The nature of oxygen in sporopollenin from the pollen of Typha angustifolia L

Native and peracetylated sporopollenin from the pollen of Typha angustifolia L. was investigated using several spectroscopic methods, inducing Fourier transform infrared spectroscopy (FTIR), solid-state 13C-nuclear magnetic resonance spectroscopy (13C-NMR) and X-ray photoelectron spectrometry (XPS). Interpretation of the experimental data shows that the greater part of oxygen found in sporopollenin originates from hydroxyl groups and must be derived from aliphatics and not from aromatics. This result indicates that not only aromatics and long unbranched aliphatics but also poly-hydroxyl aliphatic components are involved in the complex structure of the polymer. Furthermore, it is most probable that the monomers of the sporopollenin skeleton are linked by ether- and not by ester-linkage. Two possible approaches are suggested for the characterisation of sporopollenin structure.     

Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate

Poly(hydroxyalkanoates) PHAs are synthesized by many bacteria as inclusion bodies and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. A method based on FT-IR was developed to carry out rapid qualitative and quantitative analysis of PHAs in Pseudomonas, when they were grown on sodium octanoate. Using absorbance of the ester band of PHAs, a rapid method was reported to distinguish PHB and PHO and to determine polymer content in intact bacteria. Relative areas in which the C=O area was normalized to the area of the peak representing the amid group (1656 cm(-1)) characteristic of bacteria were calibrated to the polymer content which was determined after solvent extraction. Polymer contents vary from 0% to 53% and depend on the nature of the bacteria. Among 27 strains of Pseudomonas belonging to the rRNA homology group I, a very low amount of bacteria were able to produce PHB. The majority of strains were able to produce a copolymer, PHO, in which the major constituent unit is 3-hydroxyoctanoate. The FT-IR results were further confirmed by gas chromatography analysis after methanolysis of polymer, but FT-IR method requires less preparation of sample than gas chromatography and it is very useful for screening a large variety of Pseudomonas.     

Acetylation and silylation of piperidine solubilized sporopollenin from pollen of Typha angustifolia L

Silyl and acetyl derivatives of sporopollenin from the pollen of Typha angustifolia L. were prepared. The derivatized products were readily soluble in piperidine-d11 and could be investigated employing one- and two-dimensional proton and carbon NMR (nuclear magnetic resonance) spectroscopy (1H,1H-COSY and 13C,1H-HETCOR techniques). For the first time, a two dimensional 13C,1H-HETCOR NMR spectrum of a sporopollenin could be obtained. The results underline the importance of derivatization techniques for obtaining two dimensional 13C-NMR spectra of sporopollenins. Moreover, piperidine turns out to be a more suitable solvent for sporopollenins than 2-aminoethanol, as it allows for higher solubilities, being important for 2D-NMR investigations. From the HETCOR and COSY spectra of the silylated and the acetylated Typha samples the occurrence of aliphatic polyhydroxy compounds as well as phenolic OH groups became evident.     

Carbon isotopic composition of Ambrosia and Artemisia pollen: assessment of a C(3) -plant paleophysiological indicator

? There is limited evidence on how shifts in plant physiological performance influence vegetation variations in the paleorecord. ? To evaluate δ(13) C of pollen from C(3) plants as an indicator of community-level physiology, small quantities (10-30 grains) of untreated pollen and sporopollenin from herbarium specimens of Ambrosia (A.?tomentosa and A.?psilostachya) and Artemisia (A.?frigida, A.?ludoviciana and A.?dracunculus), genera abundant in grassland pollen profiles, were isolated by micromanipulation. Their δ(13) C values were measured using a spooling-wire microcombustion device interfaced with an isotope-ratio mass spectrometer. Leaf δ(13) C was also measured. Carbon isotope discrimination (Δ) for untreated pollen, sporopollenin and leaves was compared with historic records of seasonal precipitation amount, vapor pressure deficit and the Palmer Drought Severity Index (PDSI). ? Each species showed positive correlations between Δ of untreated pollen and sporopollenin. Sporopollenin Δ was most strongly correlated with PDSI. Correlations among leaf Δ and moisture indicators were stronger for Ambrosia than Artemisia. ? These results suggest that sporopollenin Δ indicates the level of moisture stress in C(3) plants. Therefore, δ(13) C analysis of pollen promises to help address important paleoecological questions, such as how community-level physiology contributes to shifts in vegetation composition.     

N-Chloroacyl-6-O-triphenylmethylchitosans: useful intermediates for synthetic modifications of chitosan

An efficient synthetic route was developed for the mild chloroacylation of chitosan with different chloroacyl chlorides. Full N-chloroacylation was obtained with this procedure without any O-acylation, and products having lower degrees of substitution can also be produced. Organo-soluble 6-O-triphenylmethylchitosan was used as a starting material for the acylation reactions. The resulting N-chloroacyl-6-O-triphenylmethylchitosan intermediates were also organo-soluble and characterized by FT-IR. N-Methylpiperazine moieties were attached to make end products that were sufficiently soluble for characterization by NMR and also to prove that the present intermediates could be used for further modifications. The end products were fully characterized by 1H NMR, 13C NMR, and 2D 1H-13C heteronuclear single-quantum correlation NMR spectroscopy. The degrees of substitution were determined by 1H NMR. Molecular weight determination by GPC-LS displayed a significant degradation of the polymer. The weight-average molar masses (M(w)) of the end products ranged from 29.6 to 49.4 kDa, when the M(w) of the starting material was 144.2 kDa.     

Comparative study of alkali- and acidic organic solvent-soluble hemicellulosic polysaccharides from sugarcane bagasse

Two-stage treatments of sugarcane bagasse with mild alkali and acidic 1,4-dioxane were performed. Pretreatment with 1M NaOH aqueous solution at 20, 25, 30, 35, and 40 degrees C for 18 h released 55.5%, 57.3%, 59.1%, 60.9%, and 62.1% of the original hemicelluloses, respectively. Post-treatment of the corresponding alkali-treated residue with 1,4-dioxane-2M HCl (9:1, v/v) at 87 degrees C for 2h, respectively, degraded 11.6%, 11.9%, 11.4%, 10.9%, and 10.6% of hemicelluloses (% dry starting material). It was found that the five alkali-soluble hemicellulosic preparations contained a much higher amounts of xylose (78.0-82.2%) and slightly higher uronic acids (4.8-5.8%), mainly 4-O-methyl-alpha-d-glucopyranosyluronic acid, but were lower in arabinose (9.3-11.7%) and glucose (2.2-4.1%) than those of the corresponding five acidic dioxane-degraded hemicellulosic fractions in which xylose (44.9-46.8%), arabinose (35.9-38.1%), and glucose (13.0-13.7%) were the major sugar constituents. The studies revealed that the five alkali-soluble hemicellulosic preparations were more linear and acidic, and had a large molecular weight (35,200-37,430 g mol(-1)) than those of the hemicellulosic fractions (12,080-13,320 g mol(-1)) degraded during the acidic dioxane post-treatment. This demonstrated that the post-treatment with acidic dioxane under the condition used resulted in substantial degradation of the hemicellulosic polymers. The 10 hemicellulosic samples were further characterized by FT-IR and 1H and 13C NMR spectroscopy, GPC and thermal analysis, and the results are reported.     

Chemolytic and solid-state spectroscopic evaluation of organic matter transformation during vermicomposting of sugar industry wastes

The molecular structure of humic acid (HA) extracted was investigated by FT-IR and (13)C CP/MAS NMR spectroscopy during the vermicomposting of sugar industry wastes, viz. pressmud, trash and bagasse for 60days. A rapid decrease in C/N and lignocellulosic (lignin, cellulose, hemicellulose) content was observed in vermicompost during early phase of the process. The FT-IR and (13)C CP/MAS NMR spectra of HA indicated a high rate of change in structure with increase in the alkyl C/O-alkyl C ratio during the process. Aromatic structures and carboxyl groups showed an initial increase but decreased after approximately 40days indicating extensive mineralization during final stages of vermicomposting.     

The resistant polymer of the walls of the hydrocarbon-rich alga Botryococcus braunii

The outer walls of the green alga Botryococcus braunii (main sites of hydrocarbon production and accumulation) show a complex constitution. They comprise a biopolymer highly resistant to non-oxidative degradation. The resistant polymer accounts for ca 9% of the cell dry wt and appears, along with hydrocarbons, as one of the major constituents of the alga. In addition to chemical resistance, B. braunii polymer exhibits other properties: mode of deposition and fluorescence, often used to identify sporopollenins. (Class of wall components generally regarded as originating from polymerization of carotenoid derivatives.) Nevertheless further studies, using IR spectroscopy and high resolution ¹³C NMR of solids, along with determination of elemental composition and unsaturation levels, indicate that the bulk of the resistant polymer from B. braunii outer walls does not derive from carotenoids; accordingly it cannot be considered, in this respect, as a sporopollenin. In fact the information obtained on the structure of this important constituent of the alga is consistent with its formation via oxidative polymerization of B. braunii dienic hydrocarbons.     

Stable isotope ratios of carbon and nitrogen in pollen grains in order to characterize plant functional groups and photosynthetic pathway types

Measurements of delta(13)C, delta(15)N and C : N ratios on modern pollen grains from temperate plants, including whole grains as well as extracted sporopollenin, were analysed in order to characterize physiological plant types at the pollen level and to determine the variation of these parameters in modern pollen grains of the same climatic area. Measurements are presented for 95 batches of whole modern pollen from 58 temperate species and on the stable fraction of modern pollen grains, chemically extracted sporopollenin, for two modern species. Fourier transform infrared (FTIR) and cross-polarization and magic-angle spinning (CP/MAS) sporopollenin spectra were conducted in parallel. C(3) and C(4) plants can be separated by delta(13)C measurements based on pollen. Probabilistic assignments to plant functional groups (herbaceous, deciduous woody, evergreen woody) of C(3) plants by the means of a discriminant analysis can be made for C : N ratios and for delta(13)C. The results are related to other studies on sporopollenin in order to use this method in future work on fossil samples. Stable isotope measurements on pollen allow improved pollen diagrams, including forms that cannot be differentiated at species level, increasing the accuracy and resolution of plant physiological type distribution in quaternary and older fossil sediments.     

Studies on sporopollenin biosynthesis in Tulipa anthers

Summary Extensive tracer experiments were carried out on Tulipa with the aim of determining the structure and biosynthesis of sporopollenin. The radiolabeled precursors were applied using an improved technique previously selected. The sporopollenin fraction was purified using either a gentle method — hydrolyzing enzymes (pronase, amylase, amyloglucosidase, cellulase, pectinase and lipase) and alkaline hydrolysis (method A) — or by a conventional aggressive procedure, where the material was enriched by alkaline hydrolysis and treated several days with 80% phosphoric acid (method B). The ¹⁴C-labeled precursors applied were mevalonate, glucose, acetate, malonic acid, phenylalanine, tyrosine, p-coumaric acid. Regardless of the method of enrichment, a higher level of incorporation into the sporopollenin fraction was always seen with [U-¹⁴C]-phenylalanine. The level of radioactivity found in sporopollenin labeled by phenylalanine or malonate was sufficiently high for the labeled polymer to be degraded and the products released analyzed for the first time. In the case of phenylalanine-labeled sporopollenin, the main degradation component, p-hydroxybenzoic acid, was also the most heavily labeled substance. This result was not dependent on the procedure used for sporopollenin enrichment. These findings are interpreted as meaning that phenylpropane metabolism via phenylalanine-ammonia lyase is involved in sporopollenin biosynthesis.     

FTIR characterisation of the chemical composition of Silurian miospores (cryptospores and trilete spores) from Gotland, Sweden

To better understand the biological affinities of cryptospores, micro-FTIR (Fourier transform infrared) spectroscopy analysis has been carried out on isolated specimens from the Upper Silurian of Gotland. The geobiochemical results have been compared to spectra of trilete spores, chitinozoans and leiospheres from the same sample. The palynomorphs are all very well preserved as attested by their pale yellow to orange colour indicative of a low thermal maturity. Micro-FTIR spectroscopy indicates that cryptospores display very similar spectra to those of the trilete spores, which are composed of sporopollenin characterised by absorption bands from aliphatic C-H in methylene (CH₂) and methyl (CH₃) groups, aromatic (C=C and C-H) groups and C=O groups of carboxylic acids. The sporopollenin composition of the cryptospore wall observed here is additional evidence demonstrating the embryophytic affinity of the cryptospores. In addition, several variations in other bands in the spectra of the different miospore morphospecies are evidenced and may be linked to their biological affinity or palaeoecological history.     

Anti-HIV-1 activity of poly(mandelic acid) derivatives

Homo- and heterochiral poly(mandelic acid)s (PMDAs) were synthesized under strongly acidic, mildly acidic, and nonacidic conditions. The water-soluble fractions of these polymers were evaluated with respect to their inhibitory activity against the human immunodeficiency virus (HIV-1). Polymers were prepared via a step-growth mechanism, yielding linear polyesters. The polymers were characterized by CHS elemental microanalysis, X-ray fluorescence (XRF), and FT-IR spectroscopy. Polymers prepared by the three methods have different structures. Both elemental microanalysis and XRF indicated the presence of S in those polymers prepared by treatment with concentrated H2SO4, which were the only ones exhibiting inhibitory and virucidal activity against HIV-1, mediated by their binding to cellular co-receptor binding sites on the virus envelope glycoprotein gp120. Additionally, FT-IR spectroscopy indicated the complete absence of C=O functionality in the H2SO4-prepared PMDA.     

Technical communication: modelling the rates of biodegradation,nitrogen fixation and uptake of fixed nitrogen during bioremediation of crude oil-contaminated environments

A biopolymer flocculant, produced by a haloalkalophilic Bacillus sp. I-471 was isolated from a sea water sample from the tidal mud flats surrounding the city of Inchon, Korea. The bioflocculant (EPS471) was produced in late logarithmic growth phase, recovered by cold ethanol precipitation of the cell-free supernatant and purified by cetylpyridinium chloride treatment. Chemical analyses of EPS471 indicated that it was an acidic polysaccharide containing neutral sugars, namely, galactose, fructose, glucose (approximate ratio of 5:2:1) and uronic acids as major and minor components, respectively. SEM studies revealed that the polymer had a porous structure with small pore size distribution indicating the compactness of the polymer. Spectroscopic analyses of the polymer by nuclear magnetic resonance (NMR) and Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of carboxyl and hydroxyl groups typical for a polysaccharide and that the polymer was a heteroglycan. The degradation temperature (T _d) analyzed by thermogravimetry was 307 °C. The rheological analysis showed the pseudoplastic nature of the polymer with shear-thinning behaviour. The polymer EPS471 may find possible application as an polymer for environmental bioremediation and other biotechnological processes.     

Structure of two new aminophospholipids from Methanobacterium thermoautotrophicum.

The methanogenic bacterium Methanobacterium thermoautotrophicum (A.T.C.C. 29183) was shown to contain two new aminophospholipids. These are 2-aminoethyl phosphate ester of diphytanylglycerol diether and a sugar containing bisdiphytanyldiglycerol tetraether. The two aminophospholipids were stable to acid methanolysis except for the sugar on the bisdiphytanyldiglycerol tetraether. Strong acid (6 M-HCl) hydrolysed the alkyl ether and aminophosphate ester bonds. The structure of the phosphate linkage was demonstrated by 31P n.m.r., and the 2-ethanolamine structure was elucidated by 1H- and 13C-n.m.r. spectroscopy and by fast-atom-bombardment m.s.     

Transesterification of soybean oil catalyzed by sulfated zirconia

Two sulfated zirconias were synthesized and characterized by X-ray diffraction and infrared spectroscopy. They were used as catalysts in the alcoholysis of soybean oil and in the esterification of oleic acid. Using sulfated zirconia prepared by the solvent-free method (S-ZrO(2)) as catalyst, the alcoholysis conversions of soybean oil under optimized conditions (120 degrees C, 1h and 5wt% of catalyst) were 98.6% (methanolysis) and 92% (ethanolysis), respectively. The esterification of oleic acid with methanol was complete after 2h. Zirconia sulfated by standard methods (SZ) had low activity in the methanolysis of soybean oil (conversion of 8.5%) and conventional zirconia (NS) was inactive for methanolysis under the conditions optimized for S-ZrO(2).     

Acidic glycolipids from kidney of suncus (Insectivora)

Lipids were extracted from the kidney of house musk shrew (Suncus murinus), which belongs to the order Insectivora. Acidic glycosphingolipids were purified from lipid extracts by mild alkaline methanolysis followed by column chromatographies on DEAE-Sephadex and silica beads (Iatrobeads). Purified glycolipids were analyzed by thin-layer chromatography, mild acid hydrolysis, gas liquid chromatography of the methyl glycosides after methanolysis and gas chromatography-mass spectrometry of the partially methylated alditol acetates. The kidney of suncus was unique in that it contained ganglioside GM2 (NeuAc type, 28.7 nmol and NeuGc type, 15.8 nmol/g tissue) as the major ganglioside. GM4 (NeuAc) (2.6 nmol/g), and GM3 (NeuAc type, 11.5 nmol and NeuGc type, 8.7 nmol/g) were also present. The content (298.9 nmol/g) of galactosyl sulfatide (GalCer-I3-sulfate) was higher than the values reported previously for other animal species. The total amount of acidic groups in glycolipids of suncus kidney was compared with the values for the kidney of 4 placental mammals to obtain an allometric correlation: log Y = 0.266 + 0.780 log X where X designates body weight, kg and Y, total acidic groups, mumol. A highly significant correlation (r = 0.999) was obtained among values from 5 representative placental mammals which live in mesic environments, suggesting that acidic glycosphingolipids are essential for the kidney function.     

Antioxidant properties of the pollen exine polymer matrix

The antioxidant properties of exine polymer matrix which forms the outer layer of pollen grain wall were studied. The main component of this matrix is sporopollenin - a unique biopolymer resistant to mechanical and chemical damage. The samples of isolated exine, purified from soluble compounds, were studied with EPR using stable nitroxyl radical TEMPO and DMPO spin trap. At the same time, we analyzed changes in fluorescence of DCFH which detected ROS in the solution. It has been established that exine effectively reduces TEMPO radical and eliminates hydroxyl radical. Also, the fluorometric analysis demonstrated that the exine eliminated H2O2, and this ability significantly decreased after treatment of exine with feruloyl esterase or mild alkaline hydrolysis (1M NaOH), i.e. after hydrolysis of hydroxycinnamic acid esters. After harsh hydrolysis (4M NaOH, 170 degrees C) of ethers bonds a large amount of hydroxycinnamic acids has been released, and exines have lost their antioxidant capacity almost completely. The obtained results point to the ability of extracellular polymer matrix of the exine to eliminate free radicals and H2O2 during crucial periods of male gametophyte development. The participation of ferulic acid and, possibly, of other hydroxycinnamic acids of sporopollenin in these processes has been demonstrated.     

Studies of low molecular weight samples of glucuronans with various acetylation degree

Partially acetylated, high molecular weight glucuronans were produced by a Sinorhizobium meliloti mutant strain. Two native glucuronan samples with various degrees of acetylation were sonicated to obtain lower molecular weight samples and with low viscosity suitable for chemical modification and (13)C NMR experiments. The average degree of substitution (DS) of the polymer was estimated by Fourier transform infrared (FTIR) and NMR. (13)C NMR spectra were obtained and used to suggest a complete assignment of the signals. The nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multi-bond coherence (HMBC) experiments were used to elucidate connectivities between the various residues and deduce the linkage of these residues within the polysaccharide.     

Novel functional biodegradable polymer IV: pH-sensitive controlled release of fibroblast growth factor-2 from a poly(gamma-glutamic acid)-sulfonate matrix for tissue engineering

The acidic pH-sensitive controlled release of fibroblast growth factor-2 (FGF-2) from a biodegradable hydrogel without any denaturation of the FGF-2 was successfully performed by a combination of FGF-2 activity and acidic pH-sensitivity. We prepared semi-interpenetrating polymer network like hetero-gels (S72-netgels) composed of poly(gamma-glutamic acid) (gamma-PGA) and 72% sulfonated gamma-PGA (gamma-PGA-S72). S72-netgels including 36 mol % sulfonic acid (S72-netgel-36) showed wide acidic pH-sensitive deswelling properties at pH = 2.0-6.0, corresponding to the isoelectric point of carboxylic acid, because of the concentration of protons due to the neighboring sulfonic acids from the carboxylic acids. The S72-netgel-36 (the volume of hydrogel is 7.85 x 10(-2) cm3) can incorporate 280 ng of FGF-2 after 24 h immersion in Tris-HCl buffer (pH = 7.4), including 1.0 microg of FGF-2. The S72-netgel-36 still retained about 60% of the FGF-2 even after 15 days of incubation in fresh Tris-HCl buffer at 37 degrees C because of the stable interaction of FGF-2 with gamma-PGA-S72 in S72-netgel-36. The release of FGF-2 from the S72-netgel-36 was successfully controlled by alternating immersion in pH = 7.4 and acidic pH buffers. Furthermore, the FGF-2 released from the S72-netgel-36 retained its activity without denaturation because the gamma-PGA-S72 in S72-netgel-36 has a protective activity. The acidic pH-sensitive FGF-2 release property of the S72-netgel-36 without denaturation of the FGF-2 may be useful for tissue engineering fields such as neovascular treatment for ischemia and inflammation.     

Structural characterization of the O-chain polysaccharide of Aeromonas caviae ATCC 15468 lipopolysaccharide

The O-chain polysaccharide produced by a mild acid degradation of Aeromonas caviae ATCC 15468 lipopolysaccharide was found to be composed of L-rhamnose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose and phosphoglycerol. Subsequent methylation and CE-ESIMS analyses and 1D/2D NMR ((1)H, (13)C and (31)P) spectroscopy showed that the O-chain polysaccharide is a high-molecular-mass acidic branched polymer of tetrasaccharide repeating units with a phosphoglycerol substituent having the following structure: [structure: see text] where Gro represents glycerol and P represents a phosphate group.