A novel ω-conotoxin Bu8 inhibiting N-type voltage-gated calcium channels displays potent analgesic activity.

ω-Conotoxins inhibit N-type voltage-gated calcium (CaV2.2) channels and exhibit efficacy in attenuating neuropathic pain but have a low therapeutic index. Here, we synthesized and characterized a novel ω-conotoxin, Bu8 from Conus bullatus, which consists of 25 amino acid residues and three disulfide bridges. Bu8 selectively and potently inhibits depolarization-activated Ba2+ currents mediated by rat CaV2.2 expressed in HEK293T cells (IC50 = 89 nmol/L). Bu8 is two-fold more potent than ω-conotoxin MVIIA, a ω-conotoxin currently used for the treatment of severe chronic pain. It also displays potent analgesic activity in animal pain models of hot plate and acetic acid writhing but has fewer side effects on mouse motor function and lower toxicity in goldfish. Its lower side effects may be attributed to its faster binding rate and higher recovery ratios. The NMR structure demonstrates that Bu8 contains a small irregular triple ß-strand. The structure-activity relationships of Bu8 Ala mutants and Bu8/MVIIA hybrid mutants demonstrate that the binding mode of CaV2.2 with the amino acid residues in loop 1 and loop 2 of Bu8 is different from that of MVIIA. This study characterizes a novel, more potent ω-conotoxin and provides new insights for designing CaV2.2 antagonists.

Enhanced production of cordycepin in Ophiocordyceps sinensis using growth supplements under submerged conditions.

Cordycepin is a crucial bioactive compound produced by the fungus Cordyceps spp. Its therapeutic potential has been recognized for a wide range of biological properties such as anticancer, anti-diabetic, antidepressant, antioxidant, immunomodulation, etc. Moreover, its human random clinical trials depicted a promising anti-inflammatory activity that reduced the airway inflammation remarkably in asthmatic patients. But its overexploitation and low production of cordycepin in naturally growing biomass are insufficient to meet its existing market demand for its therapeutic use. Therefore, strategies for enhancement of cordycepin production in Cordyceps spp. are warranted. However, specifically, wild type Ophiocordyceps sinensis possesses a very low content of cordycepin and has restricted growth in natural mycelial biomass. To overcome these limitations, this study attempted to enhance cordycepin production in its mycelial biomass in vitro under submerged conditions by adding various growth supplements. The effect of these growth supplements was evaluated by reversed-phase high-performance liquid chromatography (RP-HPLC) which demonstrated that among nucleosides- hypoxanthine and adenosine; amino acids-glycine and glutamine; plant hormones- 1-naphthaleneacetic acid (NAA) and 3-indoleacetic acid (IAA); vitamin-thiamine (B1) from each group of growth supplements yielded a higher amount of cordycepin with 466.48 ±â€¯3.88, 380.23 ±â€¯1.78, 434.97 ±â€¯2.32, 269.78 ±â€¯2.92, 227.61 ±â€¯2.34, 226.02 ±â€¯1.69 and 185.26 ±â€¯2.35 mg/L respectively as compared to control with 13.66 ±â€¯0.64 mg/L. Further, at the transcriptional level, quantitative real time-polymerase chain reaction (qRT-PCR) analysis of genes associated with metabolism and cordycepin biosynthesis depicted significant upregulation of major downstream genes- NT5E, RNR, purA, and ADEK which corroborated well with RP-HPLC analysis. Taken together, the present study identified growth supplements as potential precursors to activate the cordycepin biosynthesis pathway leading to improved cordycepin production in O. sinensis.

FT-NIR spectroscopy and RP-HPLC combined with multivariate analysis reveals differences in plant cell suspension cultures of Thevetia peruviana treated with salicylic acid and methyl jasmonate.

Plant cell suspension culture of T. peruviana is a feasible biotechnological platform for the production of secondary metabolites with anti-proliferative/cytotoxic activity, as phenolic compounds (PC); however, different in in vitro growth conditions may affect the production, demanding strategies to increase the metabolite biosynthesis, as well as the development of sensitive and rapid analytical methods for metabolite monitoring. The Fourier transform near-infrared (FT-NIR) spectroscopy and Reversed-phase high-performance liquid chromatography (RP-HPLC) combined with Multivariate analysis (MVA) were used to detect significant differences in the PC production in cultures treated with two elicitors. The results suggest that the FT-NIR-MVA is useful for discriminating samples according to the treatment, showed significant influence of the PC signal. RP-HPLC-MVA showed that the elicitor effect occurs at 72 h post-elicitation. Detection of dihydroquercetin (maximum concentration = 12.59 mg/L), a flavonoid with anti-cancer properties, is highlighted. Future studies will be aimed at scaling this culture to increase the productivity of dihydroquercetin.

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity.

Herein we describe the discovery and functional characterization of a steroidal glycosyltransferase (SGT) from Ornithogalum saundersiae and a steroidal glycoside acyltransferase (SGA) from Escherichia coli and their application in the biosynthesis of acylated steroidal glycosides (ASGs). Initially, an SGT gene, designated as OsSGT1, was isolated from O. saundersiae. OsSGT1-containing cell free extract was then used as the biocatalyst to react with 49 structurally diverse drug-like compounds. The recombinant OsSGT1 was shown to be active against both 3ß- and 17ß-hydroxyl steroids. Unexpectedly, in an effort to identify OsSGT1, we found the bacteria lacA gene in lac operon actually encoded an SGA, specifically catalyzing the acetylations of sugar moieties of steroid 17ß-glucosides. Finally, a novel enzymatic two-step synthesis of two ASGs, acetylated testosterone-17-O-ß-glucosides (AT-17ß-Gs) and acetylated estradiol-17-O-ß-glucosides (AE-17ß-Gs), from the abundantly available free steroids using OsSGT1 and EcSGA1 as the biocatalysts was developed. The two-step process is characterized by EcSGA1-catalyzed regioselective acylations of all hydroxyl groups on the sugar unit of unprotected steroidal glycosides (SGs) in the late stage, thereby significantly streamlining the synthetic route towards ASGs and thus forming four monoacylates. The improved cytotoxic activities of 3'-acetylated testosterone17-O-ß-glucoside towards seven human tumor cell lines were thus observable.

Extraction efficiency, phytochemical profiles and antioxidative properties of different parts of Saptarangi (Salacia chinensis L.) - An important underutilized plant.

The study aimed to evaluate extraction efficiency, detection and quantification of phytochemicals, minerals and antioxidative capacity of different parts of Salacia chinensis L. Continuous shaking extraction, steam bath assisted extraction, ultrasonic extraction and microwave assisted extraction with varied time intervals were employed for extraction of phenolics, flavonoids, and antioxidants. Preliminary screening revealed the presence of wide array of metabolites along with carbohydrates and starch. Steam bath assisted extraction for 10 min exposure was found most suitable for extraction phenolics (46.02 ± 2.30 mg of gallic acid equivalent per gram of dry weight and 48.57 ± 2.42 mg of tannic acid equivalent per gram of dry weight) and flavonoids (35.26 ± 1.61 mg of quercetin equivalent per gram of dry weight and 51.60 ± 2.58 mg of ellagic acid equivalent per gram of dry weight). In support, reverse phase-high performance liquid chromatography- diode array detector confirmed the presence of seven pharmaceutically important phenolic acids. Antioxidant capacity was measured by 1, 1- diphenyl-1-picryl hydrazyl (DPPH), ferric reducing antioxidant power (FRAP), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) scavenging (ABTS) and N, N-dimethyl-p-phenylenediamine (DMPD) assays and represented as trolox equivalent antioxidant capacity (TEAC) and ascorbic acid equivalent antioxidant capacity (AEAC). Antioxidant capacity ranged from 121.02 ± 6.05 to 1567.28 ± 78.36 µM trolox equivalent antioxidant capacity and 56.62 ± 2.83 to 972.48 ± 48.62 µM ascorbic acid equivalent antioxidant capacity. Roots showed higher yields of illustrated biochemical parameters, however fresh fruit pulp was found a chief source of minerals. Gas chromatography-mass spectroscopic analysis revealed the presence of a vast array of phytoconstituents associated with different plant parts. The present study revealed the amounts of minerals and diverse phytoconstituents in various parts of S. chinensis and confirmed its medicinal and nutritional implications.

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles-Part 2: Mass spectrometric methods.

To monitor the Fc glycosylation of therapeutic immunoglobulin G in bioprocess development, product characterization and release analytics, reliable techniques for glycosylation analysis are needed. Several analytical methods are suitable for this application. We recently presented results comparing detection methods for glycan analysis that are separation-based, but did not include mass spectrometry (MS). In the study reported here, we comprehensively compared MS-based methods for Fc glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods investigated were compared with respect to precision, accuracy, throughput and analysis time. Emphasis was put on the detection and quantitation of sialic acid-containing glycans. Eleven MS methods were compared to hydrophilic interaction liquid chromatography of 2-aminobenzamide labeled glycans with fluorescence detection, which served as a reference method and was also used in the first part of the study. The methods compared include electrospray MS of the heavy chain and Fc part after limited digestion, liquid chromatography MS of a tryptic digest, porous graphitized carbon chromatography MS of released glycans, electrospray MS of glycopeptides, as well as matrix assisted laser desorption ionization MS of glycans and glycopeptides. Most methods showed excellent precision and accuracy. Some differences were observed with regard to the detection and quantitation of low abundant glycan species like the sialylated glycans and the amount of artefacts due to in-source decay.

Modulation of deregulated chaperone-mediated autophagy by a phosphopeptide.

The P140 peptide, a 21-mer linear peptide (sequence 131-151) generated from the spliceosomal SNRNP70/U1-70K protein, contains a phosphoserine residue at position 140. It significantly ameliorates clinical manifestations in autoimmune patients with systemic lupus erythematosus and enhances survival in MRL/lpr lupus-prone mice. Previous studies showed that after P140 treatment, there is an accumulation of autophagy markers sequestosome 1/p62 and MAP1LC3-II in MRL/lpr B cells, consistent with a downregulation of autophagic flux. We now identify chaperone-mediated autophagy (CMA) as a target of P140 and demonstrate that its inhibitory effect on CMA is likely tied to its ability to alter the composition of HSPA8/HSC70 heterocomplexes. As in the case of HSPA8, expression of the limiting CMA component LAMP2A, which is increased in MRL/lpr B cells, is downregulated after P140 treatment. We also show that P140, but not the unphosphorylated peptide, uses the clathrin-dependent endo-lysosomal pathway to enter into MRL/lpr B lymphocytes and accumulates in the lysosomal lumen where it may directly hamper lysosomal HSPA8 chaperoning functions, and also destabilize LAMP2A in lysosomes as a result of its effect on HSP90AA1. This dual effect may interfere with the endogenous autoantigen processing and loading to major histocompatibility complex class II molecules and as a consequence, lead to lower activation of autoreactive T cells. These results shed light on mechanisms by which P140 can modulate lupus disease and exert its tolerogenic activity in patients. The unique selective inhibitory effect of the P140 peptide on CMA may be harnessed in other pathological conditions in which reduction of CMA activity would be desired.

Monitoring therapeutic monoclonal antibodies in brain tumor.

Bevacizumab induces normalization of abnormal blood vessels, making them less leaky. By binding to vascular endothelial growth factor, it indirectly attacks the vascular tumor mass. The optimal delivery of targeted therapies including monoclonal antibodies or anti-angiogenesis drugs to the target tissue highly depends on the blood-brain barrier permeability. It is therefore critical to investigate how drugs effectively reach the tumor. In situ investigation of drug distribution could provide a better understanding of pharmacological agent action and optimize chemotherapies for solid tumors. We developed an imaging method coupled to protein identification using matrix-assisted laser desorption/ionization mass spectrometry. This approach monitored bevacizumab distribution within the brain structures, and especially within the tumor, without any labeling.

Alternative preparation of inclusion bodies excludes interfering non-protein contaminants and improves the yield of recombinant proinsulin.

The goal of simple, high-yield expression and purification of recombinant human proinsulin has proven to be a considerable challenge. First, proinsulin forms inclusion bodies during bacterial expression. While this phenomenon can be exploited as a capture step, conventionally prepared inclusion bodies contain significant amounts of non-protein contaminants that interfere with subsequent chromatographic purification. Second, the proinsulin molecules within the inclusion bodies are incorrectly folded, and likely cross-linked to one another, making it difficult to quantify the amount of expressed proinsulin. Third, proinsulin is an intermediate between the initial product of ribosomal translation (preproinsulin) and the final product secreted by pancreatic beta cells (insulin). Therefore, to be efficiently produced in bacteria, it must be produced as an N-terminally extended fusion protein, which has to be converted to authentic proinsulin during the purification scheme. To address all three of these problems, while simultaneously streamlining the procedure and increasing the yield of recombinant proinsulin, we have made three substantive modifications to our previous method for producing proinsulin:.•Conditions for the preparation of inclusion bodies have been altered so contaminants that interfere with semi-preparative reversed-phase chromatography are excluded while the proinsulin fusion protein is retained at high yield.•Aliquots are taken following important steps in the procedure and the quantity of proinsulin-related polypeptide in the sample is compared to the amount present prior to that step.•Final purification is performed using a silica-based reversed-phase matrix in place of a polystyrene-divinylbenzene-based matrix.