SPM Fractions Research Articles

Scrophularia peyronii Post. from Jordan: Chemical Composition of Essential Oil and Phytochemical Profiling of Crude Extracts and Their In Vitro Antioxidant Activity.

The genus Scrophularia is one of the largest genera belonging to the Scrophulariaceae family. Different members of the genus exhibit an interesting, wide spectrum of bioactivities. Accordingly, the current study aimed to investigate, for the first time, the chemical composition of the essential oil of Scrophularia peyronii Post. from Jordan. Additionally, extracts obtained from the aerial parts with solvents of different polarities were assayed for their phytochemical constituents and in vitro antioxidant activities. The major constituents detected in the essential oil, as revealed by GC/MS analysis, contained mainly Z,Z-farnesyl acetone (11.04%), β-elemene (6.36%), n-octanal (5.98%), and spathulenol (4.58%). Each of the aqueous methanol (Sp-M) and butanol (Sp-B) extracts contained flavonoids, saponins, anthraquinone, and glycosides. Both extracts were evaluated for their total phenolic content (TPC), total flavonoid content (TFC), and their in vitro antioxidant activity, which were assayed using the DPPH radical scavenging activity and ABTS radical scavenging methods. Additionally, the two extracts were then subjected to LC-ESI-MS/MS for the qualitative determination of their secondary metabolite content, especially in flavonoids and phenolic compounds. The results showed that the Sp-B extract of S. peyronii had the highest contents of both phenolic compounds and flavonoids and showed high radical scavenging activity, as determined by the two assay methods, when compared with the Sp-M extract. The LC-ESI-MS/MS analysis resulted in the detection of 21 compounds, including 8 flavonoids, 6 phenolic acids, 6 iridoids, and 2 acids. Although the majority of compounds were detected in both extracts, it was noticed that scropolioside B, 6'-O-cinnamoylharpagide, isoferulic acid, and 6-O-methylcatapol were only detected in the Sp-M fraction.

Synoptic transects on the distribution of trace elements (Hg, Pb, Cd, Cu, Ni, Zn, Co, Mn, Fe, and Al) in surface waters of the Northern- and Southern East Atlantic

Surface seawater samples were taken in the framework of the GEOTRACES program on “POLARSTERN” expedition ANT XXIII/1 in the Eastern Atlantic in 2005 to study the distribution of the trace elements Hg (mercury), Pb (lead), Cd (cadmium), Cu (copper), Ni (nickel), Zn (zinc), Co (cobalt), Mn (manganese), Fe (iron), and Al (aluminium). With the exception of Hg, results were compared to earlier datasets from 1989 to 1990. The particulate fraction averaged over the transect was calculated to be 49% for Cd, 23% for Mn and 50% for Fe indicating a release of these TEI's (trace elements and their isotopes) from a leachable SPM fraction in the stored and acidified samples. Total Pb concentrations ranged between 5 and 20 pmol kg − 1 in 2005 with highest values in the ITCZ (intertropical convergence zone). In 1989 Pb concentrations were twice as high in the region of the ITCZ, while by a factor of 10–15 higher values were obtained in the North Atlantic. Total Cd and Co are dominated, by different seasonal upwelling regimes (Equatorial upwelling, Guinea Dome, Angola Dome). Total Cu, Ni, Fe, Mn and Al show nearly identical concentrations in 1990 and 2005. For total manganese and aluminium strong maxima (3–4 nmol kg − 1 and 55 nmol kg − 1 respectively) are observed between 23°N and 0°, while the Fe maximum (6–9 nmol kg − 1 ) is located at 7°N. Total Hg concentrations ranged between 0.5 and 4.5 pmol kg − 1 .

Hypoxia Enhances Tumor Stemness by Increasing the Invasive and Tumorigenic Side Population Fraction

Although advances have been made in understanding the role of hypoxia in the stem cell niche, almost nothing is known about a potentially similar role of hypoxia in maintaining the tumor stem cell (TSC) niche. Here we show that a highly tumorigenic fraction of side population (SP) cells is localized in the hypoxic zones of solid tumors in vivo. We first identified a highly migratory, invasive, and tumorigenic fraction of post-hypoxic side population cells (SPm([hox]) fraction) in a diverse group of solid tumor cell lines, including neuroblastoma, rhabdomyosarcoma, and small-cell lung carcinoma. To identify the SPm((hox)) fraction, we used an "injured conditioned medium" derived from bone marrow stromal cells treated with hypoxia and oxidative stress. We found that a highly tumorigenic SP fraction migrates to the injured conditioned medium in a Boyden chamber. We show that as few as 100 SPm((hox)) cells form rapidly growing tumors in vivo. In vitro exposure to hypoxia increases the SPm((hox)) fraction significantly. Quantitative real-time polymerase chain reaction and immunofluorescence studies showed that SPm((hox)) cells expressed Oct-4, a "stemness" gene having a potential role in TSC maintenance. In nude mice xenografts, SPm((hox)) cells were localized to the hypoxic zones, as demonstrated after quantum dot labeling. These results suggest that a highly tumorigenic SP fraction migrates to the area of hypoxia; this migration is similar to the migration of normal bone marrow SP fraction to the area of injury/hypoxia. Furthermore, the hypoxic microenvironment may serve as a niche for the highly tumorigenic fraction of SP cells.

Ferromagnetic resonance in granular thin films

A theoretical analysis has been done of the effects of granule size, shape, orientation, and concentration and the temperature effects on the ferromagnetic resonance (FMR) field Hr in granular thin films. The granular CoxAg1−x thin films with 0.2<x<0.85 (the volumetric filling factor 0.17<f<0.83) were prepared by electron-beam oblique coevaporation. Our room temperature FMR data show an abrupt change in the Hr(f) dependence at f=fp≈0.3, the percolation point for this system (as evidenced from the magnetization and magnetoresistance measurements). This change is associated to the development of great clusters [ferromagnetic (FM) phase] beside single granules [superparamagnetic (SPM) phase] at f>fp. For the Co–Ag system, persistence of a considerable SPM fraction is revealed by the superconducting quantum interference device data up to the highest f, and the effect at f=fp consists in a discontinuous jump of the Hr(f) slope. Otherwise, the FMR data for granular Fe–SiO2 films reveal a discontinuous jump in Hr itself at f≈0.28. The latter can indicate a 1st kind-like magnetic percolation transition in that material. Thus FMR studies can effectively probe the internal structural processes in granular magnetic systems.

Characterization and solubilization of residual redox activity in salt-washed and detergent-treated plasma membrane vesicles from spinach leaves

An NADH-hexacyanoferrate(III) oxidoreductase (N-HCF-OR) was purified from spinach leaf plasma membrane (PM) vesicles; detailed biochemical analyses, however, revealed that the purifed protein is an NADH-monodehydroascorbate oxidoreductase (N-MDA-OR) located on the cytoplasmic surface of the PM. After removing all N-MDA-OR activity from the PM vesicles by consecutive treatments with hypoosmotic shock, salt, and detergents, the remaining PM (the “stripped” PM, SPM) fraction contained about 50% of the protein and 15% of the N-HCF-OR activity of the original PM fraction. The highest redox activity (100%) of the SPM fraction was obtained with NADH as electron donor and hexacyanofer-rate(III) (HCF) as electron acceptor, although redox activity could be measured also with ubiquinone-0 (23%), dichlorophenolindophenol (16%), cytochromec (9%), and Fe3+-EDTA (2%) as electron acceptors. The followingK m values were obtained for the N-HCF-OR activity of SPM:K m(NADH)=66.5 ± 3.8 μM [with 200 μM HCF(III)],K m[HCF(III)]=11.1 ± 1.1 μM (with 150 μM NADH). NAD+ competitively inhibited the activity. Under special conditions, SB-16 (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) solubilized about 50% of the protein and more than 90% of the N-HCF-OR activity of the SPM fraction. Redox activity of the solubilized fraction with dichlorophenolindophenol as electron acceptor was 45% of that with HCF(III). The SB-16-solubilized fraction containedb-type cytochrome(s) which could be reduced by dithionite> ascorbate ≫> NADH. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SB-16-solubilized SPM fraction revealed numerous polypeptides between 17 and 95 kDa. Further purification steps are needed to match the redox activities and spectrophotometric data to one or more of the polypeptides seen on the gel.

Characterization of colloidal and particulate matter transported by the magela creek system, Northern Australia

AbstractThe composition and amount of colloidal and suspended participate matter transported during a small flood event in Magela Creek in tropical northern Australia was investigated. The flood studied constituted approximately 3 % of the total annual flow, most (90%) of which occurred between mid‐January and mid‐February of the study year. Three fractions were separated from water samples using a sequential method involving a continuous flow centrifuge to separate suspended particulate matter (SPM; nominally > 1 μm) followed by hollow fibre filtration, first using a 0.1 μm filter to separate course colloidal matter (CCM; nominal size 1–0.1 μm) and then a 0–015 μm filter to separate fine colloidal matter (FCM; nominal size 0.1–0.015 μm). The SPM was predominantly inorganic (organic matter 21 %), whereas the colloidal fractions were dominantly organic matter (CCM 60%; FCM 83%). Analysis of individual particles using electron microprobe and automated image analysis indicated that the mineral fractions in both the SPM and CCM were dominated by iron‐enriched aluminosilicates (including kaolinite) (72–82%) and quartz (9–10%), indicative of a highly weathered and extensively laterized catchment. Surprisingly there was very little difference in the composition of the SPM or CCM fractions during the flood event studied, which may indicate either that sediment availability was restricted following the major run‐off events in January and February, or that all the sediment sources within the catchment are geochemically similar.Approximately the same amounts of particulate (20 tonne), colloidal (21 tonne) and dissolved material (17 tonne) were transported during the 25 hour period of the main flood peak; over 90% of the colloidal matter was 0.1–1.0 μm in size. These data suggest that previous estimates of the amounts of particulate (and colloidal) matter transported by Magela Creek, which were based on suspended solids measurements, may have underestimated the particulate matter load by as much as 50%. It is possible that the relatively high proportion of colloidal matter is unique to Magela Creek because coagulation and aggregation of colloidal matter to particulate matter is slow due to the very low concentations of calcium and magnesium in these waters. However, if the result is more widespread, there are important implications for the global estimates of fluvially transported particulate and dissolved materials as many of the previous studies may have underestimated the particulate load and overestimated the dissolved load.

The role of small, low density particles on the partition of selected PCB congeners between water and suspended matter (North Sea area)

The dissolved and particulate suspended concentrations of five PCB congeners (with 3–7 chlorine atoms) in the Southern Bight (North Sea) are interpreted in terms of an equilibrium partitioning model. K d values for riverine/coastal samples generally increase with chlorine number of the congeners (K d = 10 4–10 6). Values are more than an order of magnitude larger at the low suspended matter concentrations offshore (< 1 mg·dm −3). This is interpreted in terms of the presence of different SPM fractions with different contents of PCB: a fraction consisting of low size/density particles carrying high contents of PCBs, dominates at low SPM concentrations. The contribution of particulate forms to the total PCB concentration per unit volume may, therefore, be significant even at low SPM concentrations.

Cell-surface antigens of developing rat cerebellar neurons: Identification with monoclonal antibodies

Three monoclonal antibodies (Mab's), named 3C5.59, 3G5.34, and 3G6.41, that recognize cell-surface antigens found on embryonic and postnatal neurons were selected for study from among a group generated against adult rat synaptic plasma membranes (SPM's). Immunofluorescence staining with these 3 antibodies showed strong reactivity of the processes and much weaker staining of the cell bodies of the small neurons cultured from early postnatal rat cerebella. Mab 3G6.41 also reacted with flat astrocyte-like cells cultured from cerebellum. In contrast Mab's 3C5.59 and 3G5.34 appeared to specifically recognize only the neurons in these cultures. In situ staining of cryostat sections with 3G5.34 and 3G6.41 demonstrated immunoreactivity that was predominantly localized to the molecular layer of the cerebellum in the early postnatal through adult stages, in agreement with the strong staining of neurites seen in primary neuronal cultures. Quantitative analysis of Mab binding to particulate protein preparations from various tissues of the adult rat indicated that each of the antigens is restricted to the nervous system in the adult. Binding studies also indicated that each antigen was enriched approximately 1.5-fold in the SPM fractions compared to total particulate fractions from cerebellum. All 3 Mab's recognize membrane-bound molecules that can be solubilized by non-ionic detergent. Mab 3G6.41 immunoprecipitated two polypeptides of 140,000 and 185,000 apparent molecular weight from detergent-solubilized cerebellar cells that were surface-iodinated in culture. These antibodies should prove useful in the further analysis of the expression and function of individual cell surface antigens during the differentiation of cerebellar granule cells.

Changes in the subcellular distribution of calmodulin-kinase II during brain development

Subcellular fractions prepared from rodent forebrain at different postnatal ages were examined for calmodulin-binding proteins using [ 125I]calmodulin and a gel overlay technique. Synaptic junction (SJ) fractions from newborn brain, which display purity comparable to adult SJ fractions, contain low but detectable amounts of 60 and 50 kdalton calmodulin-binding polypeptides; the latter being the major postsynaptic density protein. These polypeptides have recently been shown to be the calmodulin-binding protein subunits of calmodulin-dependent protein kinase II (CaM-kinase II). CaM-kinase II polypeptides represented the predominent calmodulin-binding proteins in nearly every subcellular fraction examined, regardless of postnatal age. Large increases were observed in the CaM-kinase II content of every subcellular fraction throughout postnatal development. During development, a striking shift in the subcellular distribution of CaM-kinase II was observed. Over 4 times as much CaM-kinase II was cytosolic relative to particulate in newborn brain while this ratio was completely reversed in adult brain. Large age-dependent increases in particulate-associated CaM-kinase II were observed in highly purified synaptic plasma membrane (5-fold) and SJ (14-fold) fractions. The CaM-kinase II content of SJ fractions increased approximately 70% between days 24 and 90, a period in development that follows the most active stages of synapse formation in situ. In adult brain, approximately 60% of CaM-kinase II in crude synaptosomal fractions (P 2-INT) was recovered in SJ fractions. The CaM-kinase II in SPM fractions from all developmental ages resists solubilization in Triton X-100 and greater than 90% is recovered in SJ fractions. These studies indicate that during brain development the accumulation of SJ-associated CaM-kinase II represents an important process in the molecular and enzymatic maturation of CNS postsynaptic structures.

Developmental changes in the composition of, and precursor incorporation into, polypeptides of rat brain slices

1. 1. Developmental changes in both polypeptide composition and incorporation of L-[3,4 n- 3H]-valine into rat brain slice polypeptides have been monitored by SDS polyacrylamide gel electrophoresis coupled with fluorography. 2. 2. Six polypeptide bands (mol. wt 280, 210, 117, 66, 55 and 53 k) showed developmental decreases in relative amount whilst eleven others (mol. wt 230, 156, 95, 77, 68, 62, 48, 38, 34, 28 and 18k) showed developmental increases. 3. 3. The majority of these changes were accompanied by corresponding, but not parallel changes in precursor incorporation. 4. 4. At least one polypeptide, mol. wt 44 k, showed a developmental decrease in precursor incorporation, but little change in relative amount. 5. 5. Localisation of these polypeptides in SPM, myelin and mitochondrial enriched fractions has been studied.

Synaptic junctions isolated from cerebellum and forebrain: Comparisons of morphological and molecular properties

Synaptic junction (SJ) fractions have been isolated from rat cerebellum which are similar to forebrain SJs on the basis of morphology and enrichment of synaptic structures. The polypeptide compositions of SJ fractions were analyzed by one- and two-dimensional SDS-gel electrophoresis and stained by the silver technique. Equivalent numbers of proteins that possess similar relative mobilities (Mr), isoelectric points and staining intensities were present in cerebellum and forebrain synaptic fractions. A few prominent differences were observed between cerebellum and forebrain synaptic fractions; cerebellum SJs contained a 240 K protein that was not detected in the forebrain and the 52,000 K, major PSDp protein was present in forebrain SJs in amounts that are approximately 5-fold greater than in cerebellum SJ fractions. The identity of the cerebellum mPSDp was verified by electrophoretic mobility, peptide fingerprinting and [125I]calmodulin binding. Differences between various synaptic fractions in mannose containing glycoproteins were examined by the binding of [125I]concanavalin A (Con A) to gels. On the basis of apparent molecular weights, the glycoproteins in forebrain and cerebellum SPMs were very similar. In contrast, however, the prominent glycoproteins that reside in the postsynaptic junctional membrane of forebrain SJs were undetectable in SJ fractions isolated from cerebellum. SJ fractions from cerebellum contained their own distinct group of Con A binding glycoproteins. SPM and SJ fractions from forebrain and cerebellum were examined for receptors for excitatory (aspartate, glutamate and kainic acid) and inhibitory (GABA) neurotransmitters and the benzodiazepine analog flunitrazepam. On the basis of relative receptor contents, SJ fractions isolated from either brain region were qualitatively similar and bound significant amounts of excitatory and inhibitory transmitters. These findings support the notion that SJs from cerebellum contain a distinct class of synaptic elements that are in large part derived from asymmetric, type I synapses.

Superparamagnetic and single-domain threshold sizes in magnetite

Particle size distributions have been obtained by grain counts on electron micrographs of four samples containing submicroscopic equant magnetite particles. Blocking temperature data indicate thermally unstable (superparamagnetic, SPM) magnetic behavior in one sample whose grains range from 100 to 650 A in size. The SPM is confirmed by a large increase of saturation remanence when the hysteresis of the sample is measured at 77°K. Quantitative estimates of the SPM, single-domain (SD), and multidomain (MD) magnetization fractions in this sample are made by comparing theoretical and observed values of the reduced saturation remanence at 77° and 300°K. The particle size distribution then yields a critical size ds = (290 − 360) ± 50 A for the onset of SPM in magnetite at room temperature, in close agreement with Neel's SD theory. The critical size d0 at which domain structure develops in equidimensional grains of magnetite is less well defined. It is certainly less than 480 ± 50 A and may even be less than ds; that is, a direct MD-SPM transition in magnetite is a distinct possibility. The extreme narrowness of the stable SD range in equidimensional magnetite particles suggests that either elongated SD grains or else small MD grains with pseudo-SD characteristics must be the major carriers of the high-stability natural remanence of igneous rocks.

Synaptic plasma membranes from rat brain synaptosomes: Isolation and partial characterization

We have isolated a highly purified plasma membrane fraction directly from synaptosomes prepared by centrifugation on a Ficoll-sucrose gradient. A critical feature of the preparation method is use of alkaline conditions to achieve effective osmotic shock so that membranes effectively resolve from mitochondria. If osmotic shock is carried out at alkaline pH, 70 % of the (Na +-K +)-activated-ouabain-sensitive ATPase (EC 3.6.1.4) of the synaptosomes can be recovered with only 7 % of total cytochrome oxidase (EC 1.9.3.1) in synaptosomal membrane fractions. In contrast, if osmotic shock is carried out at neutral pH, 50 % of the mitochondria overlays 85 % of the membranes. A fraction enriched in synaptic plasma membranes (SPM fraction) can be prepared on a simple discontinuous gradient in relatively good yield. The SPM fraction bands between 25–32.5 % sucrose; the yield is about 1.5 mg of protein per g tissue wet wt. An alkaline phosphatase (EC 3.1.3.1) is found in the SPM fraction. The distribution pattern of alkaline phosphatase parallels that of Na +-K +)-ATPase . 5′-Nucleotidase (EC 3.1.3.5) and acetylcholinesterase (EC 3.1.1.7) sediment at lighter densities than (Na +-K +)-ATPase or alkaline phosphatase although there is considerable overlap. The SPM fraction contains β-N- acetylglucosaminidase . This enzyme is not completely washed out of the SPM fraction by salt treatment but is released by low concentrations of Triton X-100.