Modulation of islet amyloid polypeptide induced β-cell toxicity and amyloid formation by serum albumin proteins.

Islet amyloid polypeptide (IAPP) and insulin are expressed in the beta-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human beta-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational K(ATP) channels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse beta-cell line Min6, glucose (16 mm) stimulated a 3.5-4-fold increase in the activity of a -222 to +450 IAPP promoter construct compared with values observed in 0.5 mm glucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two K(ATP) channel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 microm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent.

Study of the interaction of an anticancer drug with human and bovine serum albumin: Spectroscopic approach

Recombinant bispecific antibodies such as tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb) have shown to be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, therapeutic efficacy is hampered by a short serum half-life of these small molecules having molecule masses of 50-60 kDa. Thus, improvement of the pharmacokinetic properties of small bispecific antibody formats is required to enhance efficacy in vivo. In this study, we generated several recombinant bispecific antibody-albumin fusion proteins and analyzed these molecules for biological activity and pharmacokinetic properties. Three recombinant antibody formats were produced by fusing two different scFv molecules, bispecific scDb or taFv molecules, respectively, to human serum albumin (HSA). These constructs (scFv(2)-HSA, scDb-HSA, taFv-HSA), directed against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor complex molecule CD3, retained full binding capacity to both antigens compared with unfused scFv, scDb, and taFv molecules. Tumor antigen-specific retargeting and activation of T cells as monitored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv(2)-HSA. T cell activation could be further enhanced by a target cell-specific costimulatory signal provided by a B7-DbCEA fusion protein. Furthermore, we could demonstrate that fusion to serum albumin strongly increases circulation time of recombinant bispecific antibodies. In addition, our comparative study indicates that single chain diabody-albumin fusion proteins seem to be the most promising format for further studying cytotoxic activities in vitro and in vivo.

The interaction studies of CuII nalidixic acid–DACH chemotherapeutic drug entity, [C36H50N8O6Cu] with serum albumin proteins, viz., human serum albumin (HSA) and bovine serum albumin (BSA) employing UV–vis, fluorescence, CD, FTIR and molecular docking techniques have been carried out. Complex [C36H50N8O6Cu] demonstrated strong binding affinity towards serum albumin proteins via hydrophobic contacts with binding constants, K = 3.18 × 105 and 7.44 × 104 M–1 for HSA and BSA, respectively implicating a higher binding affinity for HSA. The thermodynamic parameters ΔG, ΔH and ΔS at different temperatures were also calculated and the interaction of complex [C36H50N8O6Cu] with HSA and BSA was found to be enthalpy and entropy favoured, nevertheless, complex [C36H50N8O6Cu] demonstrated higher binding affinity towards HSA than BSA evidenced from its higher binding constant values. Time resolved fluorescence spectroscopy (TRFS) was carried out to validate the static quenching mechanism of HSA/BSA fluorescence. The collaborative results of spectroscopic studies indicated that the microenvironment and the conformation of HSA and BSA (α–helix) were significantly perturbed upon interaction with complex [C36H50N8O6Cu]. Hirshfeld surfaces analysis and fingerprint plots revealed various intermolecular interactions viz., N–H····O, O–H····O and C–H····O linkages in a 2–dimensional framework that provide crucial information about the supramolecular architectures in the complex. Molecular docking studies were carried out to ascertain the preferential binding mode and affinity of complex [C36H50N8O6Cu] at the target site of HSA and BSA. Furthermore, only for Transmission electroscopy microscopy micrographs of HSA and BSA in presence of complex [C36H50N8O6Cu] revealed major protein morphological transitions and aggregation which validates efficient delivery of complex by serum proteins to the target site.Communicated by Ramaswamy H. Sarma

While chelation therapy was traditionally employed for the treatment of iron overload disease, several studies including clinical trials have demonstrated that certain classes of chelators possess anti-cancer activity. In an effort to develop increasingly active anti-cancer chelators, structure-activity relationship studies led to the synthesis of di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT), which possess potent in vitro and in vivo anti-tumor activity1,2. Knowledge of membrane transport is crucial in the development of successful chemotherapeutics. This is because the membrane acts as barrier preventing drug accumulation within tumor cells, inhibiting the interaction between drugs and their intracellular targets. Furthermore, cancer cells can develop drug resistance through the impairment of drug uptake. Additionally, an understanding of drug membrane transport has implications in the development of targeted drug delivery. Consequently, Bp4eT and Dp44mT were labeled with 14C to assess membrane transport through uptake experiments using human SK-N-MC neuroepithelioma cells. These studies demonstrated that these two chelators have contrasting drug delivery mechanisms despite possessing similar structures. 14C-Bp4eT enters cancer cells through passive diffusion, as demonstrated through it possessing an energy- and temperature-independent and non-saturable linear uptake. Whereas, the saturable, energy- and temperature-dependent uptake process of Dp44mT implies a carrier-mediated transport system. Albumin is known to bind drugs and hence affect drug delivery. Additionally, higher levels of albumin reside within the tumor interstitium as compared to normal tissue, a phenomena known as the enhanced permeability and retention effect3. Considering this, the concentration of albumin in the medium was altered to model the tumor microenvironment. Human and bovine serum albumin significantly (p<0.01) decreased the cellular uptake of 14C-Bp4eT. Similarly, the uptake of 14C-Dp44mT was also considerably (p<0.01) reduced in the presence of bovine serum albumin. However, human serum albumin significantly (p<0.01) increased 14C-Dp44mT uptake suggesting a specific interaction with this protein. In order to determine the mechanisms by which albumin effects membrane transport, albumin-drug binding was assessed through equilibrium dialysis, fluorescence and UV-visible spectroscopy and molecular modeling. Interestingly, these studies confirmed that both Bp4eT and Dp44mT bind directly to both bovine and human serum albumin. In conclusion, Bp4eT binds to human and bovine serum albumin, thereby decreasing the levels of free drug available to passively diffuse into cells. Conversely, the interactions of Dp44mT with human serum albumin, but not bovine, enhances chelator uptake into cancer cells. These results suggest the possibility of a novel human albumin receptor that facilitates Dp44mT uptake. Furthermore, the enhancement of Dp44mT tumor accumulation encourages the development of albumin-Dp44mT carriers in order to improve tumor drug delivery and increase its therapeutic window.

Novel flavonoid-based fluorescent probes for site-specific differentiation of human and bovine serum albumin: Visualizing quantitative research in urinary HSA.

The interaction of the immobilized triazine dye Cibacron Blue 3G-A with rat, rabbit, sheep, goat, bovine and human serum albumins was studied by affinity gel electrophoresis. Dissociation constants were estimated in each instance and showed human serum albumin to have a significantly higher affinity for the dye than did albumin from any other species. Pretreatment of the defatted proteins with bilirubin (3 mol of bilirubin/mol of protein) did not increase the dissociation constants of the serum albumins, whereas pretreatment with palmitate (7 mol of palmitate/mol of protein) increased the dissociation constant in all cases: 3-fold for human serum albumin, 15-fold for other serum albumins. Increasing the bilirubin/albumin ratio (to 7:1) did not affect the dissociation constant of the albumins studied. Decreasing the palmitate/albumin ratio decreased the dissociation constant for human serum albumin, but did not affect those of bovine and rat albumins. Altering the chain length of the presaturating fatty acid dramatically changed the dissociation constant of both human and bovine serum albumins. Butyrate, hexanoate, octanoate and decanoate did not significantly influence the dissociation constants of bovine and human serum albumins for Cibacron Blue, whereas laurate, myristate and palmitate greatly increased the dissociation constant. These data are discussed in relationship to the behaviour of albumins during dye--agarose column chromatography. In Addendum the effect of nucleotide presaturation on the interaction between Bacillus stearothermophilus 6-phosphogluconate dehydrogenase and the immobilized triazine dyes Cibacron Blue 3G-A and Procion Red HE-3B was examined, and the implications for dye--ligand chromatography are discussed.

The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.

The binding of two homologous series of oral and intravenous biliary contrast agents to human and bovine serum albumin was investigated using the gel filtration technique. All intravenous compounds are bound to human serum albumin via one high affinity and several low affinity binding sites. Within the concentration range investigated, about 3--5 high affinity binding sites for the oral compounds were found on human serum albumin. In general, the intravenous compounds have a greater affinity for human serum albumin than the oral compounds. No significant differences were found for the binding of the oral compounds to human or bovine serum albumin, while the intravenous compounds have a higher affinity for bovine than for human serum albumin. The significance of the plasma protein binding of the biliary contrast agents for the hepatic uptake is discussed.

The photobinding between riboflavin and the Trp residues from human and bovine serum albumins at two pH-dependent protein conformations was studied. At pH 7.0 both proteins showed photo-adduct formation with hyperbolic kinetics. In the bovine serum albumin this is attributed to the different locations of the two Trp residues. In the case of the human serum albumin, which has only one Trp residue, this behaviour may be related to different molecular conformations of the protein, as is also manifest in the iodide quenching experiments. At pH 3.5, the kinetics of the photo-adduct formation were found to be slower and showed a monophasic behaviour. These results are due to the conformational change of these proteins at acidic pH; the Trp residues of both proteins being now located in a more hydrophobic environment. When bovine serum albumin was anaerobically irradiated at pH 7.0 in the presence of 14C-riboflavin and then cleaved by CNBr, two peptides were obtained, containing the Trp-134 and Trp-212 residues, respectively. The incorporation of 14C-riboflavin in these samples was significantly higher at the level of the peptide containing the Trp-134 residue. Furthermore, it was demonstrated, that the energy transfer from enzymatically generated triplet acetone to riboflavin can also promote the binding of this vitamin to the Trp residues of human and bovine serum albumins.

Study question Can human Platelet Lysate (hPL) and umbilical cord plasma (UCP) be used as alternative serum sources in the culture of isolated human pre-antral follicles? Summary answer hPL significantly increased the growth and survival of human follicles cultured for 8 days compared to fetal bovine serum (FBS) and human serum albumin (HSA). What is known already Culture of human ovarian follicles is a potential new source of mature oocytes for fertility preservation and an important model system to study basic biology. However, only a few studies have produced mature human oocytes after culturing pre-antral stage follicles. Moreover, optimising culture systems is challenging due to the scarcity of human material. Platelet-rich plasma solutions have been used extensively in regenerative medicine due to their high platelet content, which upon activation, release a multitude of growth factors, hormones, and cytokines. Clinically cell-based therapies have used platelet-rich solutions, such as hPL or UCP, successfully as an animal-free serum. Study design, size, duration Human pre-antral follicles (n = 378; mean diameter: 78 µm; range: 46-237 µm) were isolated from ovarian medulla tissue. The follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with one of four sources of serum; 5% FBS (n = 74), 2.5% HSA (n = 74), 5% hPL (n = 140), and 5% UCP (n = 90). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression analysis and media hormone concentrations. Participants/materials, setting, methods Ovarian surplus tissue was donated by 7 women (aged 19-32 years) undergoing unilateral oophorectomy and ovarian tissue cryopreservation for fertility preservation. Pre-antral follicles were isolated enzymatically and growth and survival were assessed every second day during culture by microscopy. AMH and Estradiol concentrations were measured by ELISA in media collected at day 4 and 8. At day 8, surviving follicles were snap-frozen and the expression of AMH, FSHR, GDF9, and BMP15 was analysed by qPCR. Main results and the role of chance After 8 days in culture, the follicle survival rate in the hPL group (86%; n = 120/140) was significantly higher compared to the FBS group (60%; n = 45/74; p < 0.0003), but comparable to the HSA group (76%; n = 56/74; p = 0.292). On the contrary, the follicle survival rate in the UCP group (33%; n = 30/90) was significantly lower compared to any of the other groups (p < 0.0001 for all three groups). The average diameter of surviving follicles was statistically significantly larger in the hPL and UCP group compared to the FBS and HSA groups (hPL: 149 ±4.6 µm; UCP: 138 ±7.6 μm; FBS: 110 ±3.8 μm; HSA: 115 ±4.2 μm; p < 0.001 for hPL compared to FBS and HSA; p = 0.0129 for UCP compared to HSA). The relative growth of the follicles compared to their initial diameter was 45% and 33% higher in the hPL group compared to the FBS and HSA groups, respectively, which was statically significant in both groups (p < 0.001). In the UCP group, follicle growth was also significantly higher by 25% compared to the FBS group (p < 0.001) and by 14% compared to the HSA group (p = 0.0376). Hormone measurements from the culture media and follicle gene expression analysis are awaited. Limitations, reasons for caution One limitation is the variability in the initial follicle sizes. Extrapolating results according to primordial, primary, and secondary follicles would require more follicles but could provide valuable insight. Moreover, long term culture studies are needed to evaluate the effects of hPL and UCP on antral follicle development and oocyte maturation. Wider implications of the findings Our findings show that hPL can be used as an alternative serum source in the culture of human pre-antral follicles as it increased follicle growth and survival compared to FBS and HSA. UCP also increased follicle growth but had detrimental effects on follicle survival which needs further studies. Trial registration number Not applicable

Evaluation of human and bovine serum albumin on oxidation characteristics by a photosensitization reaction under complete binding of talaporfin sodium

Self-assembled small molecule based fluorescent detection of serum albumin proteins: Clinical detection and cell imaging

Quantitation of species differences in albumin–ligand interactions for bovine, human and rat serum albumins using fluorescence spectroscopy: A test case with some Sudlow's site I ligands