Pretreatment with LEDs regulates antioxidant capacity and polyphenolic profile in two genotypes of basil under salinity stress

Protoplasma - In the present study, we evaluated a pretreatment with four LED light sources (red, blue, red + blue, and white) in two genotypes (green and purple) of basil on the growth...     

AurH1: a new heptapeptide derived from Aurein1.2 antimicrobial peptide with specific and exclusive fungicidal activity

Due to the increasing incidence of fungal opportunistic infections and emergence of antibiotic‐resistant fungal strains, antimicrobial peptides (AMPs) are considered as ideal candidates for antifungal compounds. In silico methods can reduce the limitations of natural AMPs such as toxicity and instability and improve their antimicrobial properties and selectivity. In this study, we designed AurH1, a new truncated peptide, based on the six‐amino acid sequence of Aurein1.2. Further , the antimicrobial activities and toxicity effects of AurH1 on human skin fibroblast cells and red blood cells were investigated. Finally, field emission scanning electron microscopy (FE‐SEM) and flow cytometry were performed in order to study the mechanism of action of AurH1. The results indicated that AurH1 had only antifungal activity (at a minimal inhibitory concentration (MIC) of 7.3‐125 μg/mL) without any antibacterial effects on the selected bacteria, while Aurein1.2 had both antifungal and antibacterial activities as positive control. Furthermore, AurH1 did not show any toxicity on Hu02 cells and human red blood cells at its MIC range. In conclusion, it became clear that AurH1 is a selective peptide against fungi with no toxic effects on the selected bacteria and human cells.     

Circulating Level of CTRP1 in Patients with Nonalcoholic Fatty Liver Disease (NAFLD): Is It through Insulin Resistance?

Nonalcoholic fatty liver disease (NAFLD) is considered as one of the most common liver diseases. It is robustly linked to obesity and insulin resistance and is regarded as hepatic manifestation of metabolic syndrome (MetS). Adipokines are involved in the pathophysiology of liver diseases. The aim of this study was to evaluate the plasma concentrations of CTRP1 (complement-C1q TNF-related protein 1) in 22 patients with NAFLD, 22 patients with type 2 diabetes mellitus (T2DM), 22 patients with NAFLD+T2DM and 21 healthy controls, as well as their correlation with the level of metabolic and hepatic parameters. Plasma concentration of CTRP1 was measured with ELISA method. Plasma concentration of CTRP1 in patients with NAFLD, T2DM and NAFLD+T2DM were significantly higher than healthy subjects (p<0.0001). Moreover, we observed significant positive correlations between plasma level of CTRP1 and fasting blood glucose (FBG) (p<0.001), homeostasis model assessment of insulin resistance (HOMA-IR) (p<0.001), body mass index (BMI) (p = 0.001), alanine amino transferase (ALT) (p = 0.002), gamma glutamyl transferase (γ-GT) (p<0.001) and liver stiffness (LS) (p<0.001). Our results indicate the strong association of CTRP1 with insulin resistance in NAFLD. Also, it seems that CTRP1 can be considered as an emerging biomarker for NAFLD, however, more studies are necessary to unravel the role of CTRP1 in NAFLD pathogenesis.     

Designer biocatalysts for direct incorporation of exogenous galactose into globotriose

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.     

Structural elucidation of an α-1,2-mannosidase resistant oligosaccharide produced in Pichia pastoris

The N-glycosylation pathway in Pichia pastoris has been humanized by the deletion of genes responsible for fungal-type glycosylation (high mannose) as well as the introduction of heterologous genes capable of forming human-like N-glycosylation. This results in a yeast host that is capable of expressing therapeutic glycoproteins. A thorough investigation was performed to examine whether glycoproteins expressed in glycoengineered P. pastoris strains may contain residual fungal-type high-mannose structures. In a pool of N-linked glycans enzymatically released by protein N-glycosidase from a reporter glycoprotein expressed in a developmental glycoengineered P. pastoris strain, an oligosaccharide with a mass consistent with a Hexose(9)GlcNAc(2) oligosaccharide was identified. When this structure was analyzed by a normal-phase high-performance liquid chromatography (HPLC), its retention time was identical to a Man(9)GlcNAc(2) standard. However, this Hexose(9)GlcNAc(2) oligosaccharide was found to be resistant to α-1,2-mannosidase as well as endomannosidase, which preferentially catabolizes endoplasmic reticulum oligosaccharides containing terminal α-linked glucose. To further characterize this oligosaccharide, we purified the Hexose(9)GlcNAc(2) oligosaccharide by HPLC and analyzed the structure by high-field one-dimensional (1D) and two-dimensional (2D) (1)H NMR (nuclear magnetic resonance) spectroscopy followed by structural elucidation by homonuclear and heteronuclear 1D and 2D (1)H and (13)C NMR spectroscopy. The results of these experiments lead to the identification of an oligosaccharide α-Man-(1 → 2)-β-Man-(1 → 2)-β-Man-(1 → 2)-α-Man-(1 → 2) moiety as part of a tri-antennary structure. The difference in enzymatic reactivity can be attributed to multiple β-linkages on the α-1,3 arm of the Man(9)GlcNAc(2) oligosaccharide.     

Phosphate incorporation during glycogen synthesis and Lafora disease

Glycogen is a branched polymer of glucose that serves as an energy store. Phosphate, a trace constituent of glycogen, has profound effects on glycogen structure, and phosphate hyperaccumulation is linked to Lafora disease, a fatal progressive myoclonus epilepsy that can be caused by mutations of laforin, a glycogen phosphatase. However, little is known about the metabolism of glycogen phosphate. We demonstrate here that the biosynthetic enzyme glycogen synthase, which normally adds glucose residues to glycogen, is capable of incorporating the β-phosphate of its substrate UDP-glucose at a rate of one phosphate per approximately 10,000 glucoses, in what may be considered a catalytic error. We show that the phosphate in glycogen is present as C2 and C3 phosphomonoesters. Since hyperphosphorylation of glycogen causes Lafora disease, phosphate removal by laforin may thus be considered a repair or damage control mechanism.     

Sensitivity to rewarding or aversive effects of methamphetamine determines methamphetamine intake

Amphetamines have rewarding and aversive effects. Relative sensitivity to these effects may be a better predictor of vulnerability to addiction than sensitivity to one of these effects alone. We tested this hypothesis in a dose-response study in a second replicate set of mouse lines selectively bred for high vs. low methamphetamine (MA) drinking (MADR). Replicate 2 high (MAHDR-2) and low (MALDR-2) MA drinking mice were bred based on MA consumption in a two-bottle choice procedure and examined for novel tastant drinking. Sensitivities to the rewarding and aversive effects of several doses of MA (0.5, 2 and 4 mg/kg) were measured using a place conditioning procedure. After conditioning, mice were tested in a drug-free and then drug-present state for time spent in the saline- and MA-paired contexts. Similar to the first set of MADR lines, by the end of selection, MAHDR-2 mice consumed about 6 mg MA/kg/18 h, compared to nearly no MA in MALDR-2 mice, but had similar taste preference ratios. MAHDR-2 mice exhibited place preference in both the drug-free and drug-present tests, and no significant place aversion. In contrast, MALDR-2 mice exhibited no place preference or aversion during the drug-free test, but robust place aversion in the drug-present test. These data extend our preliminary findings from the first set of MADR lines and support the hypothesis that the combination of greater sensitivity to the rewarding effects of MA and insensitivity to the aversive effects of MA is genetically associated with heightened risk for MA consumption.     

Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.     

Cellular infiltration on nanofibrous scaffolds using a modified electrospinning technique

Electrospinning is currently used to fabricate nanofibrous scaffolds for tissue engineering applications. The major problem of these scaffolds is their intrinsically two-dimensional nature which inhibits cellular migration and in-growth. In this study, we have introduced a modified setup of electrospinning to produce three-dimensional nanofibrous scaffolds which allows improved infiltration of cells. An array of focused halogen light bulbs was used to localize the heat in the path of electrospun jet near the collector. The fabricated mats were then seeded with cells in order to evaluate migration and infiltration. After 14 days of culture, a homogenous distribution of cells was observed throughout the scaffolds and showed the three-dimensional architecture of nanofibrous mats. By this novel and simple setup, the prepared electrospun mats will allow the seeded cells to obtain a three-dimensional arrangement which is ideal for tissue engineering applications.     

Inhibin/activin-betaE subunit in normal and malignant human cervical tissue and cervical cancer cell lines

Inhibins are dimeric glycoproteins, composed of an alpha-subunit and one of two possible beta-subunits (betaA or betaB), with substantial roles in human reproduction and in endocrine-responsive tumours. Recently a novel beta subunit named betaE was described, although it is still unclear if normal or cancerous cervical epithelial cells as well as cervical cancer cell lines can synthesise the novel inhibin-betaE subunit. About 4 normal cervical tissue samples together with 10 specimens of well-differentiated squamous cervical cancer and adenocarcinoma of the cervix were immunohistochemical analyzed. Additionally, two cervical carcinoma cell lines (HeLa and CaSki) were analyzed by immunofluorescence and RT–PCR for the expression of this novel subunit. We demonstrated for the first time an immunolabelling of the inhibin-betaE subunit in normal and malignant cervical tissue, as well as cervical cancer cells. Although the physiological role is still quite unclear in cervical tissue, inhibin-βE might play important roles in carcinogenesis. Moreover, the synthesis of this subunit in cervical carcinoma cell lines of squamous and glandular epithelial origins also allows the use of these cell lines in elucidating its functions in cervical cancer pathogenesis. However, since the expression of the inhibin-βE is minimal in HeLa cells as assessed by immunofluorescence and RT–PCR, the CaSki cell line might be a better model for further functional experiments regarding cervical cancer pathogenesis.