Assignment of disulfide bonds in proteins by fast atom bombardment mass spectrometry

A rapid and sensitive method for assignment of disulfide bonds using fast atom bombardment mass spectrometry is described for hen egg white lysozyme and bovine ribonuclease A. The protein is initially digested to a mixture of peptides using chemical and enzymatic methods under conditions which minimize disulfide bond reduction and exchange. The digested sample is analyzed directly by fast atom bombardment mass spectrometry before and after chemical reduction of cystine residues. An important feature of the method is that it is not necessary to completely resolve the peptides in the digest chromatographically prior to analysis. The disulfide-containing peptides are also characterized directly by prolonged exposure of the sample to the high energy xenon atom beam which results in the reduction of cystine residues. Intra- as well as interchain disulfide bond assignments are made on the basis of the mass difference between the molecular ions (MH+) of the oxidized and reduced peptides. Confirmation of the mass assignments may be obtained from the mass spectra of the digests after one cycle of manual Edman degradation. Although the quantity of protein required to unambiguously assign all of the disulfide linkages will depend on the ease with which the appropriate peptide fragments can be formed, results from these studies indicate that approximately 1 nmol of protein is usually sufficient.     

Hybridization of wheat and Aegilops cylindrica: development,karyomorphology, DNA barcoding and salt tolerance of the amphidiploids

The development of salt‐tolerant genotypes is key to a better utilization of salinized irrigated lands. Given the relatively low genetic diversity within the cultivated wheats for salt tolerance, exploring the Aegilops cylindrica's genetic diversity for salt tolerance is thus crucial to breed wheat for saline environments. In the current study, wheat genotypes were hybridized with Ae. cylindrica (a hyper salt-tolerant genotype), and amphidiploid plants were produced using embryo rescue and chromosome doubling techniques. Crossability and cytological examinations of amphidiploids and BC₁ were performed before sequencing the ITS4/5 and trnE/trnF DNAs to explore the phylogenetic relationships of the amphidiploids and their parents. Finally, amphidiploids were assessed for salt tolerance. Only two common wheat cultivars (‘Chinese Spring’ and ‘Roshan’) were crossable with Ae. cylindrica. The resultant intergeneric hybrids possessed 70 chromosomes, and morphologically either were similar to the male parent in ‘Chinese Spring’ × Ae. cylindrica or tended to be intermediate between parents in ‘Roshan’ × Ae. cylindrica. The phylogenetic tree divided the genotypes into two groups, in which Clade I contained Ae. cylindrica and three amphidiploids, and Clade II consisted of female parents and one amphidiploid. Amphidiploids exhibited significantly higher tolerance to salt stress compared to the female parents (wheat cultivars) in terms of a higher dry matter, lower accumulation of Na, higher K, and higher K/Na ratio in their root and leaf tissues. Taken together, the amphiploid plants might contain valuable salt tolerance factors.

     

Effect of biotic and abiotic elicitors on production of betulin and betulinic acid in the hairy root culture of Betula pendula Roth

Abstract

Betulin (B) and betulinic acid (BA) are two triterpenoids with a wide range of biological and medicinal activities in different organs of Betula pendula. This research aimed to increase the accumulation of B and BA in the hairy root culture of B. pendula by seven biotic and abiotic elicitors. Hairy root was induced in the stem’s inner bark of B. pendula using the C58C1 strain in the WPM (Woody Plant Medium). The effects of different concentrations of elicitors and different time of root harvest in hairy root culture of B. pendula showed that highest level of growth index (GI), B, and BA was acquired in treated hairy roots with chitosan (CTS), chlorocholine chloride (CCC) and chitosan nano-fiber (CTS NF). Highest GI of B. pendula hairy roots was 13 that was obtained in the roots treated with CTS 150?mg l^?1 on the 8th day. The highest content of BA was 1.3?mg g^?1 DW after treatment with 1?mg l^?1CCC on the 4th and 6th days and 200?mg l^?1CTS NF on the 10th day. The highest B content (0.94?mg g^?1DW) was obtained in the treated hairy root by 2?mg l^?1 CCC after 4 and 6?days.     

Immobilization of HIV-1 TAT peptide on gold nanoparticles: A feasible approach for siRNA delivery

RNA interference is one of the prosperous approaches for cancer treatment. However, small interfering RNA (siRNA) delivery to cancer cells has been faced with various challenges restricting their clinical application over the decades. Since ROR1 is an onco-embryonic gene overexpressed in many malignancies, suppression of ROR1 by siRNA can potentially fight cancer. Herein, a delivery system for ROR1 siRNA based on HIV-1 TAT peptide-capped gold nanoparticles (GNPs) was developed to treat breast cancer. Besides, we introduced a new feasible method for conjugating the peptide to the nanoparticles. Since the GNPs have high affinity to the sulfur, the findings demonstrated the peptide successfully conjugated to the nanoparticles via Au–S bonds. As positively charged nanoparticles showed high cellular uptake, we could use a low concentration of nanoparticles led to high efficient gene transfection with negligible cytotoxicity that was confirmed by flow cytometry, confocal microscopy, gel retardation, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Following transfection, downregulation of ROR1 and its targeted gene, CCND1, induced apoptosis in cancer cells. In conclusion, the reported capped GNPs could be potentially utilized for delivering negatively charged therapeutic agents in particular genes.     

Inhibition of experimental autoimmune vitiligo by oral administration of mushroom tyrosinase

Experimental autoimmune vitiligo was induced by intradermal injection of mushroom tyrosinase emulsified in complete Freund's adjuvant in female C57BL/6 mice. The onset of vitiligo was characterized by hair hypopigmentation and total melanocyte depletion in the basal layer of the epidermis. Oral administration of mushroom tyrosinase prevented the expression of mushroom tyrosinase induced experimental autoimmune vitiligo. Based on the results it is likely that oral administration of mushroom tyrosinase may have practical implications in vitiligo.     

Oral administration of Escherichia coli glutamic acid decarboxylase has immunomodulatory effects in streptozotocin-induced diabetes

The extent of destruction of insulin-secreting beta cells of the Islets of Langerhans was investigated in an animal model using oral administration of glutamic acid decarboxylase (GAD) isolated from Escherichia coli. The extent of lymphocytic infiltration of the pancreatic Islet cells and the severity of diabetes were significantly reduced by oral administration of GAD to rats 14 days before intraperitoneal injections of streptozotocin (STZ, 40 mg/kg body wt on 5 consecutive days). In addition, oral administration of GAD to rats 14 days before or 3 days after STZ treatment significantly (p <0.05) reduced the levels of GAD-specific antibodies and improved the in vitro proliferative response of splenocytes to concanavalin A (Con A). These data demonstrate that oral GAD administration probably generates active cellular mechanisms which suppress the disease and therefore raise the possibility of using E. coli GAD as a new means for the immunomodulation of autoimmune diabetes.     

Antihyperlipidaemic and antihypercholesterolaemic effects of Anethum graveolens leaves after the removal of furocoumarins.

Serum triacylglycerides and total cholesterol levels in rats, with hyperlipidaemia induced by diet, were determined after oral adminstration of a water extract of Anethum graveolens leaves before and after the extraction of the furocoumarin content of the leaves. Administration of the extracts consecutively for 14 days reduced the triacylglycerides and total cholesterol levels by almost 50 and 20%, respectively. Chloroform extraction of furocoumarins from the aqueous extracts did not reduce the antihyperlipidaemic potential of the extracts to a significant degree. Oral administration of the essential oil of A. graveolens seeds, at two different doses, also reduced the triacylglyceride levels by almost 42%. The total cholesterol level was not reduced by the same doses of the essential oil.