The role of the anion in the reaction of reducing sugars with ammonium salts

Reactions of reducing sugars with ammonia and its compounds are important commercially, particularly in the preparation of flavors and caramel colors. However, such reactions generally produce a complex series of products ranging from simple molecules to complex polymeric materials, particularly since commercial systems generally involve mixtures of sugars as opposed to single sugars. This complexity has made understanding the mechanisms of such reactions difficult. Therefore, investigatory work has generally been focused on model systems. Herein we report one such study with model systems: the effects of the nature of the anion of the reactions of reducing sugars with ammonium salts. D-Glucose was reacted in aqueous solution with each of the following ammonium salts: acetate, bicarbonate, carbonate, chloride, citrate, formate, monohydrogenphosphate (DAP), sulfate, and sulfite. These reactions were carried out in a Parr bomb at 93 degrees C for 2.5 h. The initial pH of the reaction mixtures was adjusted to pH 8.0 at 25 degrees C. The resulting mixtures were analyzed by LC-MS, and the results were analyzed by comparing the product yields and distributions with those obtained with DAP. The major reaction product of interest was 2,6-deoxyfructosazine, as it had been shown to be a marker for the polymeric material formed from such reactions. It was found that ammonium salts of weak acids were much more effective in effecting the desired reactions than were those of strong acids; however, none was as effective as DAP.     

Improved chromatographic separations on anion-exchange resins: I. Partition chromatography of sugars in ethanol

A resin conditioning/regeneration procedure is described which markedly enhances separation factors in partition chromatography of sugars on a commercially available anion-exchange resin. A mixture of 18 monosaccharides is well separated in less than 3 hr. A single ethanol-water solution is used as the eluent and no column temperature changes are necessary during a run. Sugars are located with a noncorrosive reagent.     

Kinetics of the simultaneous utilization of sugar mixtures by Escherichia coli in continuous culture.

In natural environments heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present at a concentration of a few micrograms per liter or even less. Under such conditions no significant growth would be expected if cells utilized only one of the available carbon compounds, as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon compounds simultaneously. Whereas the kinetics of single-substrate and diauxic growth are well understood, little is known about how microbial growth rates depend on the concentrations of several simultaneously utilized carbon sources. In this study this question was answered for carbon-limited chemostat growth of Escherichia coli fed with mixtures of up to six sugars; the sugars used were glucose, galactose, maltose, ribose, arabinose, and fructose. Independent of the mixture composition and dilution rate tested, E. coli utilized all sugars simultaneously. Compared with growth with a single sugar at a particular growth rate, the steady-state concentrations were consistently lower during simultaneous utilization of mixtures of sugars. The steady-state concentrations of particular sugars depended approximately linearly on their contributions to the total carbon consumption rate of the culture. Our experimental data demonstrate that the simultaneous utilization of mixtures of carbon sources enables heterotrophic microbes to grow relatively fast even in the presence of low environmental substrate concentrations. We propose that the observed reductions in the steady-state concentrations of individual carbon sources during simultaneous utilization of mixtures of carbon sources by heterotrophic microorganisms reflect a general kinetic principle.     

Development of high-throughput liquid chromatography injected ion mobility quadrupole time-of-flight techniques for analysis of complex peptide mixtures

The development of a multidimensional approach involving high-performance liquid chromatography (LC), ion mobility spectrometry (IMS) and tandem mass spectrometry is described for the analysis of complex peptide mixtures. In this approach, peptides are separated based on differences in their LC retention times and mobilities (as ions drift through He) prior to being introduced into a quadrupole/octopole/time-of-flight mass spectrometer. The initial LC separation and IMS dispersion of ions is used to label ions for subsequent fragmentation studies that are carried out for mixtures of ions. The approach is demonstrated by examining a mixture of peptides generated from tryptic digestion of 18 commercially available proteins. Current limitations of this initial study and potential advantages of the experimental approach are discussed.     

Kinetic models for the growth of Escherichia coli with mixtures of sugars under carbon-limited conditions

In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the micromax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low.     

Selective cloning of hybridoma cells for enhanced immunoglobulin production using flow cytometric cell sorting and automated laser nephelometry

Techniques for selective cloning of murine hybridoma cells by flow cytometric cell sorting and use of automated laser nephelometry to determine the resultant clones' immunoglobulin secretion levels are described. Using a commercially available attachment to a fluorescence-activated cell sorter, individual hybridoma cells were successfully distributed into microtiter wells in an automated manner based on their forward angle light scatter properties and their reaction to fluorescein-conjugated anti-mouse-IgG. The techniques were used to estimate successfully the frequency of immunoglobulin-secreting cells in established cultures. In addition, heterogeneity of cell surface immunoglobulin expression was observed and utilized as a criterion for flow sorting of new hybridoma variants. In these studies, clones derived from high (anti-IgG) intensity sorting regions yielded cultures with enhanced immunoglobulin secretion levels, as determined by automated laser nephelometry. Furthermore, the surface immunoglobulin phenotype of the derived clones was conserved in subsequent progeny. Finally, it was established that inclusion of propidium iodide in the hybridoma cell sorting mixtures improved cloning efficiency by facilitating enhanced discrimination and elimination of nonviable cells. Our results indicate that flow cytometric-assisted single cell deposition provides positive attributes of several traditional hybridoma cloning techniques and, in addition, furnishes a tool for steering the cloning process toward selection of enhanced immunoglobulin producing cultures.     

A general method for quantitative separation of prostaglandins by paper chromatography.

Although prostaglandin (PG) mixtures have previously been resolved by chromatography on silica-impregnated paper, drawbacks inherent in each technique have kept them from becoming generally accepted for routine analytical separations. Singh and co-workers (1,2) obtained excellent separation of prostaglandin mixtures on silica-impregnated glass fiber paper. However, this paper was not commercially available and its preparation is tedious. On the other hand, Stamford and Unger (3) separated PGE and PGF on commercially available paper using benzene/chloroform/acetone/methanol/acetic acid as developing solvent. Nevertheless, this solvent does not resolve less polar prostaglandins and fatty acids. More generally acceptable solvent systems cannot be used quantitatively with Stamford and Unger's technique due to irreversible binding of prostaglandins at the origin. Tobias and Paulsrud (11) have separated prostaglandins on commercial silicic acid-impregnated glass fiber sheets, but these are extremely brittle and difficult to accommodate to standard paper radiochromatogram scanners.This communication describes the quantitative chromatographic separation of PGF_1α, PGE₂, PGA₁, and arachidonic acid on commercially available Whatman SG-81 silica-impregnated paper using a wide variety of developing solvents. Irreversible binding of prostaglandins at the origin, previously a serious drawback, has been eliminated by applying the sample onto premoistened paper. This method is quantitative, sensitive, reproducible, and applicable to a variety of solvent systems. In addition, it is simple and inexpensive. Although loading capacity is somewhat limited, this is no problem with prostaglandins since they can be readily concentrated in organic solvents.     

Protein separations using colloidal magnetic nanoparticles

Phospholipid-coated colloidal magnetic nanoparticles with mean magnetite core size of 8 nm are shown to be effective ion exchange media for the recovery and separation of proteins from protein mixtures. These particles have high adsorptive capacities (up to 1200 mg protein/mL adsorbent, an order of magnitude larger than the best commercially available adsorbents) and exhibit none of the diffusional resistances offered by conventional porous ion exchange media. Protein-laden particles are readily recovered from the feed solution using high-gradient magnetic filtration.     

Total synthesis of an antigenic heptasaccharide motif found in the cell-wall lipooligosaccharide of <Emphasis Type="Italic">Mycobacterium gordonae</Emphasis> strain 989

An antigenic heptasaccharide motif of the cell-wall glycolipid of Mycobacterium gordonae strain 989 has been synthesized in a linear fashion by using a general glycosylation condition and minimum number of protecting group manipulation. All suitably protected monosaccharide intermediates were prepared from commercially available reducing sugars following some novel methodologies recently developed in our laboratory. Most of the synthetic intermediates were obtained as solid compounds in excellent yields.     

Standardization of the Romanowsky staining procedure: an overview

Commerically available Romanowsky blood stains are variable mixtures of thiazein dyes and brominated fluorescein derivatives with varying degrees of metallic salt contamination in a number of different solvent systems. There is a need for standardized Romanowsky stains of constant composition, which, when used in conjunction with a carefully controlled specimen preparation technique, should give consistent performance. Such a preparation system would be of great value to hematologists in general and would be essential to the validity of data obtained by the digital processing of blood cell images. It is possible to prepare standardized Romanowsky stains as mixtures of two or three dye components, namely, eosin Y, azure B and methylene blue, although azure B has only recently become commercially available at an acceptable degree of purity. The logistic problems of stain standardization are discussed.     

Concise chemical synthesis of a tetrasaccharide repeating unit of the <Emphasis Type="Italic">O</Emphasis>-antigen of <Emphasis Type="Italic">Hafnia alvei</Emphasis> 10457

Concise chemical synthesis of a tetrasaccharide repeating unit of the O-antigen of Hafnia alvei 10457 is reported. Construction of the tetrasaccharide as its 4-methoxyphenyl glycoside was achieved by condensation of less abundant monosaccharide units such as, D-galactofuranose, N-acetyl-D-galactosamine and N-acetylneuraminic acid. The synthetic strategy consists of the preparation of suitably protected required monosaccharide intermediates from the commercially available reducing sugars and high yielding glycosylation reactions.     

A short and efficient transformation of rhamnose into activated daunosamine, acosamine, ristosamine and epi-daunosamine derivatives, and synthesis of an anthracycline antibiotic acosaminyl-epsilon-iso-rhodomycinone

3-Amino-2,3,6-trideoxyhexopyranoses are essential constituents of most anthracycline antitumour antibiotics. For an investigation of structure-activity relationships, the four diastereomeric amino sugars daunosamine, acosamine, ristosamine, and epi-daunosamine were synthesised in short and efficient routes starting from commercially available rhamnose. Several glycosyl donors were provided and their use was exemplified in the synthesis of acosaminyl-epsilon-isorhodomycinone.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

Preparative-scale enzymic synthesis of D-[14C]ribulose 1,5-bisphosphate.

A procedure is described to prepare uniformly labelled D-[14C]ribulose 1,5-bisphosphate enzymically from uniformly labelled D-[14C]glucose through the coupled reactions catalysed by hexokinase (EC 2.7.1.1), glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and 5-phosphoribulokinase (EC 2.7.1.19). All reagents utilized in the method are commercially available. The procedure is a reliable preparative-scale method for synthesizing the dibarium salt of D-[14C]ribulose 1,5-biphosphate with a specific radioactivity up to 7 mCi/mmol and a purity near 90%. The final product was free of other 14C-labelled sugars, sugar phosphate esters, Pi and nucleotides.     

Photochemical addition of 2-propanol and of acetone to methyl 5-deoxy-2,3-O-isopropylidene-β-d-erythro-pent-4-enofuranoside

High-capacity adsorbents for lectins, including Lotus tetragonolobusl-fucose-binding protein, were readily prepared by conjugation of monosaccharides with commercially available, epoxy-activated Sepharose. Purified, radioiodinated lectins were bound to cells of the mosquito Aedes aegyptii and of human KB tumour. Relative to human KB cells, mosquito cells bound less of lectins specific for the sugars (l-fucose and d-galactose) that are terminal residues in many mammalian glycoproteins, whereas the number of binding sites of lectins specific for core-region sugars (d-mannose and 2-acetamido-2-deoxy-d-glucose) were similar. Neuraminidase, which greatly enhanced binding of peanut agglutinin or soybean agglutinin to human KB cells, had negligible effects on binding of these lectins to mosquito cells. The comparative structures of surface oligosaccharides of mosquito and KB cells are discussed in relation to the lectin-binding studies.     

Flow Cytometric Analysis of Electronic Nuclear Volume and DNA Content in Normal Mouse Tissues

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE QuantaTm for measuring EV and DNA content of different normal mice tissues.Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.     

Flow cytometric analysis of electronic nuclear volume and DNA content in normal mouse tissues

Light scatter is used in flow cytometry for identification of cells based on their size and/or granularity. However, forward light scatter is not an accurate measure of cell size. The measurement of Electronic Volume (EV) by Coulter principle is more accurate. However, EV cannot be measured on most of the commercially available flow cytometers. We have described the development and applications of a flow cytometer that can simultaneously measure Electronic Nuclear Volume (ENV) and DNA content. In the present study we have used a commercially available NPE Quanta for measuring EV and DNA content of different normal mice tissues. Fresh/frozen or formalin fixed-paraffin embedded tissues from mice were processed for isolation of nuclei, which were then analyzed for EV versus DNA content. By using these two parameters, distinct sub-populations were identified in liver, thymus, small intestine and bone marrow. Dual parametric analysis of EV versus DNA content can be a valuable technique for identification of sub-populations in heterogeneous cell mixtures such as those of complex tissues like bone marrow, intestine and tumors. The methods established are rapid and can provide valuable data for identification and characterization of sub-populations for cell cycle analysis by flow cytometry.     

Efficient conversion of sugarcane stalks into ethanol employing low temperature alkali pretreatment method

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and β-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12h, achieving 88% of the theoretical yield from the sugars and cellulose.     

Utilisation of tomato pomace as a substrate for the production of vitamin B12--a preliminary appraisal

The cellulose fraction in tomato pomace was hydrolysed using Trichoderma reesei, and the resultant sugars were fermented with Propionibacterium shermanii to produce vitamin B12. A multifactorial experiment revealed that aeration of the culture of T. reesei gave substantial improvements in cellulase activity as did higher concentrations of available nitrogen, but a rapid drop in pH appeared to inhibit extensive hydrolysis; after 14 days, the maximum level of cellulose degradation was only 34.4% of the total available, and the highest level of reducing sugars achieved was 15 g l(-1). When flasks with the latter concentration of reducing sugars were inoculated with P. shermanii, 11.1 mg l(-1) of B12 were produced under optimum conditions. If the degree of hydrolysis of the cellulose could be increased, then sufficient vitamin B12 might be generated to justify extraction but, even if purification does not prove to be economically feasible, a fermented tomato pomace (dried) with 50-55 mg kg(-1) or more of B12 could prove a useful feedstuff for animals.     

Tosylated and azidated inulins as key substrates for further chemical modifications to access inulin-based advanced materials: an inulin-based glycocluster

We successfully synthesized inulin tosylates by treating commercially available inulin with tosyl chloride and triethylamine in N,N-dimethylacetoamide at the ambient temperature for 24h. The subsequent S(N)2 reactions using sodium azide afford inulin azides that can act as useful substrates for the following Huisgen cycloaddition with alkyne-terminated β-lactoside. The resultant inulin derivative having multiple β-lactosides has excellent affinity towards a β-lactoside binding lectin (RCA(120)). This synthetic strategy has various advantages, such as non-fragmentation of the inulin mainchain and wide applications for various alkyne-terminated functional units. Our strategy can be, therefore, used to develop various inulin derivatives that are applicable for food and medicinal industries.