Sesquiterpene lactones of eupatorium anomalum and eupatorium mohrii

Several new guaianolides and the previously known heliangolide eurecurvin have been isolated from Eupatorium anomalum. Eupatorium mohrii also yielded three of the new guaianolides together with eurecurvin and a new germacradienolide. The implications of these findings are discussed.     

Diterpenes from eupatorium turbinatum

The aerial parts of Eupatorium turbinatum afforded three diterpenes, a known clerodane derivative, an unusual abietane derivative and a friedolabdane, liliolide, β-farnesene and the flavonol pachypodol. The structures were elucidated by highfield NMR spectroscopy. The chemotaxonomy is discussed briefly.     

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (^·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ^·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By ³H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ^·OH in radicles and endosperm caps: the production and action of ^·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ^·OH attack on polysaccharides were evident. In vivo ^·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ^·OH is a controlled action of this type of reactive oxygen species.     

Pectic polysaccharides are attacked by hydroxyl radicals in ripening fruit: evidence from a fluorescent fingerprinting method

Background and aims Many fruits soften during ripening, which is important commercially and in rendering the fruit attractive to seed-dispersing animals. Cell-wall polysaccharide hydrolases may contribute to softening, but sometimes appear to be absent. An alternative hypothesis is that hydroxyl radicals (^•OH) non-enzymically cleave wall polysaccharides. We evaluated this hypothesis by using a new fluorescent labelling procedure to ‘fingerprint’ ^•OH-attacked polysaccharides.Methods We tagged fruit polysaccharides with 2-(isopropylamino)-acridone (pAMAC) groups to detect (a) any mid-chain glycosulose residues formed in vivo during ^•OH action and (b) the conventional reducing termini. The pAMAC-labelled pectins were digested with Driselase, and the products resolved by high-voltage electrophoresis and high-pressure liquid chromatography.Key Results Strawberry, pear, mango, banana, apple, avocado, Arbutus unedo, plum and nectarine pectins all yielded several pAMAC-labelled products. GalA–pAMAC (monomeric galacturonate, labelled with pAMAC at carbon-1) was produced in all species, usually increasing during fruit softening. The six true fruits also gave pAMAC·UA-GalA disaccharides (where pAMAC·UA is an unspecified uronate, labelled at a position other than carbon-1), with yields increasing during softening. Among false fruits, apple and strawberry gave little pAMAC·UA-GalA; pear produced it transiently.Conclusions GalA–pAMAC arises from pectic reducing termini, formed by any of three proposed chain-cleaving agents (^•OH, endopolygalacturonase and pectate lyase), any of which could cause its ripening-related increase. In contrast, pAMAC·UA-GalA conjugates are diagnostic of mid-chain oxidation of pectins by ^•OH. The evidence shows that ^•OH radicals do indeed attack fruit cell wall polysaccharides non-enzymically during softening in vivo. This applies much more prominently to drupes and berries (true fruits) than to false fruits (swollen receptacles). ^•OH radical attack on polysaccharides is thus predominantly a feature of ovary-wall tissue.     

Structure and Composition of Biological Slimes on Paper and Board Machines

Biological slimes (biofilms) collected from the wet end of paper and board machines were examined by electron microscopy and analyzed for fatty acid composition, neutral sugar composition, and ATP. Electron microscopy revealed minuscule prokaryotic organisms (diameter, 0.2 to 0.4 μm). Larger cells morphologically resembling Sphaerotilus and Leptothrix spp. were found in slimes from machines using recycled fiber or unbleached pulp. The bacteria were embedded in a slimy matrix and often contained reserve materials microscopically resembling poly-β-hydroxybutyrate and glycogen. Fatty acid analysis of the slimes revealed bacterial signature fatty acids in concentrations equivalent to the presence of 2 × 10¹⁰ to 2.6 × 10¹² (average, 7 × 10¹¹) bacterial cells (live and dead) per g (dry weight) of slime. The slimes contained several known components of bacterial polysaccharides in addition to glucose, indicating that the slime body consisted of bacterial polysaccharides. The slimes contained uronic acids equivalent to a binding capacity of 12.5 to 50 μmol of divalent cations per g (dry weight) of slime. The uronic acid-containing polysaccharides may be responsible for the accumulation of heavy metals in the slime. Calculation of the ATP contents of the slimes resulted in an estimate of 5 × 10¹² cells per g (dry weight) of slime when calibrated with pure bacterial cultures isolated from the slimes. From electron micrographs, an estimate ranging from 1 × 10¹⁰ to 1.5 × 10¹² (average, 4 × 10¹¹) cells per g (dry weight) of slime was obtained.     

Extracellular Secretion of Noncatalytic Plant Cell Wall-Binding Proteins by the Cellulolytic Thermophile Caldicellulosiruptor bescii

Caldicellulosiruptor bescii efficiently degrades cellulose, xylan, and native grasses at high temperatures above 70°C under anaerobic conditions. C. bescii extracellularly secretes multidomain glycoside hydrolases along with proteins of unknown function. In this study, we analyzed the C. bescii proteins that bind to the cell walls of timothy grass by using mass spectrometry, and we identified four noncatalytic plant cell wall-binding proteins (PWBPs) with high pI values (9.2 to 9.6). A search of a conserved domain database showed that these proteins possess a common domain related to solute-binding proteins. In addition, 12 genes encoding PWBP-like proteins were detected in the C. bescii genomic sequence. To analyze the binding properties of PWBPs, recombinant PWBP57 and PWBP65, expressed in Escherichia coli, were prepared. The PWBPs displayed a wide range of binding specificities: they bound to cellulose, lichenan, xylan, arabinoxylan, glucuronoxylan, mannan, glucomannan, pectin, oligosaccharides, and the cell walls of timothy grass. The proteins showed the highest binding affinity for the plant cell wall, with association constant (K_a) values of 5.2 × 10⁶ to 44 × 10⁶ M⁻¹ among the insoluble polysaccharides tested, as measured using depletion binding isotherms. Affinity gel electrophoresis demonstrated that the proteins bound to the acidic polymer pectin most strongly among the soluble polysaccharides tested. Fluorescence microscopic analysis showed that the proteins bound preferentially to the cell wall in a section of grass leaf. Binding of noncatalytic PWBPs with high pI values might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures.     

Prebiotic Activity of Purified Xylobiose Obtained from Ragi (Eleusine coracana, Indaf-15) Bran

The role of prebiotics in improving human health has attracted global attention and the research is mostly focused on the strains belonging to the genera Bifidobacterium and Lactobacillus. Non-digestible oligosaccharides hold significant role in recent research due to their prebiotic nature. Soluble polysaccharides (SP, 14.4%), isolated from ragi bran consisted mainly of arabinose and xylose with minor quantities of rhamnose, mannose, galactose and glucose. Ragi bran SP subjected to purified endoxylanase (from 96 h ragi malt) treatment to obtain xylo-oligosaccharides which were further purified on Biogel P-2 followed by HPLC. The purified oligosaccharide yielded (RO-1; 17.9%) was identified as xylobiose by electrospray ionization mass spectrometry (282 + 23 = 305) and ¹HNMR. In vitro studies carried out using Bifidobacterium and Lactobacillus sp. proved the prebiotic nature of the crude xylo-oligosaccharides (XOs) and RO-1. Acetate was found to be the chief short chain fatty acid released during fermentation of both crude XOs and purified xylobiose and 24 h bacterial culture showed high xylanase activity (1020–1690 μU min⁻¹).     

Immunologically active polysaccharides of acanthopanax senticosus

From the alkaline aqueous extract of Acanthopanax (Eleutherococcus) senticosus two homogeneous polysaccharides, a glucan with a mean M, of 150 000 and a heteroxylan with a mean M, of 30 000, have been isolated by DEAE-Sepharose CL-6B and Sephacryl S-400 column chromatography. Their structures were elucidated mainly by permethylation, periodate oxidation, Smith degradation, reduction experiments, partial hydrolysis and ¹³C NMR. The isolated crude polysaccharide mixture and the heteroxylan enhance phagocytosis in in vitro and in vivo immunological tests.     

Characterization of the Lipopolysaccharides and Capsules of Shewanella spp.

Electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining and ¹H, ¹³C, and ³¹P-nuclear magnetic resonance (NMR) were used to detect and characterize the lipopolysaccharides (LPSs) of several Shewanella species. Many expressed only rough LPS; however, approximately one-half produced smooth LPS (and/or capsular polysaccharides). Some LPSs were affected by growth temperature with increased chain length observed below 25°C. Maximum LPS heterogeneity was found at 15 to 20°C. Thin sections of freeze-substituted cells revealed that Shewanella oneidensis, S. algae, S. frigidimarina, and Shewanella sp. strain MR-4 possessed either O-side chains or capsular fringes ranging from 20 to 130 nm in thickness depending on the species. NMR detected unusual sugars in S. putrefaciens CN32 and S. algae BrY^DL. It is possible that the ability of Shewanella to adhere to solid mineral phases (such as iron oxides) could be affected by the composition and length of surface polysaccharide polymers. These same polymers in S. algae may also contribute to this opportunistic pathogen's ability to promote infection.     

Differential Diagnosis of Malaria on Truelab Uno?, a Portable,Real-Time,MicroPCR Device for Point-Of-Care Applications

Background

Sensitive and specific detection of malarial parasites is crucial in controlling the significant malaria burden in the developing world. Also important is being able to identify life threatening Plasmodium falciparum malaria quickly and accurately to reduce malaria related mortality. Existing methods such as microscopy and rapid diagnostic tests (RDTs) have major shortcomings. Here, we describe a new real-time PCR-based diagnostic test device at point-of-care service for resource-limited settings.

Methods

Truenat^® Malaria, a chip-based microPCR test, was developed by bigtec Labs, Bangalore, India, for differential identification of Plasmodium falciparum and Plasmodium vivax parasites. The Truenat Malaria tests runs on bigtec’s Truelab Uno^® microPCR device, a handheld, battery operated, and easy-to-use real-time microPCR device. The performance of Truenat^® Malaria was evaluated versus the WHO nested PCR protocol. The Truenat^® Malaria was further evaluated in a triple-blinded study design using a sample panel of 281 specimens created from the clinical samples characterized by expert microscopy and a rapid diagnostic test kit by the National Institute of Malaria Research (NIMR). A comparative evaluation was done on the Truelab Uno^® and a commercial real-time PCR system.

Results

The limit of detection of the Truenat Malaria assay was found to be <5 parasites/μl for both P. falciparum and P. vivax. The Truenat^® Malaria test was found to have sensitivity and specificity of 100% each, compared to the WHO nested PCR protocol based on the evaluation of 100 samples. The sensitivity using expert microscopy as the reference standard was determined to be around 99.3% (95% CI: 95.5–99.9) at the species level. Mixed infections were identified more accurately by Truenat Malaria (32 samples identified as mixed) versus expert microscopy and RDTs which detected 4 and 5 mixed samples, respectively.

Conclusion

The Truenat^® Malaria microPCR test is a valuable diagnostic tool and implementation should be considered not only for malaria diagnosis but also for active surveillance and epidemiological intervention.     

Free radical scavenging of Ganoderma lucidum polysaccharides and its effect on antioxidant enzymes and immunity activities in cervical carcinoma rats

Ganoderma lucidum are used as traditional edible and medicinal materials in China. In this study, antioxidant activities of polysaccharides from G. lucidum in China were investigated. The influence of G. lucidum polysaccharides upon activities of serum antioxidant enzymes and immunity in rats with cervical cancer. The antioxidant activity was measured by DPPH^?, O^?, and OH^? free radicals scavenging. Results showed that G. lucidum polysaccharides exhibited the higher DPPH^?, O^?, and OH^? free radicals scavenging activities. The results still showed that G. lucidum polysaccharides could significantly enhance the antioxidant enzyme activities (SOD, CAT and GPx), and reduce levels of IL-1β, IL-6 and TNF-α in rats with cervical cancer.     

Bioremoval capacity of three heavy metals by some microalgae species (Egyptian Isolates)

Three fresh water microalgal isolates [Phormidium ambiguum (Cyanobacterium), Pseudochlorococcum typicum and Scenedesmus quadricauda var quadrispina (Chlorophyta)] were tested for tolerance and removal of mercury (Hg²⁺), lead (Pb²⁺) and cadmium (Cd²⁺) in aqueous solutions as a single metal species at conc. 5–100 mg / L under controled laboratory conditions. The obtained results showed that Hg²⁺ was the most toxic of the three metal ions to the test algae even at low concentration (< 20 mg/L). While lower concentration of Pb²⁺ and Cd²⁺ (5–20 mg / L) enhanced the algal growth (chlorophyll a and protein), elevated concentrations (40–100 mg / L) were inhibitory to the growth. The results also revealed that Ph. ambiguum was the most sensitive alga to the three metal ions even at lower concentrations (5 and 10 mg / L) while P. typicum and S. quadricauda were more tolerant to high metal concentrations up to 100 mg / L. The bioremoval of heavy metal ions (Hg²⁺, Pb²⁺ and Cd²⁺) by P. typicum from aqueous solution showed that the highest percentage of metal bioremoval occurred in the first 30 min of contact recording 97% (Hg²⁺), 86% (Cd²⁺) and 70% (Pb²⁺). Transmission electron microscopy (TEM) was used to study the interaction between heavy metal ions and P. typicum cells. At ultrastructural level, an electron dense layers were detected on the algal cell surfaces when exposed to Cd, Hg and Pb. At the same time, dark spherical electron dense bodies were accumulated in the vacuoles of the algal cells exposed to Pb. Excessive accumulation of starch around the pyrenoids were recorded as well as deteriorations of the algal cell organelles exposed to the three metal ions.     

Effect of extraction temperature on the diffusion coefficient of polysaccharides from Spirulina and the optimal separation method

The extraction temperature had a significant impact on the concentration of polysaccharides derived from solid-liquid extraction of Spirulina. The polysaccharide concentration was significantly higher when the extraction was performed at 90°C than when it was performed at 80, 70, and 50°C. This result is related to the diffusion coefficients of the polysaccharides, which increased from 1.07 × 10^?12 at 50°C to 3.02 × 10^?12 m²/sec at 90°C. Using the Arrhenius equation, the pre-exponential factor (D ₀ ) and the activation energy (E _a ) for Spirulina polysaccharide extraction were calculated as 7.958 × 10^?9 m²/sec and 24.0 kJ/mol, respectively. Among the methods used for the separation of Spirulina polysaccharides, cetyltrimethylammonium bromide (CTAB, method I) and organic solvent (ethanol, in methods II and III) provided similar yields of polysaccharides. However, the separation of polysaccharides using an ultrafiltration (UF) process (method III) and ethanol precipitation was superior to separation via CTAB or vacuum rotary evaporation (method II). The use of a membrane with a molecular weight cut-off (MWCO) of 30 kDa and an area of 0.01 m² at a feed pressure of 103 kPa with a mean permeate flux of 39.3 L/m²/h and a retention rate of 95% was optimal for the UF process. The addition of two volumes (v/v) of ethanol, which gave a total polysaccharide content of approximately 4% dry weight, was found to be most suitable for polysaccharide precipitation. The results of a Sepharose 6B column separation showed that the molecular weights of the polysaccharides in fractions I and II were 212 and 12.6 kDa, respectively.     

Cellulase immobilized magnetic nanoparticles for green energy production from Allamanda schottii L: Sustainability research in waste recycling

This study presents ethanol''s fabrication by fermenting the golden trumpet flower (Allamanda schottii L) with the yeast strain Saccharomyces cerevisiae. The changes in different parameters during fermentation were studied and optimized while producing the ethanol and the end product was subjected to emission test study by blending petrol and ethanol. The Allamanda floral substrate contains 65% polysaccharides. The strain S. cerevisiae was obtained in the form of baker’s yeast from a domestic shop. For 100 ml of slurry, the highest bioethanol yield recorded was about 18.75 ml via optimization of different culture conditions, including a 1:8 ratio for slurry preparation, maintained under 35 ⁰C, 5.5 pH, 72 h. old inoculum with a quantity of 3.75 g 100 ml⁻¹, fermented for120 h. The highest yield of bioethanol was acquired under the addition of urea. This technique & design is capable of industrial-scale fabrication of bioethanol by using A. schottii floral substrates. This research was conducted to fabricate ethanol by fermentation (A. schottii L) floral substrate with S. cerevisiae. The optimum physiochemical parameters required to obtain the highest yield of bioethanol from A. schottii flower by fermentation was studied. The immobilization strategy with a cheap agricultural substrate and magnetic nanoparticles were also studied. The engine performance and emission studies were done with different blends of petrol and bio-ethanol.     

Static and dynamic light-scattering studies of pectic polysaccharides from the middle lamellae and primary cell walls of cider apples

Mild, selective, and sequential methods have been used to extract pectic polysaccharides from cider apple pomace. The extracts have been characterised by static and dynamic light-scattering. CDTA (cyclohexane-trans-1,2-diaminetetra-acetate)-soluble pectic polysaccharides from the middle lamellae were characterised as having a broad-molecular-weight distribution (M_w = 4.2 × 10⁶)_of non-free-draining stiff coils. CDTA-insoluble and Na₂CO₃-soluble pectic polysaccharides (M_w = 1.1 × 10⁶) from the primary cell walls showed properties consistent with branched, cross-linked, microgel structures.     

Significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides produced by submerged culture of Tuber melanosporum

The significance of metal ion supplementation in the fermentation medium on the structure and anti-tumor activity of Tuber polysaccharides was systematically studied in the submerged fermentation of Tuber melanosporum. The lowest weight-average molecular weight (M_w) (i.e., 115.3 × 10⁴ g/mol) of intracellular polysaccharides (IPS) was obtained when Mg²⁺ and K⁺ was added in the fermentation medium. The IPS with the lower M_w exhibited a higher inhibition ratio against S-180 tumor cells. The compact conformation of extracellular polysaccharides (EPS) was formed when only K⁺ was supplied in the fermentation medium. Interestingly, EPS with compact conformation exhibited a higher inhibition ratio (i.e., 59.2%) than EPS with branched polymer chain (i.e., 9.2%) against A549 tumor cells. The highest inhibition ratio for EPS with α-glycosidic linkages against the tumor cell line HepG2 reached 32.2% when Mg²⁺ or K⁺ was supplied in the fermentation medium. The addition of metal ion Mg²⁺, K⁺, and their combination to the fermentation medium is a vital factor affecting the structures of Tuber polysaccharides, which further determine their anti-tumor activities. The information obtained in this work will be useful for the efficient and directed production of polysaccharides with anti-tumor activities by the submerged fermentation of edible fungi mycelium.     

Frequency of ace, epa and elrA Genes in Clinical and Environmental Strains of Enterococcus faecalis

Surface proteins play an important role in the pathogenesis of enterococcal infections. Some of them are candidates for a vaccine, e.g., the frequency of endocarditis in rats vaccinated with Ace protein was 75 % as 12 opposed to 100 % in those who weren’t. However, there are other components of enterococcal cells, such as Epa antigens or internalin-like proteins, which may be used in the prophylaxis of infections caused by them. However, also other virulence factors and resistance to antibiotics are important during enterococcal infection. Therefore, the relevance of ace, epa, elrA, other virulence genes, as well as resistance to antibiotics was investigated. 161 Enterococcus faecalis strains isolated from teaching hospitals in Lodz, cultured according to standard microbiological methods, were investigated for the presence of genes encoding surface proteins by PCR. Results were analyzed with χ² test. The elrA gene was found in all clinical and environmental strains, the ace gene was also widespread among E. faecalis (96.9 %). Both tested epa genes were found in the majority of isolates (83.25 %). There was correlation between the presence of esp and ace genes (p = 0.046) as well as between epa and agg genes (p = 0.0094; χ² test). The presence of the genes encoding surface proteins investigated in our study in the great majority of isolates implies that they would appear to be required during E. faecalis infection. Therefore, they could be excellent targets in therapy of enterococcal infections or, as some studies show, candidates for vaccines.     

Controlled Release Matrix Tablets of Olanzapine: Influence of Polymers on the <Emphasis Type="Italic">In Vitro</Emphasis> Release and Bioavailability

Controlled-release (CR) tablet formulation of olanzapine was developed using a binary mixture of Methocel® K100 LV-CR and Ethocel® standard 7FP premium by the dry granulation slugging method. Drug release kinetics of CR tablet formulations F1, F2, and F3, each one suitably compressed for 9-, 12-, and 15-kg hardness, were determined in a dissolution media of 0.1 N HCl (pH 1.5) and phosphate buffer (pH 6.8) using type II dissolution apparatus with paddles run at 50 rpm. Ethocel® was found to be distinctly controlling drug release, whereas the hardness of tablets and pH of the dissolution media did not significantly affect release kinetics. The CR test tablets containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness exhibited pH-independent zero-order release kinetics for 24 h. In vivo performance of the CR test tablet and conventional reference tablet were determined in rabbit serum using high-performance liquid chromatography coupled with electrochemical detector. Bioavailability parameters including C_max, T_max, and AUC_0–48 h of both tablets were compared. The CR test tablets produced optimized C_max and extended T_max (P < 0.05). A good correlation of drug absorption in vivo and drug release in vitro (R² = 0.9082) was observed. Relative bioavailability of the test tablet was calculated as 94%. The manufacturing process employed was reproducible and the CR test tablets were stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. It was concluded that the CR test tablet formulation successfully developed may improve tolerability and patient adherence by reducing adverse effects.Key words: bioavailability, controlled release, Ethocel®, olanzapine