.25, 95% CI = 0.11–0.59, p = 0.001). Evaluating variants individually, three novel variants in ARAP1, IGF2BP2 and GCK, out of eight reaching significance singularly, remained associated after adjustment. Constructing a genetic risk model combining the effects of the three variants, only carriers of the ARAP1 and IGF2BP2 variants were significantly associated with a reduced probability to be in the lower, worst, extreme of insulin secretion (OR = 0.223, 95% CI = 0.105–0.473, p < 0.001). Observing a high number of normal glucose tolerance between carriers, a regression posthoc analysis was performed. Carriers of genetic risk model variants had higher probability to be normoglycemic, also after adjustment (OR = 2.411, 95% CI = 1.136–5.116, p = 0.022). Thus, in our southern European cohort, nonsense variants in all nine candidate genes showed association with better insulin secretion adjusted for insulin resistance, and we established the role of ARAP1 and IGF2BP2 in modulating insulin secretion.     

Metal ion binding capability of the water‐soluble poly(vinyl phosphonic acid) for mono‐, di‐, and trivalent cations

A water‐soluble polymer containing phosphonic acid groups was investigated as a polychelatogen by using the liquid‐phase polymer‐based retention technique (LPR) under different experimental conditions. The maximum retention capacity of this polymer was determined at different pHs and polymer–metal ion ratios. The metal ions investigated were Ag(I), Cu(II), Co(II), Ni(II), and Cr(III). The maximum retention capacity values of the divalent metal ions were very similar and higher than those for the trivalent cations, indicating that the polymer–metal ion interaction was basically through electrostatic type. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 2917–2922, 2004     

Metal Extraction by Sulfur‐Containing Symmetrically‐Substituted Bisphosphonic Acids. Part I. P,P′‐di(2‐ethylhexyl) Methylenebisthio‐Phosphonic Acid

Abstract

P,P′‐dialkyl methylenebisphosphonic acids are powerful metal extraction reagents exhibiting strong affinity for a variety of metal ions, especially lanthanides and actinides. While the affinity of gem‐bisphosphonic acids is generally high for most metal ions because of their relative high acidity and ability to form six‐member chelate rings, the selectivity often is low. Thus, a strategy of incorporating soft‐donor atoms such as sulfur into gem‐bisphosphonic acids has been adopted to obtain enhanced metal selectivity while retaining high extraction efficiency. To this end a new class of sulfur‐containing gem‐bisphosphonic acid solvent extraction reagents was designed, synthesized, and evaluated for heavy element separations. Specifically, the novel sulfur‐containing P,P′‐di(2‐ethylhexyl) methylenebisthiophosphonic acid, H₂DEH[MBTP], was synthesized, characterized and its aggregation, metal extraction and acid‐base behavior assessed. Vapor phase osmometry measurements indicate that H₂DEH[MBTP] is less aggregated than its P,P′‐di(2‐ethylhexyl) methylenebisphosphonic acid analogue, H₂DEH[MBP], existing in toluene primarily as an equilibrium mixture of monomer and dimer in the concentration range studied. The acid dependency data for the extraction of Am³⁺ and Eu³⁺ from aqueous perchlorate solutions by H₂DEH[MBTP] in o‐xylene exhibit slopes close to ?3 at low acidity, consistent with extraction of a trivalent metal ion. The extractant dependency data exhibit pH dependent slopes, suggesting different stoichiometry of metal extraction under different acidities.     

Insulin Receptor Isoforms Differently Regulate Cell Proliferation and Apoptosis in the Ligand-Occupied and Unoccupied State

The insulin receptor (IR) presents two isoforms (IR-A and IR-B) that differ for the α-subunit C-terminal. Both isoforms are expressed in all human cells albeit in different proportions, yet their functional properties-when bound or unbound to insulin-are not well characterized. From a cell model deprived of the Insulin-like Growth Factor 1 Receptor (IGF1-R) we therefore generated cells exhibiting no IR (R-shIR cells), or only human IR-A (R-shIR-A), or exclusively human IR-B (R-shIR-B) and we studied the specific effect of the two isoforms on cell proliferation and cell apoptosis. In the absence of insulin both IR-A and IR-B similarly inhibited proliferation but IR-B was 2–3 fold more effective than IR-A in reducing resistance to etoposide-induced DNA damage. In the presence of insulin, IR-A and IR-B promoted proliferation with the former significantly more effective than the latter at increasing insulin concentrations. Moreover, only insulin-bound IR-A, but not IR-B, protected cells from etoposide-induced cytotoxicity. In conclusion, IR isoforms have different effects on cell proliferation and survival. When unoccupied, IR-A, which is predominantly expressed in undifferentiated and neoplastic cells, is less effective than IR-B in protecting cells from DNA damage. In the presence of insulin, particularly when present at high levels, IR-A provides a selective growth advantage.     

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.     

Effect of gamma irradiation on the metal sorption and separation of some divalent metals by some new polymeric bifunctional resins

The metal sorption and separation of some divalent metals from solutions of varying acidity by a new series of gamma-irradiated ionic polymer resins have been investigated. Three polymeric resins, viz., phosphonic acid, phosphonate monoethyl ester, and phosphonate diethyl/monoethyl ester, were used in this study. The results of metal sorption and separation were compared with those of the well-known cation exchange resin Dowex X8. The results were correlated in the usual manner of distribution coefficients (D) of three divalent metals Sr(II), Co(II), and Ni(II). The results of metal sorption by the ionic resins under investigation compared with that of sulfonic acid resin, showed that Ni showed the highest affinity for the unirradiated monoethyl ester/phosphonic acid resin, while Sr showed the highest affinity for phosphonic acid resin and Co showed the highest affinity for mono/diethyl ester phosphonic acid resin. Moreover, the three metals (Sr, Co, Ni) showed the lowest affinity toward sulfonic acid resin. The calculated separation factors for the different resins indicate that the unirradiated phosphonic acid resin has the highest capacity to separate Sr from Ni, whereas mono/diethyl ester/phosphonic acid resins have the highest capacity to separate Co from Sr and Ni. Furthermore, the affinity of the ionic polymers for the metal ions was not greatly changed after gamma irradiation. © 1997 John Wiley & Sons, Inc. J Appl Polm Sci 65:1091–1101, 1997     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

A New Na+‐Dependent RNA‐Cleaving DNAzyme with over 1000‐fold Rate Acceleration by Ethanol

Enzymes working in organic solvents are important for analytical chemistry, catalysis, and mechanistic studies. Although a few protein enzymes are highly active in organic solvents, little is known regarding nucleic acid‐based enzymes. Herein, we report the first RNA‐cleaving DNAzyme, named EtNa, that works optimally in concentrated organic solvents containing only monovalent Na⁺. The EtNa DNAzyme has a rate of 2.0 h^?1 in 54 % ethanol (with 120 mm NaCl and no divalent metal ions), and a K_d of 21 mm Na⁺. It retains activity even in 72 % ethanol as well as in DMSO. With 4 mm Na⁺, the rate in 54 % ethanol is >1000‐fold higher than that in water. We also demonstrated the use of EtNa to measuring the ethanol content in alcoholic drinks. In total, this DNAzyme has three unique features: divalent metal independent activity, Na⁺ selectivity among monovalent metals, and acceleration by organic solvents.     

Chelation properties of poly(2‐hydroxy‐4‐acryloyloxybenzophenone) resins toward some divalent metal ions

The chelation behavior of poly(2‐hydroxy‐4‐acryloyloxybenzophenone) [poly(2H4ABP) or polymer I ] obtained through the free‐radical polymerization of 2‐hydroxy‐4‐acryloyloxybenzophenone monomer and for crosslinked polymers prepared from the monomer and known amounts of the crosslinker divinylbenzene (DVB) [4 mol % of DVB for polymer II, 8 mol % of DVB for polymer III, and 16 mol 16% of DVB for polymer IV ] toward the divalent metal ions Cu²⁺, Ni²⁺, Zn²⁺, and Pb²⁺ in aqueous solution was studied by a batch equilibration technique as a function of contact time and pH. The effect of the crosslinker, DVB, was also studied. The metal‐ion uptake of the polymers was determined with atomic absorption spectroscopy, and the highest uptake was achieved at pH 7.0 for polymers I, II, III, and IV. The selectivity and binding capacity of the resins toward the investigated divalent metal ions are discussed. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci 2008     

Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I

The structure and biological activities of two disulphide isomersof a C-region deletion mutant of insulin-like growth factor-I(IGF-I) which has an Asn–Gly link engineered at the junctionof the A- and B-regions were studied before and after chemicalcleavage. Circular dichroism (CD) spectra and binding affinityto IGF binding protein 3 (IGFBP3) indicated that the treatmentwith hydroxylamine did not disrupt the overall tertiary foldof the hormones. Cleavage restored some binding affinity forthe IGF-I receptor in both isomers and weakly restored the abilityto stimulate incorporation of tritiated thymidine into DNA inNIH 3T3 fibroblasts transfected with the human IGF-I receptor.Cleavage also restored metabolic capacity, as measured by theability of the isomers to promote lipogenesis in isolated ratadipocytes through the insulin receptor. These results are consistentwith the theory that binding of IGF-I to the IGF-I receptorrequires a conformational change similar to that involved ininsulin binding the insulin receptor. The weak affinity forthe IGF-I receptor after cleavage is consistent with the beliefthat residues in the C-region interact with the IGF-I receptor.This structural difference between insulin and IGF-I gives eacha higher binding affinity for its own receptor.     

Chelating resins VI: Chelating resins of formaldehyde condensed phenolic Schiff bases derived from 4,4′‐diaminodiphenyl ether with hydroxybenzaldehydes—synthesis,characterization, and metal ion adsorption studies

Phenolic Schiff bases derived from o‐, m‐, and p‐hydroxybenzaldehydes and 4, 4′‐diaminodiphenyl ether were subjected to polycondensation reaction with formaldehyde. The resins were found to form polychelates readily with several metal ions. The materials were characterized by elemental analysis, GPC, IR, UV‐Vis, ¹H‐NMR, XRD, and thermal analyses like TG, DTG, and DSC studies. The ¹H‐NMR spectra of the resins provided evidence of polycondensation with well‐defined peaks for bridging methylene and terminal methylol functions. The metal‐ligand bonds were registered in the IR spectra of the polychelates. The thermal analysis data provided the kinetic parameters like activation energy, frequency factor, and entropy changes associated with the thermal decomposition. These data indicated the resins to be more stable than the polychelates. The DSC and XRD data indicated that the incorporation of metal ions significantly enhanced the crystallinity of the polymers. The resins could adsorb several metal ions from dilute aqueous solutions. Adsorption characteristics of the resins towards Cu(II) and Ni(II) were studied spectrophotometrically both in competitive and noncompetitive conditions. The effects of pH, contact time, quantity of the sorbent, concentration of the metal ions in a suitable buffer medium were studied. The resins were found to be selective for Cu(II) leading to its separation from a mixture of Cu(II) and Ni(II). © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 967–981, 2000