Joanna Gdula-Argasińska

Nitrite binding to metmyoglobin and methemoglobin in comparison to nitric oxide binding

Abstract. Nitrite binds reversibly to the ferriheme proteins metmyoglobin and methemoglobin in aqueous buffer solution at a physiological pH of 7.4. The spectral changes recorded for the formation of metMb(NO2–) differ significantly from those observed for the nitrosylation of metMb, which can be accounted for in terms of the different reaction products. Nitric oxide binding to metMb produces a nitrosyl product with Fe(II)-NO+ character, whereas the reaction with nitrite produces an Fe(III)-NO2– complex. The kinetics of the binding and release of nitrite by metMb and metHb were investigated by stopped-flow techniques at ambient and high pressure. The kinetic traces recorded for the reaction of nitrite with metMb exhibit excellent single-exponential fits, whereas nitrite binding to metHb is characterized by double-exponential kinetics which were assigned to the reactions of the α- and β-chains of metHb with NO2–. The rate constants for the binding of nitrite to metMb and metHb were found to be much smaller than those reported for the binding of NO, such that nitrite impurities will not affect the latter reaction. The activation parameters (ΔH≠, ΔSne, ΔV≠) obtained from the temperature and pressure dependence of the reactions support the operation of a dissociative mechanism for the binding and release of nitrite, similar to that found for the binding and release of NO in metMb.

Rutabaga (Brassica napus L. var. napobrassica) seeds, roots, and sprouts: a novel kind of food with antioxidant properties and proapoptotic potential in Hep G2 hepatoma cell line.

Although rutabaga (Brassica napus L. var. napobrassica) is a popular crop, especially in North Europe and North America, its sprouts are a new kind of vegetable. Rutabaga roots, and particularly sprouts, have not been investigated so far for antioxidant and anticancer effect on human tumor cells (Hep G2). Therefore, in vitro tests were conducted to find out whether rutabaga seeds, roots, and sprouts exert a cytotoxic effect on mammalian cells and combine them with other biological properties of particular parts of the plant. Rutabaga methanol extracts were measured for total phenolic, total flavonoid concentrations, and total antioxidant activity. Cytotoxicity of the investigated extracts was measured using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay in Chinese hamster ovary cells (CHO-K1) and Hep G2 cells culture. Cell membrane integrity was assessed in CHO-K1 and Hep G2 cells by luminescence ToxiLight BioAssay. The results of the investigation have shown that sprouts have significantly higher antioxidant activity than seeds and roots, which may result from different contents of polyphenols. Rutabaga extracts (especially 8 day sprouts) inhibited the tumor cell line Hep G2 proliferation and had a slight effect on the normal mammalian CHO-K1 culture. An advanced analysis of previously observed morphological changes and cytotoxic properties demonstrated that the evaluated extracts exerted cell death via apoptosis. These findings strongly suggest that one of the biological activities of rutabaga is antiproliferative and proapoptotic potential specific to tumor cells. The obtained results demonstrate the antioxidant property of rutabaga and its potential as a nutritional supplement in cancer prevention. These findings also strongly advocate the application of rutabaga sprouts (especially harvested in conditions presented in this article) in functional food.

Input of heavy metals to the forest floor as a result of Cracow urban pollution

The influence of Cracows urban pollution on small forest ecosystems was studied during 1998-2000. Seven sites located at different distances from the Cracow conurbation and from busy road were selected. Two sites were situated 26 and 31 km from Cracow town along an eastern transect, the other five-3, 8, 14, 26 and 35 km respectively, from Cracow-along a southern transect. At the beginning of September, five litter traps were set at each site. The quantity of litter fall, along with percentage composition of each species, were estimated for an area of 1 m(2). The concentration of Cd, Pb, Cu, Zn and Fe in the litter fall and the input of these metals to the forest floor were determined. The input of cadmium ranged from 84 microg m(-2) at Kornatka in 1998 to 382 microg m(-2) at Bonarka in 2000. The input of lead was about 10 times higher than cadmium and varied from 406 microg m(-2) at Weglówka in 1998 to 3717 microg m(-2) at Bonarka in 2000. Input of Cu, Zn and Fe to the forest floor showed the similar tendency. During 1998-2000, the highest heavy metal concentrations in the litter fall and their impact on the forest floor were always found at sites close to the Cracow conurbation and, in the eastern transect, close to a steel works. Input of metals to the forest floor tended to decrease with increasing distance from the centre of Cracow.

HGF, sIL-6R and TGF-β1 Play a Significant Role in the Progression of Multiple Myeloma.

Background. In the last few years, it has been widely reported that proinflammatory and angiogenic cytokines are important for the development and progression of multiple myeloma (MM). Objectives. To further validate and acquire more insight into this view we decided to check whether plasma levels of certain cytokines and their soluble receptors differ between MM patients and healthy subjects. Patients and Methods. The study was conducted in 76 MM patients aged 22 to 77 years (60±10 years) and 35 healthy controls aged 20 to 63 years (33±10 years). Plasma levels of interleukin-6 (IL-6), b-fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1), as well as soluble receptors for IL-6 (sIL-6R) and VEGF (sVEGF-R2) were measured using enzyme-linked immunosorbent assay (ELISA). Results. Significantly higher plasma levels of IL-6 (13.65±42.61 vs. 1.04±1.12 pg/ml, p=0.006), HGF (2174±2714 vs. 648±130 pg/ml, p<0.001), b-FGF (7.92±10.78 vs. 2.54±5.38 pg/ml, p<0.001) and sIL-6R (37.1±14.2 vs. 25.3±6.4 ng/ml, p=0.003) were observed in MM patients vs. healthy controls, respectively. Plasma sVEGF-R2 was significantly lower in MM patients than in controls (7518±2119 vs. 8725±1281 pg/ml, respectively; p<0.001). We observed an inverse correlation between length of treatment and the level of sIL-6R, and TGF-β1 in plasma. Conclusions. Plasma levels of HGF, b-FGF, IL-6 and sIL-6R in MM patients were higher when compared to the control group. Antineoplastic therapy leads to a time-dependent decrease in plasma levels of sIL-6R, and TGF-β1 in MM patients. Blood plasma level of HGF is an optimal measure to differentiate patients in whom disease is progressing versus patients who respond to therapy.

Identification of predominant phytochemical compounds and cytotoxic activity of wild olive leaves (Olea europaea L. ssp. sylvestris) harvested in south Portugal.

This study has been aimed at providing a qualitative and quantitative evaluation of selected phytochemicals such as phenolic acids, flavonoids, oleuropein, fatty acids profile, and volatile oil compounds, present in wild olive leaves harvested in Portugal, as well as at determining their antioxidant and cytotoxic potential against human melanoma HTB‐140 and WM793, prostate cancer DU‐145 and PC‐3, hepatocellular carcinoma Hep G2 cell lines, as well as normal human skin fibroblasts BJ and prostate epithelial cells PNT2. Gallic, protocatechuic, p‐hydroxybenzoic, vanillic acids, apigenin 7‐O‐glucoside, luteolin 7‐O‐glucoside, and rutin were identified in olive leaves. The amount of oleuropein was equal to 22.64 g/kg dry weight. (E)‐Anethole (32.35%), fenchone (11.89%), and (Z)‐3‐nonen‐1‐ol (8%) were found to be the main constituents of the oil volatile fraction, whereas palmitic, oleic, and alpha‐linolenic acid were determined to be dominating fatty acids. Olive leaves methanol extract was observed to exerted a significant, selective cytotoxic effect on DU‐145 and PC‐3 cell lines. Except the essential oil composition, evaluated wild olive leaves, with regard to their quantitative and qualitative composition, do not substantially differ from the leaves of other cultivars grown for industrial purposes and they reveal considerable antioxidant and cytotoxic properties. Thus, the wild species may prove to be suitable for use in traditional medicine as cancer chemoprevention.

In vitro cultures of Bacopa monnieri and an analysis of selected groups of biologically active metabolites in their biomass

Abstract Context: Bacopa monnieri L. Pennell (Scrophulariaceae) is one of the most important plants in the system of Indian medicine (Ayurveda). Objective: This paper studies the optimal growth of B. monnieri for effective accumulation of metabolites. Biomass growth of this plant could be accomplished in liquid cultures on Murashige & Skoog medium. Materials and methods: Powdered shoots of in vitro cultures of B. monnieri were extracted by methanol for indole compounds, phenolic compounds and bacosides for RP-HPLC analysis. Fatty acid analysis was performed via gas chromatography. Anti-inflammatory effect of B. monnieri extracts was evaluated in the A549 cells. COX-2 and cPGES expression was analyzed using Western blots. Results: l-Tryptophan and serotonin were found in biomass from in vitro cultures of B. monnieri on MS medium and in biomass from the MS mediums enriched with the different additions such as of 0.1 g/L magnesium sulphate, 0.1 g/L zinc hydroaspartate, 0.1 g/L l-tryptophan, 0.25 g/L serine, 0.5 g/L serine and 0.5 mg/L anthranilic acid. The content of l-tryptophan and serotonin compounds was significant in biomass from medium with the addition of 0.1 g/L zinc hydroaspartate (0.72 mg/g dry weight and 1.19, respectively). Phenolic compounds identified in biomass from the same variants of MS medium were chlorogenic acid (ranging from 0.20 to 0.70 mg/g dry weight), neochlorogenic acid (ranging from 0.11 to 0.40 mg/g dry weight) and caffeic acid (ranging from 0.01 to 0.04 mg/g dry weight). The main group of fatty acids in biomass was saturated fatty acids (53.4%). The predominant fatty acid was palmitic acid. A significant decrease of COX-2 and cPGES expression was observed in the A549 cells activated with LPS and treated with B. monnieri extracts. Discussion and conclusions: As far as we know, this is the first analysis of indole compounds and phenolic acids in this plant. The multi-therapeutic effect of B. monnieri is expressed by the activity of bacosides. Information about the presence of indole and phenolic compounds, and fatty acids in this plant is limited, but the content of these compounds might participate in the physiological activity of B. monnieri.

Anti-inflammatory activities of garlic sprouts, a source of α-linolenic acid and 5-hydroxy-l-tryptophan, in RAW 264.7 cells

The purpose of this study was to analyze the indolic, phenolic, and fatty acid content and antioxidant activity of garlic sprouts growing in the dark and in the daylight. The pro- or anti-inflammatory properties of the garlic sprout extract were investigated by evaluating the cyclooxygenase-2 (COX-2), prostaglandin E synthase (cPGES), glutathione S transferase (GSTM1), nuclear factor NF-κB, peroxisome proliferator-activated receptors (PPARs), and aryl hydrocarbon receptor (AhR) protein levels in the RAW 264.7 cells activated with lipopolysaccharide (LPS). The highest amount of total indolic (73.56 mg/100 g f.w.) and phenolic compounds (36.23 mg/100 g f.w.) was detected in garlic sprouts grown in the daylight. Studies on antioxidant activity (the FRAP and DPPH method) of garlic sprouts showed that this activity is significantly higher for sprouts grown in full access to light when compared to those grown in the dark. In garlic sprout extracts, α-linolenic acid (ALA) was found to be in greater amount. COX-2 and cPGES level was lower when compared to LPS alone activated cells. After garlic extract treatment, higher level of GSTM1, PPARΥ, cytosolic p50 and p65 protein, as well as a lower NF-ĸB p50/p65 activity was noted in the RAW 264.7 cells which suggested PPARs and AhR transrepression mechanism of NF-ĸB signalling. The obtained results indicate Allium sativum sprouts are a rich source of n-3 fatty acids, indolic and phenolic compounds characterized by anti-inflammatory and antioxidative activity, which may support their high therapeutic and dietary potential.

Comparative Study of Predominant Phytochemical Compounds and Proapoptotic Potential of Broccoli Sprouts and Florets

The study compares lyophilized broccoli sprouts and florets in terms of their chemical composition, cytotoxic and proapoptotic potential against hepatocellular carcinoma HepG2, colorectal cancer SW480, and skin fibroblast BJ cells. Sinapic and isochlorogenic acids were predominant phenolics in the sprouts and florets, respectively. The amount of sulforaphane in the sprouts was significantly higher vs. florets. Oleic and linoleic acids dominated in the sprouts, while caproic, stearic and oleic acids in the florets. Broccoli sprouts were selectively cytotoxic on HepG2 and SW480 cells, with proapoptotic effect for the latter, while the florets were less selective, but more active, with profound proapoptotic effect for HepG2 cells (77.4%). Thus, lyophilized broccoli sprouts may be effectively used in dietary chemoprevention.

The role of the bone marrow microenvironment in the pathogenesis of multiple myeloma

Multiple myeloma (MM) is one of the most common hematologic malignancies. It remains an incurable disease, so far. The mainstay of treatment for decades was pointless therapy with cytostatic agents and immunosuppressants. Because myeloma is most common in the elderly population, vulnerable to aggressive therapy, non-specific treatment approaches led to poor patient survival. Intensive study of MM, allowed identification of the molecular interactions between myeloma cells and bone marrow tumour microenvironment, responsible for the development of the disease and associated complications, such as osteolytic bone lesions. Understanding the molecular mechanisms of action of adhesion molecules, cytokines and signalling pathways involved in the development of myeloma, has led to develop of novel, targeted therapies to improve the quality of patients life and significantly prolong the median survival time. This paper discusses the current state of knowledge of signalling pathways involved in the progression of cancer and the destruction of bone tissue, with particular emphasis on interactions with the bone marrow microenvironment of the tumour.