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d-Arabitol, an alternative sweetener to sugar, has low calorie content, high sweetness, low glycemic index, and insulin resistance-improving ability. In this study, d-arabitol-producing yeast strains were isolated from various commercial types of miso, and strain Gz-5 was selected among these strains. Phylogenetic tree analysis of the internal transcribed spacer sequence revealed that strain Gz-5 was distinct from Zygosaccharomyces rouxii, a major fermenting yeast of miso. The strain, identified as Zygosaccharomyces sp. Gz-5, grew better than other Z. rouxii in 150 g/L NaCl and produced 114 g/L d-arabitol from 295 g/L glucose in a batch culture for 8 days (0.386 g/g-consumed glucose). In a fed-batch culture, the yeast produced 133 g/L d-arabitol for 14 days, and the total d-arabitol amount increased by 1.75-fold. These results indicated that Zygosaccharomyces sp. Gz-5, a non-genetically modified strain, has excellent potential for the industrial production of d-arabitol., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

We characterized the pyrroloquinoline quinone (PQQ)-dependent dehydrogenase 9 (PQQ-DH9) of Gluconobacter sp. strain CHM43, which is a homolog of PQQ-dependent glycerol dehydrogenase (GLDH). We used a plasmid construct to express PQQ-DH9. The expression host was a derivative strain of CHM43, which lacked the genes for GLDH and the membrane-bound alcohol dehydrogenase and consequently had minimal ability to oxidize primary and secondary alcohols. The membranes of the transformant exhibited considerable d-arabitol dehydrogenase activity, whereas the reference strain did not, even if it had PQQ-DH9-encoding genes in the chromosome and harbored the empty vector. This suggests that PQQ-DH9 is not expressed in the genome. The activities of the membranes containing PQQ-DH9 and GLDH suggested that similar to GLDH, PQQ-DH9 oxidized a wide variety of secondary alcohols but had higher Michaelis constants than GLDH with regard to linear substrates such as glycerol. Cyclic substrates such as cis-1,2-cyclohexanediol were readily oxidized by PQQ-DH9., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

D-Arabitol is an important pentitol that is widely used in the food, pharmaceutical and chemical industries. It is mainly produced by yeasts during the biotransformation of glucose. To obtain strains with high D-arabitol production, Candida parapsilosis was mutated using atmospheric and room temperature plasma (ARTP). Among the screened mutants, mutant A6 had the highest yield at 32.92 g/L, a 53.98% increase compared with the original strain (21.38 g/L). Furthermore, metabolic regulators were added to the medium to improve D-arabitol production. Pyrithioxin dihydrochloride increased D-arabitol production by 34.4% by regulating glucose-6-phosphate dehydrogenase, and 4-methylpyrazole increased D-arabitol production by 77.4% compared with the control group by inhibiting alcohol dehydrogenase activity. Amphotericin B and Triton X-100 increased D-arabitol production by 23.8% and 42.2% by improving the membrane permeability and dissolved oxygen content, respectively. This study may provide important implications for obtaining high-yield D-arabitol strains.

Despite the increasing relevance, ranging from academic research to industrial applications, only a limited number of non-conventional, oleaginous Yarrowia lipolytica strains are characterized in detail. Therefore, we analyzed three strains in regard to their metabolic and physiological properties, especially with respect to important characteristics of a production strain. By investigating different cultivation conditions and media compositions, similarities and differences between the distinct strain backgrounds could be derived. Especially sugar alcohol production, as well as an agglomeration of cells were found to be connected with growth at high temperatures. In addition, sugar alcohol production was independent of high substrate concentrations under these conditions. To investigate the genotypic basis of particular traits, including growth characteristics and metabolite concentrations, genomic analysis were performed. We found sequence variations for one third of the annotated proteins but no obvious link to all phenotypic features., (© FEMS 2019.)

Six 1',5'-anhydrohexitol uridine triphosphates were synthesized with aromatic substitutions appended via a carboxamide linker to the 5-position of their bases. An improved method for obtaining such 5-substituted hexitol nucleosides and nucleotides is described. The incorporation profile of the nucleotide analogues into a DNA duplex overhang using recently evolved XNA polymerases is compared. Long, mixed HNA sequences featuring the base modifications are generated. The apparent binding affinity of four of the nucleotides to the enzyme, the rate of the chemical step and of product release, plus the specificity constant for the incorporation of these modified nucleotides into a DNA duplex overhang using the HNA polymerase T6G12_I521L are determined via pre-steady-state kinetics. HNA polymers displaying aromatic functional groups could have significant impact on the isolation of stable and high-affinity binders and catalysts, or on the design of nanomaterials., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Non-Saccharomyces yeasts impact wine fermentations and can diversify the flavor profiles of wines. However, little information is available on the metabolic networks of most of these species. Here we show that unlike the main wine yeast Saccharomyces cerevisiae, Torulaspora delbrueckii and to a lesser extent Lachancea thermotolerans produce significant concentrations of C5 and C6 polyols under wine fermentation conditions. In particular, D-arabitol, D-sorbitol and D-mannitol were produced at significant levels. Their release into the extracellular matrix started when that of glycerol ceased. The data also show that polyol production is influenced by initial sugar concentration, repressed by acetic acid and induced in ethanol supplemented media. Moreover, unlike glycerol and sorbitol, mannitol was partially re-assimilated when populations started to decline. The findings suggest that polyol synthesis is a physiological adaptation to stressful conditions characteristic of alcoholic fermentation and that these polyols may serve a similar purpose as glycerol production in S. cerevisiae, including osmoadaptation and redox balancing.

Arabitol belongs to the pentitol family and is used in the food industry as a sweetener and in the production of human therapeutics as an anticariogenic agent and an adipose tissue reducer. It can also be utilized as a substrate for chemical products such as arabinoic and xylonic acids, propylene, ethylene glycol, xylitol and others. It is included on the list of 12 building block C3-C6 compounds, designated for further biotechnological research. This polyol can be produced by yeasts in the processes of bioconversion or biotransformation of waste materials from agriculture, the forest industry (l-arabinose, glucose) and the biodiesel industry (glycerol). The present review discusses research on native yeasts from the genera Candida, Pichia, Debaryomyces and Zygosaccharomyces as well as genetically modified strains of Saccharomyces cerevisiae which are able to utilize biomass hydrolysates to effectively produce L- or D-arabitol. The metabolic pathways of these yeasts leading from sugars and glycerol to arabitol are presented. Although the number of reports concerning microbial production of arabitol is rather limited, the research on this topic has been growing for the last several years, with researchers looking for new micro-organisms, substrates and technologies., (© 2015 The Society for Applied Microbiology.)

An engineered strain for the conversion of D-fructose to allitol was developed by constructing a multi-enzyme coupling pathway and cofactor recycling system in Escherichia coli. D-Psicose-3-epimerase from Ruminococcus sp. and ribitol dehydrogenase from Klebsiella oxytoca were coexpressed to form the multi-enzyme coupling pathway for allitol production. The cofactor recycling system was constructed using the formate dehydrogenase gene from Candida methylica for continuous NADH supply. The recombinant strain produced 10.62 g/l allitol from 100 mM D-fructose. To increase the intracellular concentration of the substrate, the glucose/fructose facilitator gene from Zymomonas mobilis was incorporated into the engineered strain. The results showed that the allitol yield was enhanced significantly to 16.53 g/l with a conversion rate of 92 %. Through optimizing conversion conditions, allitol was produced effectively on a large scale by the whole-cell biotransformation system; the yield reached 48.62 g/l when 500 mM D-fructose was used as the substrate.

A new strain producing high yield of D-arabitol was isolated from hyperosmotic environments and the ITS rDNA sequencing analysis revealed it as Zygosaccharomyces rouxii. In addition, using a pH control and repeated-batch fermentation strategy in a 5-L reactor, the maximum yield and the highest volumetric productivity of D-arabitol were 93.48 ± 2.79 g/L and 1.143 g/L h, respectively. Volumetric productivity was successfully improved from 0.86 to 1.143 g/L h, which was increased by 32.9 % after 72 h of fermentation. Z. rouxii JM-C46 has potential to be used for D-arabitol and xylitol production from glucose via D-arabitol route.

Sugar alcohols have been widely applied in the field of food and medicine for their unique properties. Compared to chemical production, microbial production of sugar alcohol has become attractive for its environmental and sustainable pattern. In this study, a potential yeast isolated from soil of Beijing suburbs was identified as Pichia anomala TIB-x229, and its key enzyme of D-arabitol dehydrogenase for microbial production of sugar alcohols was functionally characterized. This yeast could simultaneously produce D-arabitol, xylitol, and/or ribitol from a different ratio of sugar substrates at a high efficiency by bioconversion, and no glucose repression happened when mixed sugars of xylose and glucose were used as the substrates during the bioconversion. This yeast could also efficiently convert complicated feedstock such as xylose mother liquor to D-arabitol, xylitol, and ribitol with 55 % yields. To elucidate the conversion relationship of the sugar alcohols, especially D-arabitol and xylitol, the key D-arabitol dehydrogenase gene from P. anomala was cloned, expressed and purified for further in vitro characterization. The results showed that this D-arabitol dehydrogenase could catalyze arabitol to xylulose further, which is significant for xylitol production from glucose. Our study laid the foundation for improving the production of sugar alcohols by metabolic and fermentation engineering strategies.

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

When an M31 strain of Agrobacterium tumefaciens was grown in a mineral salt medium at 30 °C containing 1.0% lactitol as sole carbon source, a keto-sugar was efficiently accumulated in the supernatant. This oxidation from lactitol to the keto-sugar was caused by M31 cells grown with medium containing a disaccharide unit, including sucrose, lactitol, lactose, maltose, or maltitol, suggesting that the enzyme is inducible. M31 also demonstrated good growth characteristics in Tryptic Soy Broth (TSB) medium containing 1.0% sucrose, and cells grown under these conditions showed strong lactitol transformation activity. The keto-sugar product was reduced by chemical hydrogenation and the resulting product was hydrolyzed to D-gulose, D-galactose, and D-sorbitol by acid hydrolysis, revealing that the reduced products are lactitol and D-gulosyl-(β-1,4)-D-sorbitol. Taken together, these results indicate that M31 can convert lactitol to 3-ketolactitol and thus provide access to the rare sugar D-gulose.

An endophytic yeast, Rhodotorula mucilaginosa strain PTD3, that was isolated from stems of hybrid poplar was found to be capable of production of xylitol from xylose, of ethanol from glucose, galactose, and mannose, and of arabitol from arabinose. The utilization of 30 g/L of each of the five sugars during fermentation by PTD3 was studied in liquid batch cultures. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. PTD3 demonstrated an exceptional ability to ferment the hydrolysate, overcome hexose repression of xylose utilization with a short lag period of 10 h, and tolerate sugar degradation products. In direct comparison, PTD3 had higher xylitol yields from the mixed sugar hydrolysate compared with the widely studied and used xylitol producer Candida guilliermondii.

D-arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, D: -arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in D-arabitol production. Within temperatures ranging from 25 to 39°C, the highest D-arabitol concentration of 99.2 mmol l(-1) was obtained from 555 mmol l(-1) of lactose after 120 h of batch cultivation at 37°C. The yield of D-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the D-arabitol concentration further increased to 168 mmol l(-1). According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for D-arabitol production.

A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.

Aims: The objective of this study was to examine the induction of thermotolerance in the biocontrol agent Candida sake CPA-1 cells by mild heat treatments to enhanced survival of formulations using spray-drying. The possible role of heat-shock proteins (HSPs) biosynthesis in induced thermotolerance and the role of sugars and sugar alcohols were also determined., Methods and Results: Studies were conducted on C. sake cells grown in molasses medium and exposed to mild temperatures of 30 and 33 degrees C during mid- (16 h), late-exponential (24 h), early- (30 h) and mid-stationary (36 h) growth phases. The effect on viability was determined both before and after spray-drying. Cycloheximide and chloramphenicol were used to examine the role of HSPs and HPLC was used to analyse the accumulation of sugar and sugar alcohols. The results indicate that both temperatures induced thermotolerance in cells of C. sake. Mild heat-adapted cells at 33 degrees C in the early- or mid-stationary phases had survival values after spray-drying significantly higher (P

The conversion specificity of Bacillus pallidus Y25 for polyols, including elusive rare sugar alcohols, was investigated. B. pallidus cells showed transformation potential for several rare polyols, including allitol, L-mannitol, D/L-talitol, and D-iditol, and converted them to their corresponding ketoses. This indicates that the bacterium had two polyol dehydrogenases specific for polyols that have D-erythro and D-threo configurations. By combination with intrinsic isomerases, polyols were converted directly to various aldoses, including L-xylose, L-talose, D-altrose, and L-glucose.

D-psicose, a new alternative sweetener, was produced from allitol by microbial oxidation of the newly isolated strain Enterobacter aerogenes IK7. Cells grown in tryptic soy broth medium (TSB) supplemented with D-mannitol at 37 degrees C were found to have the best oxidation potential. The cells, owing to broad substrate specificity, oxidized various polyols (tetritol, pentitol, and hexitol) to corresponding rare ketoses. By a resting cell reaction, 10% of allitol was completely transformed to the product D-psicose, which thus becomes economically feasible for the mass production of D-psicose. Finally, the product was crystallized and confirmed to be D-psicose by analytical methods.

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced D-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30 degrees C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 +/- 1.1 g D-arabitol from 175 +/- 1.1 g glucose per liter at pH 5.0, 30 degrees C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced D-arabitol from fructose, galactose, and mannose. The yeast produced D-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 +/- 1.9 g D-arabitol from 175 +/- 1.8 g glucose per liter in 210 h at pH 5.0, 30 degrees C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via D-arabitol route.

The accumulation of compatible solutes is often regarded as a basic strategy for the protection and survival of plants under abiotic stress conditions, including both salinity and oxidative stress. In this work, a possible causal link between the ability of contrasting barley genotypes to accumulate/synthesize compatible solutes and their salinity stress tolerance was investigated. The impact of H(2)O(2) (one of the components of salt stress) on K(+) flux (a measure of stress 'severity') and the mitigating effects of glycine betaine and proline on NaCl-induced K(+) efflux were found to be significantly higher in salt-sensitive barley genotypes. At the same time, a 2-fold higher accumulation of leaf and root proline and leaf glycine betaine was found in salt-sensitive cultivars. The total amino acid content was also less affected by salinity in salt-tolerant cultivars. In these, potassium was found to be the main contributor to cytoplasmic osmolality, while in salt-sensitive genotypes, glycine betaine and proline contributed substantially to cell osmolality, compensating for reduced cytosolic K(+). Significant negative correlations (r= -0.89 and -0.94) were observed between Na(+)-induced K(+) efflux (an indicator of salt tolerance) and leaf glycine betaine and proline. These results indicate that hyperaccumulation of known major compatible solutes in barley does not appear to play a major role in salt-tolerance, but rather, may be a symptom of salt-susceptibility.

Salt stress is one of the most important factors limiting plant cultivation. Many investigations of plant response to high salinity have been performed using conventional transcriptomics and/or proteomics approaches. However, transcriptomics and proteomics techniques are not all-encompassing methods that can achieve exclusive insights into the metabolite networks contributing to biochemical reactions. Hence, the functions of the complex stress response pathways are yet to be determined, especially at the metabolic level. A time-course metabolic profiling with Arabidopsis thaliana cell cultures after the imposition of salt stress is reported in this study. Analyses of primary metabolites, especially small polar metabolites such as amino acids, sugars, sugar alcohols, organic acids, and amines, was performed by GC/MS and LC/MS at 0.5, 1, 2, 4, 12, 24, 48, and 72 h after a salt-stress treatment with 100 mM NaCl being the final concentration. The mass chromatographic data were converted into matrix data sets, which were subjected to data mining processes, including principal component analysis (PCA) and batch-learning self-organizing mapping analysis (BL-SOM). The mining results suggest that the methylation cycle for the supply of methyl groups, the phenylpropanoid pathway for lignin production, and glycinebetaine biosynthesis are synergetically induced as a short-term response against salt-stress treatment. The results also suggest the the co-induction of glycolysis and sucrose metabolism as well as co-reduction of the methylation cycle as long-term responses to salt stress.

Aims: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance., Methods and Results: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied., Conclusions: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance., Significance and Impact of Study: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.

A NADP-dependent d-arabitol dehydrogenase gene was cloned from Gluconobacter oxydans CGMCC 1.110 and functionally expressed in Escherichia coli. With d-arabitol as sole carbon source, E. coli transformants grew rapidly in minimal medium, and produced d-xylulose. The enzymatic properties of the 29kDa enzyme were documented. The DNA sequence surrounding the gene suggested that it is part of an operon with several components of a sugar alcohol transporter system, and the d-arabitol dehydrogenase gene belongs to the short-chain dehydrogenase family.

The accumulation of sugars in grape berries requires the co-ordinate expression of sucrose transporters, invertases, and monosaccharide transporters. A monosaccharide transporter homologue (VvHT1, Vitis vinifera hexose transporter 1) has previously been isolated from grape berries at the veraison stage, and its expression was shown to be regulated by sugars and abscisic acid. The present work investigates the function and localization of VvHT1. Heterologous expression in yeast indicates that VvHT1 encodes a monosaccharide transporter with maximal activity at acidic pH (pH 4.5) and high affinity for glucose (K(m)=70 muM). Fructose, mannose, sorbitol, and mannitol are not transported by VvHT1. In situ hybridization shows that VvHT1 transcripts are primarily found in the phloem region of the conducting bundles. Immunofluorescence and immunogold labelling experiments localized VvHT1 in the plasma membrane of the sieve element/companion cell interface and of the flesh cells. The expression and functional properties of VvHT1 suggests that it retrieves the monosaccharides needed to provide the energy necessary for cell division and cell growth at an early stage of berry development.

Six genes of the Arabidopsis thaliana monosaccharide transporter-like (MST-like) superfamily share significant homology with polyol transporter genes previously identified in plants translocating polyols (mannitol or sorbitol) in their phloem (celery [Apium graveolens], common plantain [Plantago major], or sour cherry [Prunus cerasus]). The physiological role and the functional properties of this group of proteins were unclear in Arabidopsis, which translocates sucrose and small amounts of raffinose rather than polyols. Here, we describe POLYOL TRANSPORTER5 (AtPLT5), the first member of this subgroup of Arabidopsis MST-like transporters. Transient expression of an AtPLT5-green fluorescent protein fusion in plant cells and functional analyses of the AtPLT5 protein in yeast and Xenopus oocytes demonstrate that AtPLT5 is located in the plasma membrane and characterize this protein as a broad-spectrum H+-symporter for linear polyols, such as sorbitol, xylitol, erythritol, or glycerol. Unexpectedly, however, AtPLT5 catalyzes also the transport of the cyclic polyol myo-inositol and of different hexoses and pentoses, including ribose, a sugar that is not transported by any of the previously characterized plant sugar transporters. RT-PCR analyses and AtPLT5 promoter-reporter gene plants revealed that AtPLT5 is most strongly expressed in Arabidopsis roots, but also in the vascular tissue of leaves and in specific floral organs. The potential physiological role of AtPLT5 is discussed.

Sorbitol is a major photosynthetic product and a major phloem-translocated component in Rosaceae (e.g. apple, pear, peach, and cherry). We isolated the three cDNAs, MdSOT3, MdSOT4, and MdSOT5 from apple (Malus domestica) source leaves, which are homologous to plant polyol transporters. Yeasts transformed with the MdSOTs took up sorbitol significantly. MdSOT3- and MdSOT5-dependent sorbitol uptake was strongly inhibited by xylitol and myo-inositol, but not or only weakly by mannitol and dulcitol. Apparent K(m) values of MdSOT3 and MdSOT5 for sorbitol were estimated to be 0.71 mM and 3.2 mM, respectively. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), strongly inhibited the sorbitol transport. MdSOT3 was expressed specifically in source leaves, whereas MdSOT4 and MdSOT5 were expressed in source leaves and also in some sink organs. MdSOT4 and MdSOT5 expressions were highest in flowers. Fruits showed no or only weak MdSOT expression. Although MdSOT4 and MdSOT5 were also expressed in immature leaves, MdSOT expressions increased with leaf maturation. In addition, in situ hybridization revealed that all MdSOTs were expressed to high levels in phloem of minor veins in source leaves. These results suggest that these MdSOTs are involved in sorbitol loading in Rosaceae.

Fifty-five bacterial strains isolated from soil were screened for efficient poly-3-hydroxybutyrate (P3HB) biosynthesis from xylose. Three strains were also evaluated for the utilization of bagasse hydrolysate after different detoxification steps. The results showed that activated charcoal treatment is pivotal to the production of a hydrolysate easy to assimilate. Burkholderia cepacia IPT 048 and B. sacchari IPT 101 were selected for bioreactor studies, in which higher polymer contents and yields from the carbon source were observed with bagasse hydrolysate, compared with the use of analytical grade carbon sources. Polymer contents and yields, respectively, reached 62% and 0.39 g g(-1) with strain IPT 101 and 53% and 0.29 g g(-1) with strain IPT 048. A higher polymer content and yield from the carbon source was observed under P limitation, compared with N limitation, for strain IPT 101. IPT 048 showed similar performances in the presence of either growth-limiting nutrient. In high-cell-density cultures using xylose plus glucose under P limitation, both strains reached about 60 g l(-1) dry biomass, containing 60% P3HB. Polymer productivity and yield from this carbon source reached 0.47 g l(-1) h(-1) and 0.22 g g(-1), respectively.

In the previous X-ray crystallographic study, it was found that beta-amylase from Bacillus cereus var. mycoides has three carbohydrate-binding sites aside from the active site: two (Site2 and Site3) in domain B and one (Site1) in domain C. To investigate the roles of these sites in the catalytic reaction and raw starch-binding, Site1 and Site2 were mutated. From analyses of the raw starch-binding of wild-type and mutant enzymes, it was found that Site1 contributes to the binding affinity to raw-starch more than Site2, and that the binding capacity is maintained when either Site1 or Site2 exists. The raw starch-digesting ability of this enzyme was poor. From inhibition studies by maltitol, GGX and alpha-CD for hydrolyses of maltopentaose (G5) and amylose ( (n) = 16) catalyzed by wild-type and mutant enzymes, it was found that alpha-CD is a competitive inhibitor, while, maltitol behaves as a mixed-type or competitive inhibitor depending on the chain length of the substrate and the mutant enzyme. From the analysis of the inhibition mechanism, we conclude that the bindings of maltitol and GGX to Site2 in domain B form an abortive ESI complex when amylose ( (n) = 16) is used as a substrate.

Aims: The effect of osmotic and matric potential stress on growth and sugar alcohols (polyols: glycerol, erythritol, arabitol and mannitol) and sugars (trehalose and glucose) accumulation in toxigenic and nontoxigenic colonies of Aspergillus flavus and A. parasiticus was evaluated., Methods and Results: Growth of Aspergillus section Flavi with significant reductions at 20 and 30 degrees C was more sensitive to changes in matric potential, between 60 and 100% in the range of -7 to -14 MPa. No significant differences were found between toxigenic and nontoxigenic strains for both species. Total polyol accumulation in unamended maize meal agar medium (-0.75 MPa water potential) was higher at 30 than 20 degrees C. The major change in concentrations of endogenous sugars and total polyols was in matrically amended medium (with PEG 8000) at -7 and -10 MPa. Accumulation of glucose, arabitol, mannitol and erythritol content of A. flavus and A. parasiticus mycelial colonies was greater in normal unstressed maize meal agar medium (-0.75 Mpa) at 20 degrees C. This was modified by solute and matric stress., Conclusions: The data showed relative sensitivity to osmotic and matric potential, and temperature, and the impact on growth rates, polyol and sugar accumulation in mycelia of A. flavus and A. parasiticus., Significance and Impact of the Study: The matric potential effects on growth may be of particular importance for growth and survival in environments with low-matric potential stress. The tolerance of spoilage fungi such as Aspergillus section Flavi to such modifications could increase the potential for spoilage and mycotoxin production in such substrates. This knowledge is important for understanding the relative ecological fitness of these aflatoxigenic species and in the development of prevention strategies for their control.

Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated. Two proteins in the soluble fraction were found to have the ability to increase xylitol production. Both of these xylitol-increasing factors were purified, and on the basis of their NH(2)-terminal amino acid sequences the genes encoding both of the factors were cloned. Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolase/glucose-6-phosphate isomerase, and the other is ribulokinase. Using membrane and soluble fractions of G. oxydans, 3.8 g/l of xylitol were produced from 10 g/l D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolase/glucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 g/l respectively, confirming the identity of the xylitol-increasing factors.

A potent producer of D-arabitol was isolated by screening of natural sources and identified as Metschnikowia reukaufii AJ14787. Resting cells of this strain can efficiently produce D-arabitol from D-glucose with a weight yield of more than 60%, and can also produce D-arabitol from several other types of sugars such as polyols, ketoses, and aldoses. To improve productivity, various culture conditions such as temperature and the concentrations of D-glucose and nitrogen sources were examined. Under optimal conditions, 206 g/l of D-arabitol was produced from D-glucose with a weight yield of 52% in 100 hours.

Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity.

Microorganisms capable of producing xylitol from D-arabitol were screened for. Of the 420 strains tested, three bacteria, belonging to the genera Acetobacter and Gluconobacter, produced xylitol from D-arabitol when intact cells were used as the enzyme source. Among them, Gluconobacter oxydans ATCC 621 produced 29.2 g/l xylitol from 52.4 g/l D-arabitol after incubation for 27 h. The production of xylitol was increased by the addition of 5% (v/v) ethanol and 5 g/l D-glucose to the reaction mixture. Under these conditions, 51.4 g/l xylitol was obtained from 52.4 g/l D-arabitol, a yield of 98%, after incubation for 27 h. This conversion consisted of two successive reactions, conversion of D-arabitol to D-xylulose by a membrane-bound D-arabitol dehydrogenase, and conversion of D-xylulose to xylitol by a soluble NAD-dependent xylitol dehydrogenase. Use of disruptants of the membrane-bound alcohol dehydrogenase genes suggested that NADH was generated via NAD-dependent soluble alcohol dehydrogenase.

The D-sorbitol dehydrogenase gene, sldA, and an upstream gene, sldB, encoding a hydrophobic polypeptide, SldB, of Gluconobacter suboxydans IFO 3255 were disrupted in a check of their biological functions. The bacterial cells with the sldA gene disrupted did not produce L-sorbose by oxidation of D-sorbitol in resting-cell reactions at pHs 4.5 and 7.0, indicating that the dehydrogenase was the main D-sorbitol-oxidizing enzyme in this bacterium. The cells did not produce D-fructose from D-mannitol or dihydroxyacetone from glycerol. The disruption of the sldB gene resulted in undetectable oxidation of D-sorbitol, D-mannitol, or glycerol, although the cells produced the dehydrogenase. The cells with the sldB gene disrupted produced more of what might be signal-unprocessed SldA than the wild-type cells did. SldB may be a chaperone-like component that assists signal processing and folding of the SldA polypeptide to form active D-sorbitol dehydrogenase.

The hybridising potential of anhydrohexitol nucleoside analogues (HNAs) is well documented, but tedious synthesis of the monomers hampers their development. In a search for better analogues, the synthesis of two new methylated anhydrohexitol congeners 1 and 2 was accomplished and the physico-chemical properties of their respective oligomers were evaluated. Generally, oligonucleotides (ONs) containing the 3'-O-methyl derivative 1 showed a small increase in thermal stability towards complementary sequences as compared to HNA. Compared to the altritol modification, 3'-O-methylation seems to cause a small decrease in thermal stability of duplexes, especially when targeting RNA. These results suggest the possibility of derivatisation of the 3'-hydroxyl group of altritol-containing congeners without significantly affecting the thermal stability of the duplexes. The methyl glycosidic analogues 2 likewise increased the affinity for RNA in comparison with well-known HNA, while at the same time being economically more favorable monomers. However, homopolymers of 2 displayed self-pairing, but not so homopolymers of 1. Upon incorporation of the hexitols within RNA sequences in an effort to induce a beneficial pre-organised structure, the positive effect of the 3'-O-methyl derivative 1 proved larger than that of 2.

To identify the enzyme responsible for pentitol oxidation by acetic acid bacteria, two different ribitol oxidizing enzymes, one in the cytosolic fraction of NAD(P)-dependent and the other in the membrane fraction of NAD(P)-independent enzymes, were examined with respect to oxidative fermentation. The cytoplasmic NAD-dependent ribitol dehydrogenase (EC 1.1.1.56) was crystallized from Gluconobacter suboxydans IFO 12528 and found to be an enzyme having 100 kDa of molecular mass and 5 s as the sedimentation constant, composed of four identical subunits of 25 kDa. The enzyme catalyzed a shuttle reversible oxidoreduction between ribitol and D-ribulose in the presence of NAD and NADH, respectively. Xylitol and L-arabitol were well oxidized by the enzyme with reaction rates comparable to ribitol oxidation. D-Ribulose, L-ribulose, and L-xylulose were well reduced by the enzyme in the presence of NADH as cosubstrates. The optimum pH of pentitol oxidation was found at alkaline pH such as 9.5-10.5 and ketopentose reduction was found at pH 6.0. NAD-Dependent ribitol dehydrogenase seemed to be specific to oxidoreduction between pentitols and ketopentoses and D-sorbitol and D-mannitol were not oxidized by this enzyme. However, no D-ribulose accumulation was observed outside the cells during the growth of the organism on ribitol. L-Ribulose was accumulated in the culture medium instead, as the direct oxidation product catalyzed by a membrane-bound NAD(P)-independent ribitol dehydrogenase. Thus, the physiological role of NAD-dependent ribitol dehydrogenase was accounted to catalyze ribitol oxidation to D-ribulose in cytoplasm, taking D-ribulose to the pentose phosphate pathway after being phosphorylated. L-Ribulose outside the cells would be incorporated into the cytoplasm in several ways when need for carbon and energy sources made it necessary to use L-ribulose for their survival. From a series of simple experiments, membrane-bound sugar alcohol dehydrogenase was concluded to be the enzyme responsible for L-ribulose production in oxidative fermentation by acetic acid bacteria.

Aim: To evaluate the effect of modifications of water activity (aw 0. 996-0.92) of a molasses medium with different solutes (glycerol, glucose, NaCl, proline or sorbitol) on growth, intracellular water potentials (psi(c)) and endogenous accumulation of polyols/sugars in the biocontrol yeast Candida sake., Methods and Results: Modification of solute stress significantly influenced growth, psi(c) and accumulation of sugars (glucose/trehalose) and polyols (glycerol, erythritol, arabitol and mannitol) in the yeast cells. Regardless of the solute used to modify aw, growth was always decreased as water stress increased. Candida sake cells grew better in glycerol- and proline-amended media, but were sensitive to NaCl. The psi(c) measured using psychrometry showed a significant effect of solutes, aw and time. Cells from the 0.96 aw NaCl treatment presented the lowest psic value (- 5.20 MPa) while cells from unmodified media (aw = 0. 996) had the highest value (- 0.30 MPa). In unmodified medium, glycerol was the predominant reserve accumulated. Glycerol and arabitol were the major compounds accumulated in media modified with glucose or NaCl. In proline media, the concentration of arabitol increased. In glycerol- and sorbitol-amended media, the concentration of glycerol rose. Some correlations were obtained between compatible solutes and psi(c)., Conclusions and Significance: This study demonstrates that subtle changes in physiological parameters significantly affect the endogenous contents of C. sake cells. It may be possible to utilize such physiological information to develop biocontrol inocula with improved quality.

In this study the ability of various sugars and sugar alcohols to induce aldose reductase (xylose reductase) and xylitol dehydrogenase (xylulose reductase) activities in the yeast Candida tenuis was investigated. Both enzyme activities were induced when the organism was grown on D-xylose or L-arabinose as well as on the structurally related sugars D-arabinose or D-lyxose. Mixtures of D-xylose with the more rapidly metabolizable sugar D-glucose resulted in a decrease in the levels of both enzymes formed. These results show that the utilization of D-xylose by C. tenuis is regulated by induction and catabolite repression. Furthermore, the different patterns of induction on distinct sugars suggest that the synthesis of both enzymes is not under coordinate control.

Free energy simulations have been employed to rationalize the binding differences between A-74704, a pseudo C2-symmetric inhibitor of HIV-1 protease and its diester analog. The diester analog inhibitor, which misses two hydrogen bonds with the enzyme active site, is surprisingly only 10-fold weaker. The calculated free energy difference of 1.7 +/- 0.6 kcal/mol is in agreement with the experimental result. Further, the simulations show that such a small difference in binding free energies is due to (1) weaker hydrogen bond interactions between the two (P1 and P1') NH groups of A-74704 with Gly27/Gly27' carbonyls of the enzyme and (2) the higher desolvation free energy of A-74704 compared with its ester analog. The results of these calculations and their implications for design of HIV-1 protease inhibitors are discussed.

The accumulation and excretion of polyols was investigated under various growth conditions and at different stages of the life cycle of Aspergillus niger. Glycerol was found to be the major solute in osmotic adjustment of the hyphae. Conidiospores contain large amounts of mannitol, which are rapidly metabolized during early germination, leading to the accumulation of glycerol. Glycerol is the major polyol in young mycelium, whereas in older mycelium mannitol and erythritol predominate. In all experiments, polyols were also excreted. The mechanism and function of this process is unknown, but it might be a way to control the levels of the intracellular polyol pools. Polyols are rapidly taken up again upon starvation. In a glycerol kinase mutant the synthesis of glycerol is unaffected but the excreted level of the polyol is higher. This glycerol is taken up again upon starvation, and accumulates intracellularly as it can not be metabolized further.

The degradation pathway for L-arabinose, which consists of a sequence of alternating reduction and oxidation reactions prior to ultimate phosphorylation, was studied in Aspergillus nidulans wild-type as well as in an L-arabinose non-utilizing mutant. The inability of the mutant to use L-arabinose was caused by the absence of L-arabitol dehydrogenase activity. The effect of the mutation on polyol accumulation patterns was studied upon growth on various carbon sources. The presence of L-arabinose resulted in intracellular accumulation of arabitol in this mutant. Moreover, the mutant secreted arabitol under these conditions and, in contrast to the wild-type, featured enhanced expression of enzymes involved in L-arabinose catabolism as well as of extracellular glycosyl hydrolases involved in degradation of the plant cell wall polysaccharide L-arabinan.

Lysine residues have been suggested to be involved in the hysteretic decrease of the activity of spinach ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the binding of ribulose 1,5-bisphosphate to its regulatory sites [Yokota, A. & Tsujimoto, N. (1992) Eur. J. Biochem. 204, 901-909]. This work identifies the lysine residues and investigates the effects of their chemical modification on the course of RuBisCO reaction. The carbamylated form of RuBisCO reacted with trinitrobenzene sulfonate in three phases; an initial rapid, second slow, and final non-specific reaction. Lys-334 in loop 6, Lys-21, and Lys-128, all from the large subunits, were trinitrophenylated in the first 60-min reaction. Lys-305 of the large subunits was labeled in the next step. The modification of these residues was strongly suppressed in the enzyme form that had undergone hysteretic conformational change after binding 2-carboxyarabinitol 1,5-bisphosphate at its catalytic sites. Instead, Lys-450 of the large subunits and Lys-71 from the small subunits were newly modified in the quaternary complex. A higher concentration of 2-carboxyarabinitol 1,5-bisphosphate reduced the trinitrophenylation of the two residues to half. The modification of the carbamylated form of the enzyme for 30 min was expected to arylate Lys-21, Lys-128, and Lys-334 at random, and the course of the reaction of the partially modified enzyme was expected to deviate from that of the unmodified enzyme. Experimental results showed that this was the case.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of seventeen yeast strains by 13C-NMR spectroscopy has confirmed the significance of glycerol as the sole osmoregulatory solute under salt-stressed conditions, and has shown arabitol to be present in most of the osmotolerant species. Ribitol was detected in some species, including Debaryomyces hansenii, although ribitol accumulation did not correlate with the osmotic pressure of the medium. Relative amounts of arabitol and ribitol decreased in relation to glycerol when the external osmotic pressure was increased. Trehalose was present during exponential growth of some species.

Ribulose bisphosphate carboxylase (RuBisCO) binds carboxyarabinitol bisphosphate (CABP) on its regulatory sites [Yokota, A. (1991) J. Biochem. 110, 246-252]. The characteristics of the equilibrium binding of CABP to the sites were examined by the gel-filtration method. Since RuBisCO binds CABP on the substrate sites with a dissociation constant of less than 10 pM, CABP bound exclusively to the substrate sites at less than 5 microM. Plotting the number of CABP bound to the sites other than the substrate sites against the concentration of CABP gave a typical "bumpy" curve; the binding number in the intermediate plateau at 20 to 40 microM CABP was 3.7 to 4.4 mol per mol of RuBisCO and that at the saturating concentration of CABP was 7.6 to 7.8 mol per mol of RuBisCO. The Hill plot of their relationship gave a line which bent strongly at 20 to 40 microM CABP. The best fitting of the data to the equations derived from the binding model constructed according to the reported model [Teipel, J. & Koshland, D.E., Jr. (1969) Biochemistry 8, 4656-4663] showed that the binding of CABP to the regulatory sites proceeded with positive cooperativity both before and after the plateau. The dissociation constant decreased from 31 to 14 microM by the factor of 1/1.3 in the former group and 490 to 0.7 microM by the factor of 1/8.9 in the latter with increasing binding number of CABP.

To obtain information about the effects of lenticular polyols on the prevention, initial stages, and development of diabetic cataracts, we identified and determined with gas-liquid chromatography or gas-liquid chromatography/mass spectrometry eight polyols in cataractous lenses of non-insulin-dependent diabetes mellitus patients and nondiabetic subjects. In the diabetics' lenses, the concentrations of polyols (e.g., sorbitol, fructose, mannitol, and adonitol) were higher than in the nondiabetics' lenses, whereas the concentration of 1-deoxyglucose was lower. The mean concentration of myo-inositol in lenses of diabetics was lower than that of nondiabetics, but this difference was statistically not significant. The total content of eight polyols in the diabetics' lenses did not differ significantly from that in the nondiabetics. In the lenses of diabetics, the content of glucose correlated positively with that of adonitol, fructose, and sorbitol. In the lenses of nondiabetics, the content of glucose correlated positively with that of mannitol and inversely with that of 1-deoxyglucose and myo-inositol. In diabetics, hemoglobin A1 (%) correlated positively with the concentration of adonitol in the lenses and inversely with the concentration of lens myo-inositol; however, it did not correlate with the concentration of glucose in lenses. Regulation of both the metabolism of lenticular polyols and the pattern of polyols in serum may be necessary for normalizing lenticular polyol content.

Serum arabinitol concentrations and arabinitol/creatinine ratios were determined in 25 cancer patients with invasive candidiasis, 73 severely ill cancer patients who did not have invasive candidiasis, and 15 uninfected patients with chronic renal failure. Elevated arabinitol concentrations were found in patients with renal failure and invasive candidiasis. Serum arabinitol concentrations exceeded 1.19 micrograms/ml in 13 of 18 patients with invasive candidiasis who were studied when renal function was normal and in three of 52 control patients. Among patients with renal failure, 10 of 14 with invasive candidiasis but only four of 36 control patients had serum arabinitol concentrations of greater than 5.85 micrograms/ml. Serum arabinitol and creatinine concentrations were strongly correlated. The arabinitol/ creatinine ratio exceeded 1.5 in 16 of 25 patients with invasive candidiasis but in only three of 88 control patients. Increased serum arabinitol concentrations appear to reflect increased production of arabinitol by yeast in individuals with invasive candidiasis.

The effects of renal function on serum concentrations of the candidal metabolite arabinitol were studied by examining the accumulation and elimination of arabinitol in animals and humans. Serum concentrations of arabinitol rose sharply and in direct proportion to creatine concentrations after nephrectomy in rats. The serum half-life of exogenous arabinitol was 20.8 hr in anephric rats but only 0.62 hr in sham-nephrectomized control animals. The mean +/- SD apparent space of distribution of arabinitol was 419 +/- 26 ml/kg. The fraction of exogenously administered arabinitol recovered in the urine was 0.73 +/- 0.13 in uremic rats, 0.85 +/- 0.28 in control rats, and 0.95 +/- 0.10 in normal dogs. The arabinitol/creatinine clearance ratio was 0.99 +/- 0.08 in normal dogs and 0.99 +/- 0.25 in 22 critically ill patients with cancer. Thus arabinitol is eliminated by nearly quantitative urinary excretion and its cleared at virtually the same rate as creatinine. Therefore, the rate of arabinitol appearance in the body from any source equals the urinary arabinitol excretion rate and is directly proportional to the concentration ratio of arabinitol to creatinine in serum or urine.

Renal colony counts, total arabinitol appearance, and the arabinitol/creatinine ratios in the serum and urine of rats with lethal candidiasis were measured. Total arabinitol appearance and the arabinitol/creatinine ratios increased significantly after infection. The serum arabinitol/creatinine ratio was high (2 SD above the mean value in normal animals) in five of six infected rats at 24 hr, in eight of eight at 48 hr, and in none of the control animals. The urinary arabinitol/creatinine ratio was high in 14 of 19 infected rats by 24 hr, in 11 of 11 by 48 hr, and in none of the control animals. Total arabinitol appearance and the arabinitol/creatinine ratios were highly correlated with renal colony counts. Arabinitol production in vitro was sufficient to account for the increased arabinitol appearance observed in vivo in 12 of 14 rats. Thus it is concluded that the excess arabinitol that appeared in vivo was produced by the infecting fungus and that arabinitol is a quantitative marker substance for candidiasis.

Dianhydrogalactitol (DAG; NSC-132313), a hexitol epoxide, was used to treat intracerebral rodent tumors. DAG was most active against the murine ependymoblastoma [treated/controls (T/C)greater than 440%], less active against murine glioma 26 (T/C approximately 112-150%), and least active against rat 9L gliosarcoma (T/C approximately 100%). Application of a two-compartment open model for plasma disappearance of 14C-DAG in rats gave a volume of distribution at steady state of approximately 872 ml, a clearance of approximately 9.4 ml/minute, and an elimination constant of 0.025/minute. Entry of 14C-DAG was more rapid into the 9L tumor than into the normal brain. When a two-compartment series model for brain and tumor entry was applied, the t1/2 (half-time) for compartmental equilibrium was approximately 22 and 105 minutes in the brain, and 4 and 56 minutes in the 9L tumor. The drug rapidly entered the brain and tumor intracellular compartments. Binding to RNA was linear with time, and the absolute amount of binding was approximately six times greater for RNA than for DNA.

The pharmacokinetics of dianhydrogalactitol (DAG), NSC-132313, were studied in the beagle dog at doses of 3 mg - kg-1 and 6 mg - kg-1. DAG concentrations in plasma were determined by a gas chromatographic method capable of specifically detecting the parent drug and differentiating between it and products of its degradation or metabolism. Plasma disappearance time curves were generated and shown to follow simple two-compartment model behavior after iv administration of DAG. Distribution and elimination of DAG appeared to be dose-independent in the limited dose range studied. After iv administration, the drug was rapidly distributed throughout extracellular fluids (volume of the central compartment = 462 ml - kg-1) and subsequently was rapidly cleared (total body clearance = 23.4 ml - min-1 - kg-1) and eliminated (t1/2, b = 26.2 min) from the animal. Experiments (in vitro) with the use of radiolabeled DAG indicated that the drug binds reversibly and irreversibly to red blood cells.