Non-active Fraction Research Articles

Salinity-induced changes in plastoquinone pool redox state in halophytic Mesembryanthemum crystallinum L.

We have analyzed the effect of salinity on photosystem II (PSII) photochemistry and plastoquinone (PQ) pool in halophytic Mesembryanthemum crystallinum plants. Under prolonged salinity conditions (7 or 10 days of 0.4 M NaCl treatment) we noted an enlarged pool of open PSII reaction centers and increased energy conservation efficiency, as envisaged by parameters of the fast and slow kinetics of chlorophyll a fluorescence. Measurements of oxygen evolution, using 2,6-dichloro-1,4-benzoquinone as an electron acceptor, showed stimulation of the PSII activity due to salinity. In salt-acclimated plants (10 days of NaCl treatment), the improved PSII performance was associated with an increase in the size of the photochemically active PQ pool and the extent of its reduction. This was accompanied by a rise in the NADP+/NADPH ratio. The presented data suggest that a redistribution of PQ molecules between photochemically active and non-active fractions and a change of the redox state of the photochemically active PQ pool indicate and regulate the acclimation of the photosynthetic apparatus to salinity.

New cytotoxic chalcone derivatives from Astragalus ponticus Pall.

Astragalus ponticus Pall. species was investigated for its antiproliferative effects on HeLa cells. Two new chalcones (B5 and B8) along with eight known compounds (B1, B2, B3, B6, B7, B10, B14 and B15) were isolated by following bioactivity guided isolation methods. In addition, from non-active fraction, three cycloartane glycosides (B11, B12 and B13) were isolated. Molecular structures of these isolated compounds were revealed by using spectroscopic methods like MS, 1D and 2D NMR and a single crystal X-ray diffraction analysis. New compounds B5 and B8 showed the highest antiproliferative activities against HeLa cells (IC50 values of 36.6 and 20.6 µM, respectively) while the rest showed high and low activities. Non-endemic species attract relatively low attention from the scientific community but this study demonstrates that valuable new compounds, which might be used as ingredients in medicinal preparations, can be obtained from these materials.

Characterization of individual ice residual particles by the single droplet freezing method: a case study in the Asian dust outflow region

Abstract. In order to better characterize ice nucleating (IN) aerosol particles in the atmosphere, we investigated the chemical composition, mixing state, and morphology of atmospheric aerosols that nucleate ice under conditions relevant for mixed-phase clouds. Five standard mineral dust samples (quartz, K-feldspar, Na-feldspar, Arizona test dust, and Asian dust source particles) were compared with actual aerosol particles collected from the west coast of Japan (the city of Kanazawa) during Asian dust events in February and April 2016. Following droplet activation by particles deposited on a hydrophobic Si (silicon) wafer substrate under supersaturated air, individual IN particles were located using an optical microscope by gradually cooling the temperature to −30 ∘C. For the aerosol samples, both the IN active particles and non-active particles were analyzed individually by atomic force microscopy (AFM), micro-Raman spectroscopy, and scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX). Heterogeneous ice nucleation in all standard mineral dust samples tested in this study was observed at consistently higher temperatures (e.g., −22.2 to −24.2 ∘C with K-feldspar) than the homogeneous freezing temperature (−36.5 ∘C). Meanwhile, most of the IN active atmospheric particles formed ice below −28 ∘C, i.e., at lower temperatures than the standard mineral dust samples of pure components. The most abundant IN active particles above −30 ∘C were predominantly irregular solid particles that showed clay mineral characteristics (or mixtures of several mineral components). Other than clay, Ca-rich particles internally mixed with other components, such as sulfate, were also regarded as IN active particle types. Moreover, sea salt particles were predominantly found in the non-active fraction, and internal mixing with sea salt clearly acted as a significant inhibiting agent for the ice nucleation activity of mineral dust particles. Also, relatively pure or fresh calcite, Ca(NO3)2, and (NH4)2SO4 particles were more often found in the non-active fraction. In this study, we demonstrated the capability of the combined single droplet freezing method and thorough individual particle analysis to characterize the ice nucleation activity of atmospheric aerosols. We also found that dramatic changes in the particle mixing states during long-range transport had a complex effect on the ice nucleation activity of the host aerosol particles. A case study in the Asian dust outflow region highlighted the need to consider particle mixing states, which can dramatically influence ice nucleation activity.

Highly active fractions of the medicinal leech recombinant destabilase-lysozyme

From the highly purified but lowly active recombinant protein Destabilas-Lysozyme (Dest-Lys) by use cation-exchange column TSK CM 3-SW chromatography, it was separated non-active fraction IV, contained 90% of protein. Fractions I, II and III, represented proteins with lysozyme and isopeptidase activities. Their lysozyme activity correlates with the activity of natural Des-Lys. The ratio of the activities in fractions I - III is such, that maximal lysozyme activity is concentrated in fraction III, isopeptidase - in fraction I. It is discussed the possibility of Dest-Lys different functions regulation is depended on the formation of protein complex forms.

First Characterization of Bioactive Components in Soybean Tempe That Protect Human and Animal Intestinal Cells against Enterotoxigenic Escherichia coli (ETEC) Infection

Tempe extracts can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells and thereby can play a role in controlling ETEC-induced diarrhea. The component responsible for this adhesion inhibition activity is still unknown. This research describes the purification and partial characterization of this bioactive component of tempe. After heating, defatting, and protease treatment, the extracts were found to remain active. However, after treatment with polysaccharide-degrading enzyme mixtures the bioactivity was lost. Ultrafiltration revealed the active component to be >30 kDa. Further purification of the bioactive tempe extracts yielded an active fraction with an increased carbohydrate content of higher arabinose content than the nonactive fractions. In conclusion, the bioactive component contains arabinose and originates from the arabinan or arabinogalactan side chain of the pectic cell wall polysaccharides of the soybeans, which is probably released or formed during fermentation by enzymatic modifications.

Plastoquinol is the Main Prenyllipid Synthesized During Acclimation to High Light Conditions in Arabidopsis and is Converted to Plastochromanol by Tocopherol Cyclase

Plants have evolved various strategies to acclimate to high light conditions at different levels of organization. High light stress stimulates synthesis of different antioxidant enzymes and low molecular weight antioxidants, mainly in chloroplasts. In the present studies we showed that plastoquinol, in addition to alpha-tocopherol, is the main lipid-soluble antioxidant synthesized during acclimation of Arabidopsis plants to high light conditions. The level of plastoquinol increased >10-fold and independently of tocopherols, as revealed using tocopherol biosynthetic mutants. The high light-induced increase in plastoquinol level was mainly attributable to the photochemically non-active fraction of this compound localized in plastoglobuli, which are the storage site of prenyllipids for their antioxidant action. Our data also revealed that tocopherol cyclase is required for plastochromanol biosynthesis from plastoquinol in vivo. Plastochromanol accumulated in increasing amounts in leaves during growth and it was also identified in seeds. The obtained data suggest that plastochromanol may, similarly to other prenyllipids, fulfill antioxidant function in leaves and seeds, especially during aging.

Screening of binding activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis to berries and juices

Antiadhesion therapy is a promising approach to the fight against pathogens. Antibiotic resistance and the lack of effective vaccines have increased the search for new methods to prevent infectious diseases. Previous studies have shown the antiadhesion activity of juice from cultivated cranberries (Vaccinium macrocarpon Ait.) against bacteria, especially E. coli. In this study, the binding of two streptococcal strains, Streptococcus pneumoniae and Streptococcus agalactiae, to molecular size fractions (FI, FII and FIII, <10 kDa, 10-100 kDa, and >100 kDa, respectively) of berries and berry and fruit juices from 12 plant species were studied using a microtiter well assay. For Streptococcus suis a hemagglutination inhibition assay was used. In general, binding activity was detected especially to wild cranberry (Vaccinium oxycoccos L.) and to other Vaccinium species. S. pneumoniae cells bound most to cranberry juice fraction FI and S. agalactiae cells to cranberry fraction FIII. Hemagglutination induced by S. suis was most effectively inhibited by cranberry fraction FII. NMR spectra of some characteristic active and non-active fractions were also measured. They indicate that fractions FII and FIII contained proanthocyanidins and/or other phenolic compounds. The results suggest Vaccinium berries as possible sources of antiadhesives against bacterial infections.

Using EGFP fusions to monitor the functional expression of GPCRs in the Drosophila Schneider 2 cells

In combining fluorescence measurements with ligand binding assays, the versatility of the EGFP C-terminally fused to the human mu opioid receptor (EGFP-hMOR) has been exploited to notably improve the expression level of functional G protein-coupled receptors in Drosophila S2 cells. A selected array of efficient optimization approaches is presented herein, ranging from a cell-sorting method, allowing for a substantial enrichment in EGFP-hMOR expressing cells, to the addition of chemical and pharmacological chaperones, significantly enhancing the yield and the activity of the expressed receptors. Consistent with previous studies, significant discrepancies were observed between the total amounts of fluorescent receptors over a limited subpopulation capable of ligand binding, even after expression optimization. Subsequently, membrane isopycnic centrifugation experiments allowed to separate the ligand binding active from the non-active membrane fraction, the latter most probably containing misfolded receptors. Taken together, these results illustrate a coherent set of advantageous productive and preparative methods for the production of GPCRs in the highly valuable Drosophila S2 expression system.

Evaluation of Polygonum bistorta for Anticancer Potential Using Selected Cancer Cell Lines

The chloroform and hexane fractions and their sub-fractions of Polygonum bistorta (Polygonaceae) were evaluated for their cytotoxic activity against P338 (Murine lymphocytic leukaemia), HepG2 (Hepatocellular carcinoma), J82 (Bladder transitional carcinoma), HL60 (Human leukaemia), MCF7 (Human breast cancer) and LL2 (Lewis lung carcinoma) cancer cell lines in culture. Both the chloroform and hexane fractions and a few of their sub-fractions showed moderate to very good activity against P388, HL60 and LL2 cancer cell lines. Both active and non-active fractions were further investigated for their chemical constituents. A total of nine compounds, viz. 24(E)-ethylidenecycloartanone (1), 24(E)-ethylidenecycloartan-3alpha-ol (2), cycloartane-3,24-dione (3), 24-methylenecycloartanone (4), friedelin (5), 3beta-friedelinol (6), beta-sitosterol (7), gamma-sitosterol (8) and beta-sitosterone (9) were isolated. One of the pure compounds, 24(E)-ethylidenecycloartanone 1, which was obtained in sufficient quantity, was tested for its cytotoxicity against P388, LL2, HL60 and WEHI164 (Murine fibrosarcoma) cancer cell lines but was found to have no activity even at a concentration of 100 microg/mL.

Detection of griseofulvin in a marine strain of Penicillium waksmanii by ion trap mass spectrometry

A marine strain of Penicillium waksmanii Zaleski was isolated from a sample of seawater from shellfish-farming area in the Loire estuary (France). The in vitro marine culture showed an important antifungal activity. Bioassay-guided fractionation was used to purify the crude extract. Dereplication by electrospray-ion trap/mass spectrometry (ESI-IT/MS) afforded the identification of the antifungal compound, after a semi-purification consisting of two stages. A comparison of the ionic composition between the active and the non-active fractions allowed the detection of a monocharged ion at m/ z 353 containing a chlorine atom, which could be attributed to the antifungal griseofulvin [C 17H 17ClO 6+H] +. Multi-stage fragmentation (MS n) confirmed the identity of the m/ z 353 ion of the antifungal fraction as griseofulvin. It is the first description of griseofulvin production by a strain of P. waksmanii and the first chemical study of a strain of this species isolated from marine temperate cold water.

Optical Properties of Dissolved Organic Matter in Finnish and Estonian Lakes

The main objective of the present study is to test various methods for describing the absorption spectra of coloured dissolved organic matter (CDOM) and to determine the numerical values of some optical parameters of CDOM in lakes with diverse water quality. First, the parameters of an exponential model in different spectral intervals were determined. In addition, the suitability of some other models for the approximation of CDOM spectra was estimated. Specific absorption coefficients of CDOM were calculated from the absorption coefficients and dissolved organic carbon (DOC) concentrations. The experimental initial data were differences between spectral attenuation coefficients of filtered and distilled water. Two datasets were used: 1) for 13 Estonian and 7 Finnish lakes (altogether 404 spectra between 350 and 700 nm) measured by the Estonian Marine Institute (EMI); 2) for 10 Finnish lakes (73 spectra) measured by the Finnish Environment Institute (FEI). The spectra of CDOM absorption coefficients (aCDOM) were calculated from experimental data taking into account the correction due to scattering properties of colloids in the filtered water. The total content of CDOM in natural waters of Estonian and Finnish lakes was expressed by means of aCDOM at the wavelength of 380 nm. It varied significantly, from 0.71 to 19.5 m−1, the mean value (of all the investigated lakes) being around 6.6 m−1. Slopes of the exponential approximation varied widely, from 0.006 to 0.03 nm−1. Averaged over all lakes values of slope for the interval 380-500 nm obtained from the EMI dataset are close to those obtained from the FEI dataset: from 0.014 nm−1 (without correction) to 0.016-0.017 nm-1 (with different types of correction). These results are in good correspondence with most published data. Attempts to describe the spectra in the region of 350-700 nm by means of hyperexponential functions (∽ exp(-αλη)) show that: (1) η &amp;lt; 1 (in the case of traditional exponential approximation η = 1); (2) a promising idea is to seek the best fit only for wavelengths λ &amp;gt; λ1, where λ1 will be chosen taking into account the real shape of aCDOM spectra. The mean value of the specific absorption coefficient (a*CDOM) at the wavelength 380 nm obtained in this study (0.44 L mg−1 m−1) is close to the values published in the literature, if we assume that a*CDOM (380) is calculated using the data of dissolved organic matter (DOM). The optically non-active fraction of DOM in our study was high and therefore a*CDOM (380) was considerably higher (1.01 L mg−1 m−1) than a*CDOM (380). The results of the present work could be used in the modeling of underwater light field as well as in the interpretation of radiation measurements and optical remote sensing results.

Density gradient separation of active and non-active cells from natural environments.

We present a method for the selective, physical separation of active and non-active bacterial cells from natural communities. The method exploits the reduction of tetrazolium salts to form insoluble formazan crystals intracellularly in response to the addition of different oxidisable substrates. The intracellular deposition of formazan alters the bouyant density of active cells enabling them to be separated by density gradient centrifugation. The method has been successfully applied to the fractionation and collection of large whole cell sub-populations of active and non-active cells from sea-water samples. Removal of the bands from the density gradient, followed by PCR amplification and DGGE analyses showed distinct differences in the PCR amplicon diversity associated with the active and non-active cell fractions; an indication of changes in bacterial community structure in response to the addition of oxidisable substrate. Thus, based on their in situ respiration potential, the approach enables the cytochemical enrichment and molecular characterisation of mixed bacterial populations in natural environments.

Size-exclusion chromatography of plasma proteins with high molecular masses

Two different hydrophilic materials with large pores, Superose 6 and Fractogel EMD BioSec (S), which are designed for size-exclusion chromatography (SEC) of plasma proteins with high molecular masses, are tested for their performance on a preparative scale. The model mixtures are preparations of the clotting factors VIII (FVIII) and IX (FIX). A combination of a Fractogel EMD BioSec (S) column and a Superose 6 column has proved to be particularly effective for separations in a wide molecular size range, from several millions down to about 20 000. Superose 6 showed good results on a small scale as well as on a large scale, even in the molecular mass range over 1 000 000. However, recovery of FVIII clotting activity was less than 70% with this material and therefore not satisfactory. Fractogel did not perform well in terms of separation on a small scale. However, in the case of biopolymers with high molecular masses, separation was improved by using larger columns. With Fractogel, recovery of activity of the two clotting factors FVIII and FIX was satisfactory, above 80%. On a large scale, the active fraction in the clotting factor concentrate was successfully separated from the non-active fraction with either size exclusion (SE) material. In the preparation under investigation, the clotting factor VIII is found in a complex with the von Willebrand factor (vWF). The FVIII–vWF complex has a molecular mass of several millions. It dissociates in the presence of high concentrations of Ca 2+ ions. Under such conditions FVIII and vWF were successfully separated with both SEC columns.

Soluble guanylate cyclase of platelets: Function and regulation in normal and pathological states

Chromatography of 105,000 × g supernatants of human and rat platelets on DEAE-cellulose yielded identical elution profiles containing 2 protein fractions (peaks I and II). Only peak II was found to possess guanylate cyclase activity. In the spectrum of the 105,000 × g supernatant of human platelets the absorption maximum was specified at 410 nm (the Soret band) which disappeared from the spectrum of the active protein fraction (peak II) but was detected in the nonactive fraction (peak I). The enzyme preparation was obtained in the heme-deficient form. In the experiments with rat platelets, the Soret band was absent from the corresponding spectra and the enzyme was not activated by sodium nitroprusside; i.e., in soluble guanylate cyclase of rat platelets, unlike the generally accepted notion, the heme is not a prosthetic group of the enzyme. It was shown that carnosine (β-alanyl- l-histidine), a water-soluble antioxidant, inhibits guanylate cyclase activation by sodium nitroprusside. This inhibitory effect is caused by the interaction of carnosine with the guanylate cyclase heme and can be used for evaluating the degree of saturation of the enzyme with the heme. ADP-induced aggregation of human platelets (donors) is accompanied by a fall in the basal guanylate cyclase activity (with Mg 2+) and the enhancement of the enzyme stimulation with sodium nitroprusside, protoporphyrin IX, arachidonic acid and l-arginine with simultaneous cGMP elevation in platelets. A hypothetic scheme of the regulatory role of cGMP in platelet aggregation is proposed. In the experiments with the acute myocardial ischemia of rats, 15 min after the surgery a sharp fall in the platelet guanylate cyclase activity accompanied by a decrease in the enzyme activity in the ischemic zone of the left ventricle of heart took place. The results provided evidence of the high sensitivity of platelet guanylate cyclase to pathological changes occurring in the myocardium at the earliest stages of the development of pathology.

Effect of Candida albicans cell wall components on the adhesion of the fungus to human and murine vaginal mucosa.

In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance. Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions. Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory.

Studies on the solvophobic nature of porphyrin self aggregation vs. the lipophylic nature of porphyrin-membrane binding.

Fractionation of hematoporphyrin derivative by chromatography has been performed by several methods such as TLC, HPLC and gel exclusion (Dougherty, 1983; Dougherty et al., 1983; Grossweiner et al., 1983; Kessel, 1983; Evensen et al., 1984). The pattern, number and nature of the fractions depend obviously on the method of separation. Yet, common to all methods, not all fractions retain the ability of the drug to act as a photodynamic agent for tumor treatment and there are also indications that the active and nonactive fractions differ in terms of aggregation (Dougherty et al., 1983; Kessel, 1984; Evensen et al., 1984).

Non-enzymic Proteinaceous Inhibitor of IAA Oxidase in Stem Cuttings of Ipomoea fistulosa

This paper deals with the investigations on IAA oxidase system in stem cuttings of Ipomoea fistulosa. The proteins precipitated from the crude extract by acetone consist of two fractions; (i) an ‘active fraction’ with high IAA oxidase activity that constitutes 9% of the acetone-precipitated proteins and is soluble at pH 2.6; (ii) the ‘non-active fraction’ that dissolves at higher pHs and does not show IAA oxidase activity. The ‘non-active fraction’ inhibits the activity of ‘active fraction’ at higher pHs. Evidence is presented to show that the inhibitor (s) in the ‘non-active fraction’ is (are) non-enzymic protein(s) and that the ‘active fraction’ oxidizes IAA even in the absence of co-factors. It appaers that the auxin is bound to the proteins in the ‘ non-active fraction’ and is released during the period of incubation.