Preparation and characterization of mucilage polysaccharide for biomedical applications

In the present investigation, the polysaccharide/mucilage from waste of Abelmoscus esculentus by modification in hot extraction using two different solvents (Acetone, Methanol) were extracted, characterized and further compared with seaweed polysaccharide for their potential applications. The percentage yield, emulsifying capacity and swelling index of this mucilage were determined. The macro algae and okra waste, gave high % yield (22.2% and 8.6% respectively) and good emulsifying capacity (EC% = 52.38% and 54.76% respectively) with acetone, compared to methanol (11.3% and 0.28%; EC% = 50%) (PH = 7) while swelling index was greater with methanol than acetone extracts respectively. The infrared (I.R.) spectrum of the samples was recorded to investigate the chemical structure of mucilage. Thermal analysis of the mucilage was done with TGA (Thermal Gravimetric Analyzer) and DSC (Differential Scanning Calorimeter) which showed both okra and algal polysaccharide were thermostable hydrogels.     

The fine structure of mucilage secreting cells ofHibiscus esculentus pods

Summary Large secretory cells are found in the pericarp of okra capsules. In early developmental stages these cells are conspicuous because of the great quantity of Golgi apparatus-derived, PAS positive substance stored between the protoplast and the cell wall. It is suggested that these cells may play a major role in the formation of okra mucilage.     

Aspects of the Differentiation of the Egg of the Moss Physcomitrium coorgense Broth

Oogenesis in Physcomitrium coorgense follows the course seenin lower archegoniates generally. However no evidence was foundof cytoplasmic autophagy, or, during maturation of the egg,of nucleo-cytoplasmic interaction of the kind reported in liverwortsand ferns. The internal lamellar system of the plastids dedifferentiatesalmost entirely, and there is a corresponding increase in thefrequency of osmiophilic droplets. Starch also disappears, andsimultaneously large quantities of mucilage appear in the cytoplasm.The mucilage is secreted into the venter of the archegoniumand envelopes the egg. The egg lacks a special osmiophilic membrane.     

A biological active artificial saliva formulation containing flower mucilage from Ceylon Spinach (Basella alba Linn.)

Ceylon Spinach (Basella albe) is an edible perennial vine found in tropical Asia and Africa, known as vegetables containing mucilage. Its mucilage from flowers was extracted by microwaving and precipitated with 95% ethanol. Five artificial saliva formulations composing of mucilage from Ceylon Spinach, calcium chloride (CaCl₂), potassium chloride (KCl) and sodium fluoride (NF) were developed. The best formulation No.5 containing 0.61% of the mucilage with the non-Newtonian pseudoplastic flow (8.9 ± 0.2 cP) and the wetting time (12.50 ± 2.24 min) similar to the normal human saliva was selected. This artificial saliva formulation exhibited biological activities including an antioxidative activity by DPPH free radical scavenging with the SC₅₀ of 14.26 ± 2.00 mg/ml (0.05 folds of ascorbic acid), and the adhesion inhibition of S. mutans on hydroxyapatite beads at 17.01 ± 7.75%, while the natural human saliva exhibited an increase bacterial adhesion of 33.10 ± 9.70%. The safety of this formulation which gave no cytotoxicity on normal human gingival fibroblasts at 99.20 ± 21.09% cell viability was also demonstrated. The results from this study have indicated high biological activity and safety of the developed formulation containing mucilage from Ceylon Spinach which is potential to be used as artificial saliva for xerostomia patients.     

Liquid crystal-type assembly of native cellulose-glucuronoxylans extracted from plant cell wall.

In numerous plant cell walls, the cellulose microfibrils are arranged in a helicoidal pattern which has been considered as an analog to a cholesteric order. Here, we report on the spontaneous helicoidal organization which occurs in acellular conditions from aqueous suspensions of cellulose. The cellulosic mucilage of mature seeds of quince (Cydonia oblonga L) was studied both in situ (pre-release mucilage) and after water extraction and in in vitro re-assembly (prolonged high speed ultracentrifugation, further progressive dehydration and embedding in LR White methacrylate or hydrosoluble melamine resin). The cellulosic component was characterized by the use of cellobiohydrolase (CBH1) bound to colloidal gold, and the glucuronic acid residues of the xylan matrix were characterized by the use of cationised gold. Inside the seeds, the pre-release mucilage is mostly helicoidal, with the occurrence of more or less ordered domains, which indicate a fluid organization relevant to an actual liquid crystal state. Cytochemical tests revealed the tight association between cellulose and glucuronoxylans, the latter constituting a charged coat around each microfibril. Following the hydration of the seed, a cellulosic suspension was extracted in which microfibrils were totally dispersed. The progressive dehydration of the suspension gave rise to concentrated viscous drops. Ultrastructural observations revealed the occurrence of multidomain organization, from non-ordered to cholesteric-like regions, revealing that the mucilage is at the same time crystalline and liquid. This constitutes the first demonstration that liquid crystal type assemblies can arise from crystalline and biological cellulose in aqueous suspension. It strengthens the hypothesis that a transient liquid crystal state must occur during the cellulose ordering. The possible morphogenetic role of the glucuronoxylans in the cholesteric organization of the cellulose is discussed.     

Comparison of the effect of the linseed extract Salinum® and a methyl cellulose preparation on the symptoms of dry mouth

The effect of a linseed extract Salinum® and a sodium carboxymethyl cellulose preparation called MAS-84 was compared with regard to its effect on the symptoms of dry mouth. Twenty patients with xerostomia, who had been treated for cancer in the head and neck by radiation were recruited from the clinic for maxillofacial surgery, Malmö University Hospital. Following radiation treatment the salivation was severely reduced. The symptoms of a general feeling of a dry mouth, difficulties in chewing and swallowing, taste disturbances, problems with speech and mouth burning were registered on a subjective verbal rating scale. In addition plaque index and gingival bleeding index were determined. The study design was crossover and performed single blind. The experimental period was 7 weeks. The patients were randomly divided into 2 groups, One group used Salinum® and the other MAS-84 for 3 weeks. The fourth week was a wash out period and for the next three weeks the patients shifted preparation. Each of the preparations was used ad libitium. Registrations of the various parameters were undertaken on days 0, 7 and 21 of the respective period. At the initial examination all patients reported considerable disturbances from mouth-dryness. These symptoms were reduced in 15 patients during the Salinum® period and in 9 during the MAS-84 period. The relief was significantly more pronounced during the use of Salinum® compared to that during the use of the methyl cellulose preparation. On day 21 plaque and gingival bleeding were significantly reduced during the Salinum® period but not during the MAS-84 period. The results of the present study confirm those of a previous pilot study and indicate that the linseed mucilage significantly reduced the symptoms of dry mouth. This effect increased with increasing time of saliva substitute use. The linseed mucilage Salinum® appeared to be a suitable saliva replacement in mouth dry patients.     

Dynamics of microcystin-degrading bacteria in mucilage of <Emphasis Type="Italic">Microcystis</Emphasis>

To reveal the process of degradation of hepatotoxic microcystin produced in Microcystis cells during the Microcystis bloom period, we used fluorescence in situ hybridization (FISH) to analyze the population dynamics of microcystin-degrading bacteria in Microcystis mucilage. We designed and applied an oligonucleotide probe targeted to the 16S rRNA sequence of strain Y2 of a microcystin-degrading bacterium (MCD-bacterium), which was isolated from Lake Suwa, Japan. In both the 1998 and 1999 tests, FISH clearly showed that MCD-bacteria existed in the mucilage and that, when a high concentration of cell-bound microcystin was detected, MCD-bacteria exceeded 10% of the sum of bacteria hybridized with group-specific probes. The concentration of MCD-bacteria was highest in summer 1998, when a toxic species, M. viridis, was dominant. There was a high correlation between the number of MCD-bacteria in the mucilage and the concentration of cell-bound microcystin in the lake. Our results suggest that MCD-bacteria responded to changes in the concentration of microcystin and degraded the microcystin when it was released from Microcystis cells. We also analyzed changes in the bacterial community structure associated with the Microcystis colonies by using domain- and group-specific oligonucleotide probes. Changes in the concentrations of the Cytophaga/Flavobacterium group and -Proteobacteria, which can degrade macromolecules derived from Microcystis cells, were synchronized with changes in the concentration of Microcystis. The results not only suggest the significant role of MCD-bacteria in detoxification, but also demonstrate a possible sequence of degradation from Microcystis cells to microcystin maintained in the cell, which is then carried out by bacterial consortia in the mucilage.     

Fluorescence Spectroscopy as a Tool to Unravel the Dynamics of Protein Nanoparticle Formation by Liquid Antisolvent Precipitation

Protein-based particles are very promising colloidal systems for protection and controlled release applications in the food, cosmetics and pharmaceutical sector. One technique to produce these protein colloidal particles is liquid antisolvent precipitation (LAS). Despite the simplicity and versatility of LAS, not much is known about the protein conformational changes and interactions that are at the basis of the particle formation process. In this study, steady state fluorescence experiments using intrinsic fluorophores were evaluated as a tool to unravel the dynamics of the protein nanoparticle formation. Colloidal whey protein isolate and gliadin particles were produced by LAS. Changes in particle diameter (distribution), polydispersity index and photophysical properties of intrinsic fluorophores were monitored as a function of antisolvent concentration. By combining dynamic light scattering with photophysical data, a model of the changes occurring during particle formation and disintegration could be proposed. The results suggest that particle formation and disintegration are fully reversible processes during which the main changes in protein conformation (around the fluorescent probes) occur at the same antisolvent concentrations. In principle, steady state fluorescence measurements using intrinsic probes can indeed be used to effectively report on (part of the) conformational changes for both protein systems under study.     

Calcium measurements in organelles with Ca2+-sensitive fluorescent proteins

The recent improvement in the design and use of genetically encoded fluorescent Ca2+ indicators should foster major progress in three aspects of Ca2+ signaling. At the subcellular level, ratiometric probes with expanded dynamics are now available to measure accurately the local Ca2+ changes occurring within specific cell compartments. These tools will allow to determine precisely the role of organelles and of cellular microdomains in Ca2+ handling. At the cellular level, the permanent labeling offered by the genetic probes enables large-scale, long-term Ca2+ measurements with robotic multiplexing technologies such as fluorescence plate readers or automated microscopes. This opens the way to large-scale pharmacological or genetic screens based on organelle-specific functional assays. At the whole animal level, probes with improved dynamics and reduced interference with endogenous proteins will allow to generate transgenic animals bearing Ca2+ sensitive indicators in specific cells and tissues. With this approach, Ca2+ signals can be recorded in neurons, heart, and pancreas of live animals during physiological and pathological stimulations. In this chapter, I will review the progress made in the design and use of genetic Ca2+ indicators, and discuss how these new tools provide an opportunity to challenge several unsolved questions in Ca2+ signaling.     

Climate Change and the Potential Spreading of Marine Mucilage and Microbial Pathogens in the Mediterranean Sea

Background

Marine snow (small amorphous aggregates with colloidal properties) is present in all oceans of the world. Surface water warming and the consequent increase of water column stability can favour the coalescence of marine snow into marine mucilage, large marine aggregates representing an ephemeral and extreme habitat. Marine mucilage characterize aquatic systems with altered environmental conditions.

Methodology/Principal Findings

We investigated, by means of molecular techniques, viruses and prokaryotes within the mucilage and in surrounding seawater to examine the potential of mucilage to host new microbial diversity and/or spread marine diseases. We found that marine mucilage contained a large and unexpectedly exclusive microbial biodiversity and hosted pathogenic species that were absent in surrounding seawater. We also investigated the relationship between climate change and the frequency of mucilage in the Mediterranean Sea over the last 200 years and found that the number of mucilage outbreaks increased almost exponentially in the last 20 years. The increasing frequency of mucilage outbreaks is closely associated with the temperature anomalies.

Conclusions/Significance

We conclude that the spreading of mucilage in the Mediterranean Sea is linked to climate-driven sea surface warming. The mucilage can act as a controlling factor of microbial diversity across wide oceanic regions and could have the potential to act as a carrier of specific microorganisms, thereby increasing the spread of pathogenic bacteria.     

The FEI2-SOS5 pathway and CELLULOSE SYNTHASE 5 are required for cellulose biosynthesis in the Arabidopsis seed coat and affect pectin mucilage structure

A common adaptation in angiosperms is the deposition of hydrophilic mucilage into the apoplast of seed coat epidermal cells during the course of their differentiation. Upon imbibition, seed mucilage, composed mainly of carbohydrates (i.e. pectins, hemicelluloses and glycans) expands rapidly, encapsulating the seed and aiding in seed dispersal and germination. The FEI1/FEI2 receptor-like kinases and the SOS5 extracellular GPI-anchored protein were previously shown to act on a pathway regulating cellulose biosynthesis during Arabidopsis root elongation. In the highlighted study, we demonstrated that FEI2 and SOS5 regulate the production of the cellulosic rays deposited across the inner adherent-layer of seed mucilage. Mutations in either fei2 or sos5 disrupted the formation of rays, which was associated with an increase in the soluble, outer layer of pectin mucilage and accompanied by a reduction in the inner adherent-layer. Mutations in CELLULOSE SYNTHASE 5 also led to reduced rays and mal-partitioning of the pectic component of seed mucilage, further establishing a structural role for cellulose in seed mucilage. Here, we show that FEI2 expressed from a CaMV 35S promoter complemented both root and seed mucilage defects of the fei1 fei2 double mutant. In contrast, expression of FEI1 from a 35S promoter complemented the root, but not the seed phenotype of the fei1 fei2 double mutant, suggesting that unlike in the root, FEI2 plays a unique and non-redundant role in the regulation of cellulose synthesis in seed mucilage. Altogether, these data suggest a novel role for cellulose in anchoring the pectic component of seed mucilage to the seed surface and indicate that the FEI2 protein has a function distinct from that of FEI1, despite the high sequence similarity of these RLKs.     

Local Evolution of Seed Flotation in Arabidopsis

Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 β-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed.     

Extraction of geminiviral DNA from a highly mucilaginous plant (Abelmoschus esculentus)

A protocol is described for the extraction of geminiviral DNA from bhendi yellow vein mosaic virus-infectedAbelmoschus esculentus (known as bhendi or okra) containing high amounts of mucilage and other phenolic compounds. This method involves extraction with a buffer containing sodium citrate at pH 6 and PEG precipitation of the virus followed by alkali lysis. The extraction buffer eliminates the mucilage and other polyphenols, PEG precipitates the viral particles and DNA and the alkali lysis enriches the replicative forms of the viral DNA. The extracted DNA could be digested with restriction enzymes and cloned without any interference from chromosomal DNA. The quality of the DNA extracted by this method was compared to three other common plant DNA extraction protocols and was found superior. This method was used for PCR amplification and cloning of the 2.7 kbp DNA-A of BYVMV.     

Cellulose synthesis via the FEI2 RLK/SOS5 pathway and cellulose synthase 5 is required for the structure of seed coat mucilage in Arabidopsis

The seeds of Arabidopsis thaliana and many other plants are surrounded by a pectinaceous mucilage that aids in seed hydration and germination. Mucilage is synthesized during seed development within maternally derived seed coat mucilage secretory cells (MSCs), and is released to surround the seed upon imbibition. The FEI1/FEI2 receptor-like kinases and the SOS5 extracellular GPI-anchored protein were shown previously to act on a pathway that regulates the synthesis of cellulose in Arabidopsis roots. Here, we demonstrate that both FEI2 and SOS5 also play a role in the synthesis of seed mucilage. Disruption of FEI2 or SOS5 leads to a reduction in the rays of cellulose observed across the seed mucilage inner layer, which alters the structure of the mucilage in response to hydration. Mutations in CESA5, which disrupts an isoform of cellulose synthase involved in primary cell wall synthesis, result in a similar seed mucilage phenotype. The data indicate that CESA5-derived cellulose plays an important role in the synthesis and structure of seed coat mucilage and that the FEI2/SOS5 pathway plays a role in the regulation of cellulose synthesis in MSCs. Moreover, these results establish a novel structural role for cellulose in anchoring the pectic component of seed coat mucilage to the seed surface.     

Lubricating Properties of the Initial Salivary Pellicle — an AFM Study

The role of saliva in the oral cavity is manifold; an important function is to serve as lubricant between hard (enamel) and soft (mucosal) tissues. Intraoral lubrication is of crucial importance in order to maintain functions such as deglutition, mastication and the faculty of speech. A large number of people suffer from impaired salivary functions, displaying symptoms such as ‘dry mouth’. This results in a need for methods to assess the lubricating properties of both native saliva and potential artificial saliva formulations. Here, normal as well as lateral forces, acting between adsorbed salivary films, have been measured for the first time by means of colloidal probe atomic force microscopy (AFM). It was found that the presence of salivary pellicles between hard surfaces reduces the friction coefficient by a factor of 20. This reduction of friction is consistent with the long-range purely repulsive nature of the normal forces acting between the salivary films. The lubricating mechanism is presumably based on a full separation of the sliding surfaces by the salivary films. The friction between salivary films has been investigated at normal loads that cover the clinical jaw closing forces, and it can be concluded that the lubricating properties are maintained within this load interval. The present study indicates the usefulness of colloidal probe AFM, which offers a direct and quantitative measure of lubrication at a molecular level, in the study of biotribological phenomena. In particular, the results obtained here may have implications for the development of saliva substitutes.     

Lubricating properties of the initial salivary pellicle--an AFM study

The role of saliva in the oral cavity is manifold; an important function is to serve as lubricant between hard (enamel) and soft (mucosal) tissues. Intraoral lubrication is of crucial importance in order to maintain functions such as deglutition, mastication and the faculty of speech. A large number of people suffer from impaired salivary functions, displaying symptoms such as 'dry mouth'. This results in a need for methods to assess the lubricating properties of both native saliva and potential artificial saliva formulations. Here, normal as well as lateral forces, acting between adsorbed salivary films, have been measured for the first time by means of colloidal probe atomic force microscopy (AFM). It was found that the presence of salivary pellicles between hard surfaces reduces the friction coefficient by a factor of 20. This reduction of friction is consistent with the long-range purely repulsive nature of the normal forces acting between the salivary films. The lubricating mechanism is presumably based on a full separation of the sliding surfaces by the salivary films. The friction between salivary films has been investigated at normal loads that cover the clinical jaw closing forces, and it can be concluded that the lubricating properties are maintained within this load interval. The present study indicates the usefulness of colloidal probe AFM, which offers a direct and quantitative measure of lubrication at a molecular level, in the study of biotribological phenomena. In particular, the results obtained here may have implications for the development of saliva substitutes.     

The emergence of okra enation leaf curl virus—an important begomovirus,infecting okra in several states across India

Okra enation leaf curl virus (OELCuV), a begomovirus, is an emerging serious constraint for okra production in India. OELCuV was earlier reported in Haryana, Gujarat and Karnataka. In the current study, a survey was conducted on okra crop in the other predominant okra growing regions of India and identified for the first time, a widespread symptomology of OELCuV. The disease incidence was recorded between 5 and 74% in all the surveyed regions. The diseased samples collected from all the locations were amplified with coat protein specific new primer in PCR. The amplicons were sequenced and deposited to NCBI Gene Bank. The finding could be highly useful in okra breeding programs against OELCuV.     

抱茎独行菜种皮粘液质相关基因TTG1的克隆、表达分析及功能鉴定

抱茎独行菜（Lepidium perfoliatum L.）为十字花科具典型粘液质繁殖体植物,而TTG1基因（Transpa-rent testa glabra 1）所编码的蛋白是调控种皮细胞分化并影响粘液质释放的转录因子。目前关于TTG1基因在粘液质繁殖体植物中的研究报道较少,为探究TTG1基因在抱茎独行菜粘液质发育中的作用,本研究利用同源克隆技术获得抱茎独行菜TTG1基因cDNA开放阅读框（ORF）序列,命名为LpTTG1。序列分析表明,该基因ORF全长为1032 bp,编码343个氨基酸,含有WD40基序;qRT-PCR分析结果显示,该基因在抱茎独行菜各组织中均有表达,反映了该基因功能的多样性;免疫组织化学定位结果表明,LpTTG1在种子发育过程中内珠被和外珠被的表达水平变化与外珠被粘液质的合成过程相一致,推测该基因可能参与调控抱茎独行菜种皮的发育及粘液质的形成。将LpTTG1基因转化拟南芥,该基因的过量表达显著促进了粘液质合成途径下游基因AtMUM4在角果中的表达,表明该基因有可能参与粘液质合成途径调控,并促进下游产物MUM4的产生。然而,对LpTTG1转基因拟南芥与野生型植株表型的比较发现,两者种子形态及粘液质分泌与释放方式均无显著差异,这可能是因为抱茎独行菜种皮发育和粘液质形成是一个多基因调控的复杂过程,某一基因的过量表达也许不会引起明显的表型变化。     

Shear field mapping in actin networks by using magnetic tweezers

An improved magnetic bead microrheometer based on phase contrast microscopy allowing high resolution measurements of local deformations within macromolecular networks is applied to study local viscoelastic properties of cross-linked actin networks. By embedding non-magnetic colloidal beads as probes into the networks, the spatial variation of the strain field within cross-linked actin networks can be mapped. Moreover, the Poisson ratio and shear modulus can be measured locally.