Horseradish esterases: detection,purification and identification

Our goal is to characterize esterases from horseradish tissues and assign their physiological roles. In the present study we focused on isolation, purification and identification of esterases from different horseradish tissues: plantlets and two tumor tissue lines. Horizontal IEF system enabled separation of six esterase isoforms with quite different pI values as well as with pronounced differences in expression levels among analyzed tissues. Esterases were extracted, fractionated by means of cation exchange chromatography, and analyzed by planar gel electrophoresis (SDS–PAGE) and isoelectrical focusing (IEF), UV/Vis spectroscopy, MALDI mass spectrometry (MS) and MALDI-MS/MS. Several chromatographic strategies were applied for esterase purification and characterization. Two subsequent cation exchange chromatographic steps based on SP-Sepharose FF material, followed by in-solution digestion combined with MALDI-MS and MS/MS proved to be the best strategy for identification of two esterase proteins, namely Pectinesterase/pectinesterase inhibitor 18 and GDSL esterase/lipase ESM1.     

Fructose consumption enhances glucocorticoid action in rat visceral adipose tissue

The rise in consumption of refined sugars high in fructose appears to be an important factor for the development of obesity and metabolic syndrome. Fructose has been shown to be involved in genesis and progression of the syndrome through deregulation of metabolic pathways in adipose tissue. There is evidence that enhanced glucocorticoid regeneration within adipose tissue, mediated by the enzyme 11beta-hydroxysteroid dehydrogenase Type 1 (11βHSD1), may contribute to adiposity and metabolic disease. 11βHSD1 reductase activity is dependent on NADPH, a cofactor generated by hexose-6-phosphate dehydrogenase (H6PDH). We hypothesized that harmful effects of long-term high fructose consumption could be mediated by alterations in prereceptor glucocorticoid metabolism and glucocorticoid signaling in the adipose tissue of male Wistar rats. We analyzed the effects of 9-week drinking of 10% fructose solution on dyslipidemia, adipose tissue histology and both plasma and tissue corticosterone level. Prereceptor metabolism of glucocorticoids was characterized by determining 11βHSD1 and H6PDH mRNA and protein levels. Glucocorticoid signaling was examined at the level of glucocorticoid receptor (GR) expression and compartmental redistribution, as well as at the level of expression of its target genes (GR, phosphoenolpyruvate carboxyl kinase and hormone-sensitive lipase). Fructose diet led to increased 11βHSD1 and H6PDH expression and elevated corticosterone level within the adipose tissue, which was paralleled with enhanced GR nuclear accumulation. Although the animals did not develop obesity, nonesterified fatty acid and plasma triglyceride levels were elevated, indicating that fructose, through enhanced prereceptor metabolism of glucocorticoids, could set the environment for possible later onset of obesity.     

Anxiolytic‐Like Action of Selected 4‐(Alkylamino)‐3‐nitrocoumarin Derivatives in BALB/c Mice

In this work, we explored the possible polypharmacological potential of the already established antimicrobials against gastrointestinal pathogens, 4‐(alkylamino)‐3‐nitrocoumarins, as antianxiety agents, using a battery of in vivo experiments. Three chosen coumarin derivatives, differing in the substituent (sec‐butylamino, hexadecylamino, or benzylamino) at position 4, at the doses of 25, 50 and 100 mg kg^–1, were evaluated in light/dark, open‐field, horizontal wire and diazepam‐induced sleep models using male BALB/c mice. Depending on the applied dose, all three tested coumarins displayed a noteworthy anxiolytic‐like effect. 4‐(sec‐Butylamino)‐3‐nitro‐2H‐chromen‐2‐one and 4‐(hexadecylamino)‐3‐nitro‐2H‐chromen‐2‐one could be recognized as true anxiolytics in the lowest applied dose, based on three tests, without exerting any sedative effects. Thus, the 3‐nitrocoumarin core deserves further chemical diversity exploration in the ‘antianxiety’ direction.     

Potentiometric stripping analysis of zinc and copper in human teeth and dental materials.

Potentiometric stripping analysis (PSA) with oxygen as the oxidant has been used to determine soluble zinc and copper levels in exfoliated human teeth (all of which required extraction for orthodontic reasons) and commercial dental materials. The soluble zinc and copper contents of teeth were slightly below the zinc and copper contents in whole teeth reported by other researchers, except in the case of tooth with removed amalgam filling. Soluble zinc and copper concentrations of the dental materials and metal ceramic crowns were 0.50-6.30, and of 2.00-4.30 microg/g, respectively. The results of this work suggest that PSA may be a good method for zinc and copper leaching studies during the investigation of dental prosthetic materials' biocompatibility. Corrosive action of acidic media as evidenced by SEM micrographs caused the leaching of metal ions from teeth.     

Antioxidant properties of <Emphasis Type="Italic">Galium verum</Emphasis> L. (Rubiaceae) extracts

The antioxidant properties of methanol extracts of Lady’s Bedstraw (Galium verum L., Rubiaceae) herb from two different localities in Serbia were evaluated. Antioxidant activity was assessed in four different model systems. Free radical scavenging capacity (RSC) was examined by measuring the scavenging activity of extracts on 2,2-diphenyl-1-pycrylhydrazil (DPPH) and hydroxyl radical (OH), as well as on hydrogen peroxide. In addition, the protective effects of lipid peroxidation (LP) in corn oil were evaluated by the TBA-assay using the Fe²⁺/ascorbate system of induction. The amount of dried extract, the content of total phenolics, flavonoids and chlorophylls was also determined. Extracts from both locations expressed very strong scavenger activity, reducing the DPPH^⊙ (IC₅₀=3.10 μg/mland 8.04 μg/ml) and OH radical formation (IC₅₀=0.05 μg/ml and 0.54 μg/ml) and neutralising H₂O₂ (IC₅₀=4.98 μg/ml and 3.80 μg/ml), in a dose dependant manner. Also, examined extracts showed notable inhibition of LP (IC₅₀=11.69 μg/ml and 19.47 μg/ml). The observed differences in antioxidant activity could be partially explained by the levels of phenolics (2.44–4.65 mg and 4.57–5.16 mg gallic acid equivalents/g dry extract), flavonoids (6.38–10.70 μg and 15.56–17.96 μg quercetin equivalents/g dry extract) and chlorophylls in the investigated Lady’s Bedstraw extracts.     

CysK from Lactobacillus casei encodes a protein with O-acetylserine sulfhydrylase and cysteine desulfurization activity

A gene encoding an O-acetyl-L-serine sulfhydrylase (cysK) was cloned from Lactobacillus casei FAM18110 and expressed in Escherichia coli. The purified recombinant enzyme synthesized cysteine from sulfide and O-acetyl-L-serine at pH 5.5 and pH 7.4. At pH 7.4, the apparent K(M) for O-acetyl-L-serine (OAS) and sulfide were 0.6 and 6.7 mM, respectively. Furthermore, the enzyme showed cysteine desulfurization activity in the presence of dithiothreitol at pH 7.5, but not at pH 5.5. The apparent K(M) for L-cysteine was 0.7 mM. The synthesis of cystathionine from homocysteine and serine or OAS was not observed. When expressed in a cysMK mutant of Escherichia coli, the cloned gene complemented the cysteine auxotrophy of the mutant. These findings suggested that the gene product is mainly involved in cysteine biosynthesis in L. casei. Quantitative real-time PCR and a mass spectrometric assay based on selected reaction monitoring demonstrated that L. casei FAM18110 is constitutively overexpressing cysK.     

Protein expression of ubiquitin in interscapular brown adipose tissue during acclimation of rats to cold: the impact of ∙NO

Molecular and Cellular Biochemistry - In this study, the effects of l-arginine-nitric-oxide (∙NO)-producing pathway on protein content of ubiquitin, as an important component of...     

Staphylococcus aureus DinG, a helicase that has evolved into a nuclease

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase.