Tannins present in Cichorium intybus enhance glucose uptake and inhibit adipogenesis in 3T3-L1 adipocytes through PTP1B inhibition

Insulin resistance is a fundamental aspect for the etiology of non-insulin dependent diabetes mellitus (NIDDM) and has links with a wide array of secondary disorders including weight gain and obesity. The present study analyzes the effect of Cichorium intybus methanolic (CME) extract on glucose transport and adipocyte differentiation in 3T3-L1 cells by studying the radiolabelled glucose uptake and lipid accumulation assays, respectively. By performing detannification (CME/DT), the role of tannins present in CME on both the activities was evaluated. CME and CME/DT exhibited significant glucose uptake in 3T3-L1 adipocytes with a dose-dependent response. Glucose uptake profile in the presence of PI3K and IRTK inhibitors (Wortmannin and Genistein) substantiates the mechanism used by both the extracts. CME inhibited the differentiation of 3T3-L1 preadipocytes but failed to show glucose uptake in inhibitor treated cells. The activity exhibited by CME/DT is exactly vice versa to CME. Furthermore, the findings from PTP1B inhibition assay, mRNA and protein expression analysis revealed the unique behavior of CME and CME/DT. The duality exhibited by C. intybus through adipogenesis inhibition and PPARgamma up regulation is of interest. Current observation concludes that the activities possessed by C. intybus are highly desirable for the treatment of NIDDM because it reduces blood glucose levels without inducing adipogenesis in 3T3-L1 adipocytes.     

MspI Allelic Pattern of Bovine Growth Hormone Gene in Indian Zebu Cattle (Bos indicus) Breeds

The MspI allelic variation in intron III of the bovine growth hormone (bGH) gene was explored using PCR-RFLP in 750 animals belonging to 17 well-recognized breeds of Indian zebu cattle (Bos indicus) reared in different geographic locations of the country. Restriction digestion analysis of a 329-bp PCR fragment of the bGH intron III region with MspI restriction enzyme revealed two alleles (MspI− and MspI+) and two genotypes (−/− and +/−) across the 17 cattle breeds studied. The allelic frequency varied from 0.67 to 0.94 for MspI (−) and from 0.06 to 0.33 for MspI (+) across the 17 breeds, with a combined average frequency of 0.87 and 0.13, respectively. No animal with +/+ genotype was detected across the samples analyzed. The chi-square test showed that the difference in MspI allelic frequency was not significant (p > 0.05), regardless of the geographic origin, coat color, or utility of the cattle breed. The high MspI (−) allele frequencies obtained for Indian zebu cattle in this study are in sharp contrast to those reported for taurine breeds from northern Europe, Mediterranean countries, and America. Findings of this study further substantiate the hypothesis that the MspI (−) allele has an Indian origin.     

New approach to the study of hormone-protein interaction using the microcalorimetric method.

Binding enthalpies of various hormones to bovine serum albumin (BSA) and human serum albumin (HSA) in 50 mM phosphate buffer, pH 7.4, at 37 degrees C have been determined by direct microcalorimetry. The observed enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to BSA were found to be -13.24 plus or minus 0.11 -10.31 plus or minus 0.02, -2.37 plus or minus 0.46, -17.64 plus or minus 0.32 and -17.14 plus or minus 0.36 kcal/mol of hormone, respectively. under the same experimental conditions the enthalpies of binding of progesterone, testosterone, dihydrotestosterone, corticosterone and estriol to HSA were found to be -23.94 plus or minus 0.32, -18.88 plus or minus 0.49, -11.14 plus or minus 0.02, -9.88 plus or minus 0.14 and -20.85 plus or minus 0.39 kcal/mol of hormone, respectively.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Demonstration of the existence of a second,non-lysosomal glucocerebrosidase that is not deficient in Gaucher disease

In addition to the lysosomal glucocerebrosidase, a distinct β-glucosidase that is also active towards glucosylceramide could be demonstrated in various human tissues and cell types. Subcellular fractionation analysis revealed that the hitherto undescribed glucocerebrosidase is not located in lysosomes but in compartments with a considerably lower density. The non-lysosomal glucocerebrosidase differed in several respects from lysosomal glucocerebrosidase. The non-lysosomal isoenzyme proved to be tightly membrane-bound, whereas lysosomal glucocerebrosidase is weakly membrane-associated. The pH optimum of the non-lysosomal isoenzyme is less acidic than that of lysosomal glucocerebrosidase. Non-lysosomal glucocerebrosidase, in contrast to the lysosomal isoenzyme, was not inhibited by low concentrations of conduritol B-epoxide, was markedly inhibited by taurocholate, was not stimulated in activity by the lysosomal activator protein saposin C, and was not deficient in patients with Gaucher disease. Non-lysosomal glucocerebrosidase proved to be less sensitive to inhibition by castanospermine or deoxynojirimycin but more sensitive to inhibition by D-gluconolactone than the lysosomal glucocerebrosidase. The physiological function of this second, non-lysosomal, glucocerebrosidase is as yet unknown.     

The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase

Glutathione S-transferase (GST) and multidrug resistance-associated proteins (MRPs) play major roles in drug resistance in melanoma. In this study, we investigated caffeic acid phenethyl ester (CAPE) as a selective GST inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. Tyrosinase bioactivates CAPE to an o-quinone, which reacts with glutathione to form CAPE-SG conjugate. Our findings indicate that 90% CAPE was metabolized by tyrosinase after a 60-min incubation. LC–MS/MS analyses identified a CAPE-SG conjugate as a major metabolite. In the presence of tyrosinase, CAPE (10–25 μM) showed 70–84% GST inhibition; whereas in the absence of tyrosinase, CAPE did not inhibit GST. CAPE-SG conjugate and CAPE-quinone (25 μM) demonstrated ?85% GST inhibition via reversible and irreversible mechanisms, respectively. Comparing with CDNB and GSH, the non-substrate CAPE acted as a weak, reversible GST inhibitor at concentrations >50 μM. Furthermore, MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, decrease the IC₅₀ of CAPE (15 μM) by 13% and 21%, apoptotic cell death by 3% and 13%, and mitochondrial membrane potential in human SK-MEL-28 melanoma cells by 10% and 56%, respectively. Moreover, computational docking analyses suggest that CAPE binds to the GST catalytic active site. Caffeic acid, a hydrolyzed product of CAPE, showed a similar GST inhibition in the presence of tyrosinase. Although, as controls, 4-hydroxyanisole and l-tyrosine were metabolized by tyrosinase to form quinones and glutathione conjugates, they exhibited no GST inhibition in the absence and presence of tyrosinase. In conclusion, both CAPE and caffeic acid selectively inhibited GST in the presence of tyrosinase. Our results suggest that intracellularly formed quinones and glutathione conjugates of caffeic acid and CAPE may play major roles in the selective inhibition of GST in SK-MEL-28 melanoma cells. Moreover, the inhibition of MRP enhances CAPE-induced toxicity in the SK-MEL-28 melanoma cells.     

Nuclear factor-κB is a common upstream signal for growth differentiation factor-5 expression in brown adipocytes exposed to pro-inflammatory cytokines and palmitate

We have previously demonstrated that genetic and acquired obesity similarly led to drastic upregulation in brown adipose tissue (BAT), rather than white adipose tissue, of expression of both mRNA and corresponding protein for the bone morphogenic protein/growth differentiation factor (GDF) member GDF5 capable of promoting brown adipogenesis. In this study, we evaluated expression profiles of GDF5 in cultured murine brown pre-adipocytes exposed to pro-inflammatory cytokines and free fatty acids (FFAs), which are all shown to play a role in the pathogenesis of obesity. Both interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were effective in up-regulating GDF5 expression in a concentration-dependent manner, while similar upregulation was seen in cells exposed to the saturated FFA palmitate, but not to the unsaturated FFA oleate. In silico analysis revealed existence of the putative nuclear factor-κB (NF-κB) binding site in the 5′-flanking region of mouse GDF5, whereas introduction of NF-κB subunits drastically facilitated both promoter activity and expression of GDF5 in brown pre-adipocytes. Chromatin immunoprecipitation analysis confirmed significant facilitation of the recruitment of NF-κB to the GDF5 promoter in lysed extracts of BAT from leptin-deficient ob/ob obese mice. Upregulation o GDF5 expression was invariably inhibited by an NF-κB inhibitor in cultured brown pre-adipocytes exposed to IL-1β, TNF-α and palmitate. These results suggest that obesity leads to upregulation of GDF5 expression responsible for the promotion of brown adipogenesis through a mechanism relevant to activation of the NF-κB pathway in response to particular pro-inflammatory cytokines and/or saturated FFAs in BAT.     

Quasi‐spherical silver nanoparticles with high dispersity and uniform sizes: preparation,characterization and bioactivity in their interaction with bovine serum albumin

A stepwise seeded growth route for the preparation of silver nanoparticles (AgNPs) is reported. In the process, silver nitrate was used as a precursor, with sodium borohydride as a reducing agent and trisodium citrate as both a reductant and stabilizer. The AgNPs were characterized using several methods, including UV–vis spectroscopy, X‐ray diffraction and transmission electron microscopy. The prepared AgNPs were quasi‐spherical and crystalline, with an average diameter of 21 nm. Interactions between the AgNPs and bovine serum albumin (BSA) were investigated using UV–vis, fluorescence spectroscopy and synchronous fluorescence spectroscopy (SFS). It was proved that the quenching mechanism is a static process. The binding constants and number of binding sites were calculated. The thermodynamic parameters implied that the binding process was spontaneous and the main driving force of the interaction was electrostatic. The results of the SFS indicated that the conformational change of BSA was induced by AgNPs. Copyright © 2016 John Wiley & Sons, Ltd.     

Construction of transgenic mice producing CAT to milk by co-injection of two overlapping fragments of bovine αs1-casein-CAT gene

Two αs1-casein/chloramphenicol acetyltransferase (CAT) gene constructs overlapping by 3.0kb were constructed and co-injected into murine zygotes. In 9 of 10 lines of transgenic mice obtained, based on analysis of structure and expression of the transgene, accurate extrachromosomal homologous recombination (ECR) between the two overlapping DNA fragments was found. Different levels of CAT activity were detected in milk from these lines. The highest CAT activity was about 25-50μg/mL milk. In some mice. CAT activity was found in salvia gland, thymus and spleen extracts. The high frequency and accuracy of ECR reported here will be applicable in the experimental manipulation for generation of relatively large transgene.