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Galactitol, a rare sugar alcohol, has promising potential in the food industry and pharmaceutical field. The available industrial production methods rely on harsh hydrogenation processes, which incur high costs and environmental concerns. It is urgent to develop environmentally friendly and efficient biosynthesis technologies. In this study, a xylose reductase named AnXR derived from Aspergillus niger CBS 513.88 was identified and characterized for the enzymatic properties. AnXR exhibited the highest activity at 25 ℃ and pH 8.0, and it belonged to the NADPH-dependent aldose reductase family. To engineer a strain for galactitol production, we deleted the galactokinase (GAL1) gene in Saccharomyes cerevisiae by using the recombinant gene technology, which significantly reduced the metabolic utilization of D-galactose by host cells. Subsequently, we introduced the gene encoding AnXR into this modified strain, creating an engineered strain capable of catalyzing the conversion of D-galactose into galactitol. Furthermore, we optimized the whole-cell catalysis conditions for the engineered strain, which achieved a maximum galactitol yield of 12.10 g/L. Finally, we tested the reduction ability of the strain for other monosaccharides and discovered that it could produce functional sugar alcohols such as xylitol and arabinitol. The engineered strain demonstrates efficient biotransformation capabilities for galactitol and other functional sugar alcohols, representing a significant advancement in environmentally sustainable production practices.

The smo locus (sorbitol mannitol oxidation) is found on the chromosome of S. meliloti 's tripartite genome. Mutations at the smo locus reduce or abolish the ability of the bacterium to grow on several carbon sources, including sorbitol, mannitol, galactitol, d-arabitol and maltitol. The contribution of the smo locus to the metabolism of these compounds has not been previously investigated. Genetic complementation of mutant strains revealed that smoS is responsible for growth on sorbitol and galactitol, while mtlK restores growth on mannitol and d-arabitol. Dehydrogenase assays demonstrate that SmoS and MtlK are NAD + -dependent dehydrogenases catalysing the oxidation of their specific substrates. Transport experiments using a radiolabeled substrate indicate that sorbitol, mannitol and d-arabitol are primarily transported into the cell by the ABC transporter encoded by smoEFGK . Additionally, it was found that a mutation in either frcK , which is found in an operon that encodes the fructose ABC transporter, or a mutation in frk , which encodes fructose kinase, leads to the induction of mannitol transport.

Background: Gluconobacter oxydans, is used in biotechnology because of its ability to oxidize a wide variety of carbohydrates, alcohols, and polyols in a stereo- and regio-selective manner by membrane-bound dehydrogenases located in periplasmic space. These reactions obey the well-known Bertrand-Hudson's rule. In our previous study (BBA-General Subjects, 2021, 1865:129740), we discovered that Gluconobacter species, including G. oxydans and G. cerinus strain can regio-selectively oxidize the C-3 and C-5 hydroxyl groups of D-galactitol to rare sugars D-tagatose and L-xylo-3-hexulose, which represents an exception to Bertrand Hudson's rule. The enzyme catalyzing this reaction is located in periplasmic space or membrane-bound and is PQQ (pyrroloquinoline quinine) and Ca 2+ -dependent; we were encouraged to determine which type of enzyme(s) catalyze this unique reaction., Methods: Enzyme was identified by complementation of multi-deletion strain of Gluconobacter oxydans 621H with all putative membrane-bound dehydrogenase genes., Results and Conclusions: In this study, we identified this gene encoding the membrane-bound PQQ-dependent dehydrogenase that catalyzes the unique galactitol oxidation reaction in its 3'-OH and 5'-OH. Complement experiments in multi-deletion G. oxydans BP.9 strains established that the enzyme mSLDH (encoded by GOX0855-0854, sldB-sldA) is responsible for galactitol's unique oxidation reaction. Additionally, we demonstrated that the small subunit SldB of mSLDH was membrane-bound and served as an anchor protein by fusing it to a red fluorescent protein (mRubby), and heterologously expressed in E. coli and the yeast Yarrowia lipolytica. The SldB subunit was required to maintain the holo-enzymatic activity that catalyzes the conversion of D-galactitol to L-xylo-3-hexulose and D-tagatose. The large subunit SldA encoded by GOX0854 was also characterized, and it was discovered that its 24 amino acids signal peptide is required for the dehydrogenation activity of the mSLDH protein., General Significance: In this study, the main membrane-bound polyol dehydrogenase mSLDH in G. oxydans 621H was proved to catalyze the unique galactitol oxidation, which represents an exception to the Bertrand Hudson's rule, and broadens its substrate ranges of mSLDH. Further deciphering the explicit enzymatic mechanism will prove this theory., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing financial interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol to fructose and tagatose, respectively, using NAD + as a cofactor. The main objective of this study was to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon-optimized for heterologous expression in Escherichia coli BL21 (DE3) Gold cells and the protein was subsequently overexpressed and purified. Size-exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetramer. SmoS was crystallized, and crystals obtained in the absence of substrate diffracted to 2.1 Å resolution and those of a complex with sorbitol diffracted to 2.0 Å resolution. SmoS was characterized kinetically and shown to have a preference for sorbitol despite having a higher affinity for galactitol. Computational ligand-docking experiments suggest that tagatose binds the protein in a more energetically favourable complex than fructose, which is retained in the active site over a longer time frame following oxidation and reduces the rate of the reaction. These results supplement the inventory of biomolecules with potential for industrial applications and enhance the understanding of metabolism in the model organism S. meliloti.

Background: In acetic acid bacteria such as Gluconobacter oxydans or Gluconobacter cerinus, pyrroloquinoline quinone (PQQ) in the periplasm serves as the redox cofactor for several membrane-bound dehydrogenases that oxidize polyhydric alcohols to rare sugars, which can be used as a healthy alternative for traditional sugars and sweeteners. These oxidation reactions obey the generally accepted Bertrand Hudson's rule, in which only the polyhydric alcohols that possess cis d-erythro hydroxyl groups can be oxidized to 2-ketoses using PQQ as a cofactor, while the polyhydric alcohols excluding cis d-erythro hydroxyl groups ruled out oxidation by PQQ-dependent membrane-bound dehydrogenases., Methods: Membrane fractions of G. oxydans were prepared and used as a cell-free catalyst to oxidize galactitol, with or without PQQ as a cofactor., Results: In this study, we reported an interesting oxidation reaction that the polyhydric alcohols galactitol (dulcitol), which do not possess cis d-erythro hydroxyl groups, can be oxidized by PQQ-dependent membrane-bound dehydrogenase(s) of acetic acid bacteria at the C-3 and C-5 hydroxyl groups to produce rare sugars l-xylo-3-hexulose and d-tagatose., Conclusions: This reaction may represent an exception to Bertrand Hudson's rule., General Significance: Bertrand Hudson's rule is a well-known theory in polyhydric alcohols oxidation by PQQ-dependent membrane-bound dehydrogenase in acetic acid bacteria. In this study, galactitol oxidation by a PQQ-dependent membrane-bound dehydrogenase represents an exception to the Bertrand Hudson's rule. Further identification of the associated enzymes and deciphering the explicit enzymatic mechanism will prove this theory., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Interorgan immunological communication is critical to connect the local-systemic innate immune response and orchestrate a homeostatic host defense. However, the factors and their roles in this process remain unclear. We find Drosophila IMD response in guts can sequentially trigger a systemic IMD reaction in the fat body. Sugar alcohols of the polyol pathway are essential for the spatiotemporal regulation of gut-fat body immunological communication (GFIC). IMD activation in guts causes elevated levels of sorbitol and galactitol in hemolymph. Aldose reductase (AR) in hemocytes, the rate-limiting enzyme of the polyol pathway, is necessary and sufficient for the increase of plasma sugar alcohols. Sorbitol relays GFIC by subsequent activation of Metalloprotease 2, which cleaves PGRP-LC to activate IMD response in fat bodies. Thus, this work unveils how GFIC relies on the intermediate activation of the polyol pathway in hemolymph and demonstrates that AR provides a critical metabolic checkpoint in the global inflammatory response., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The legume endosymbiont Sinorhizobium meliloti can utilize a broad range of carbon compounds to support its growth. The linear, six-carbon polyol galactitol is abundant in vascular plants and is metabolized in S. meliloti by the contribution of two loci SMb21372-SMb21377 and SMc01495-SMc01503 which are found on pSymB and the chromosome, respectively. The data suggest that several transport systems, including the chromosomal ATP-binding cassette (ABC) transporter smoEFGK, contribute to the uptake of galactitol, while the adjacent gene smoS encodes a protein for oxidation of galactitol into tagatose. Subsequently, genes SMb21374 and SMb21373, encode proteins that phosphorylate and epimerize tagatose into fructose-6-phosphate, which is further metabolized by the enzymes of the Entner-Doudoroff pathway. Of note, it was found that SMb21373, which was annotated as a 1,6-bis-phospho-aldolase, is homologous to the E. coli gene gatZ, which is annotated as encoding the non-catalytic subunit of a tagatose-1,6-bisphosphate aldolase heterodimer. When either of these genes was introduced into an Agrobacterium tumefaciens strain that carries a tagatose-6-phosphate epimerase mutation, they are capable of complementing the galactitol growth deficiency associated with this mutation, strongly suggesting that these genes are both epimerases. Phylogenetic analysis of the protein family (IPR012062) to which these enzymes belong, suggests that this misannotation is systemic throughout the family. S. meliloti galactitol catabolic mutants do not exhibit symbiotic deficiencies or the inability to compete for nodule occupancy.

Background: Lactase is an enzyme that hydrolyzes lactose into glucose and galactose in the small intestine, where they are absorbed. Hypolactasia is a common condition, primarily caused by genetic programming, that leads to lactose maldigestion and, in certain cases, lactose intolerance. Galactitol and galactonate are 2 products of hepatic galactose metabolism that are candidate markers for the intake of lactose-containing foods., Objectives: The primary objective of the study was to explore the changes in serum and urine metabolomes during postprandial dairy product tests through the association between lactase persistence genotype and the postprandial dynamics of lactose-derived metabolites., Methods: We characterized the 6-h postprandial serum kinetics and urinary excretion of lactose, galactose, galactitol, and galactonate in 14 healthy men who had consumed a single dose of acidified milk (800 g) which contained 38.8 g lactose. Genotyping of LCT-13910 C/T (rs4988235) was performed to assess primary lactase persistence., Results: There were 2 distinct postprandial responses, classified as high and low metabolite responses, observed for galactose, and its metabolites galactitol and galactonate, in serum and urine. In all but 1 subject, there was a concordance between the high metabolite responses and genetic lactase persistence and between the low metabolite responses and genetic lactase nonpersistence (accuracy 0.92), galactitol and galactonate being more discriminative than galactose., Conclusions: Postprandial galactitol and galactonate after lactose overload appear to be good proxies for genetically determined lactase activity. The development of a noninvasive lactose digestion test based on the measurement of these metabolites in urine could be clinically useful. This trial was registered at clinicaltrials.gov as NCT02230345.

The evolution of new strains within the gut ecosystem is poorly understood. We used a natural but controlled system to follow the emergence of intraspecies diversity of commensal Escherichia coli, during three rounds of adaptation to the mouse gut (∼1,300 generations). We previously showed that, in the first round, a strongly beneficial phenotype (loss-of-function for galactitol consumption; gat-negative) spread to >90% frequency in all colonized mice. Here, we show that this loss-of-function is repeatedly reversed when a gat-negative clone colonizes new mice. The regain of function occurs via compensatory mutation and reversion, the latter leaving no trace of past adaptation. We further show that loss-of-function adaptive mutants reevolve, after colonization with an evolved gat-positive clone. Thus, even under strong bottlenecks a regime of strong-mutation-strong-selection dominates adaptation. Coupling experiments and modeling, we establish that reverse evolution recurrently generates two coexisting phenotypes within the microbiota that can or not consume galactitol (gat-positive and gat-negative, respectively). Although the abundance of the dominant strain, the gat-negative, depends on the microbiota composition, gat-positive abundance is independent of the microbiota composition and can be precisely manipulated by supplementing the diet with galactitol. These results show that a specific diet is able to change the abundance of specific strains. Importantly, we find polymorphism for these phenotypes in indigenous Enterobacteria of mice and man. Our results demonstrate that natural selection can greatly overwhelm genetic drift at structuring the strain diversity of gut commensals and that competition for limiting resources may be a key mechanism for maintaining polymorphism in the gut., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Premature ovarian insufficiency (POI) is a frequent long-term complication of classic galactosemia. The majority of women with this disorder develop POI, however rare spontaneous pregnancies have been reported. Here, we evaluate the effect of D-galactose and its metabolites, galactitol and galactose 1-phosphate, on oocyte quality as well as embryo development to elucidate the mechanism through which these compounds mediate oocyte deterioration. Metaphase II mouse oocytes (n = 240), with and without cumulus cells (CCs), were exposed for 4 hours to D-galactose (2 μM), galactitol (11 μM) and galactose 1-phosphate (0.1 mM), (corresponding to plasma concentrations in patients on galactose-restricted diet) and compared to controls. The treated oocytes showed decreased quality as a function of significant enhancement in production of reactive oxygen species (ROS) when compared to controls. The presence of CCs offered no protection, as elevated ROS was accompanied by increased apoptosis of CCs. Our results suggested that D-galactose and its metabolites disturbed the spindle structure and chromosomal alignment, which was associated with significant decline in oocyte cleavage and blastocyst development after in-vitro fertilization. The results provide insight into prevention and treatment strategies that may be used to extend the window of fertility in these patients.

Galactitol degradation by salmonellae remains underinvestigated, although this metabolic capability contributes to growth in animals (R. R. Chaudhuri et al., PLoS Genet 9:e1003456, 2013, https://doi.org/10.1371/journal.pgen.1003456). The genes responsible for this metabolic capability are part of a 9.6-kb gene cluster that spans from gatY to gatR (STM3253 to STM3262) and encodes a phosphotransferase system, four enzymes, and a transporter of the major facilitator superfamily. Genome comparison revealed the presence of this genetic determinant in nearly all Salmonella strains. The generation time of Salmonella enterica serovar Typhimurium strain ST4/74 was higher in minimal medium with galactitol than with glucose. Knockout of STM3254 and gatC resulted in a growth-deficient phenotype of S Typhimurium, with galactitol as the sole carbon source. Partial deletion of gatR strongly reduced the lag phase of growth with galactitol, whereas strains overproducing GatR exhibited a near-zero growth phenotype. Luciferase reporter assays demonstrated strong induction of the gatY and gatZ promoters, which control all genes of this cluster except gatR, in the presence of galactitol but not glucose. Purified GatR bound to these two main gat gene cluster promoters as well as to its own promoter, demonstrating that this autoregulated repressor controls galactitol degradation. Surface plasmon resonance spectroscopy revealed distinct binding properties of GatR toward the three promoters, resulting in a model of differential gat gene expression. The cyclic AMP receptor protein (CRP) bound these promoters with similarly high affinities, and a mutant lacking crp showed severe growth attenuation, demonstrating that galactitol utilization is subject to catabolite repression. Here, we provide the first genetic characterization of galactitol degradation in Salmonella, revealing novel insights into the regulation of this dissimilatory pathway., Importance: The knowledge of how pathogens adapt their metabolism to the compartments encountered in hosts is pivotal to our understanding of bacterial infections. Recent research revealed that enteropathogens have adapted specific metabolic pathways that contribute to their virulence properties, for example, by helping to overcome limitations in nutrient availability in the gut due to colonization resistance. The capability of Salmonella enterica serovar Typhimurium to degrade galactitol has already been demonstrated to play a role in vivo, but it has not been investigated so far on the genetic level. To our knowledge, this is the first molecular description of the galactitol degradation pathway of a pathogen., (Copyright © 2017 American Society for Microbiology.)

Objective: To seek efficient aldose reductase inhibitors (ARIs) with excellent in vitro and in vivo biological activities against rat galactosemic cataract., Methods: The method was firstly optimized to screen strong ARIs from nonoriented synthetic compounds and natural extracts. Then, diosgenin was assessed on osmotic expansion of primarily cultured lens epithelial cells (LECs) induced by galactose (50 mM). Diosgenin was administered to galactosemic rats by oral (100 and 200 mg/kg) or direct drinking (0.1%) to evaluate its anticataract effects., Results: Diosgenin was found as the strongest ARI with IC 50 of 4.59 × 10 -6  mol/L. Diosgenin (10  μ M) evidently inhibited the formation of tiny vacuoles and upregulation of AR mRNA in LECs. In vivo, diosgenin delayed lens opacification, inhibited the increase of ratio of lens weight to body weight, and decreased AR activity, galactitol level, and AR mRNA expression, especially in the diosgenin drinking (0.1%) group., Conclusions: Diosgenin was an efficient ARI, which not only significantly decreased the LECs' osmotic expansion in vitro but also markedly delayed progression of rat galactosemic cataract in vivo. Thus, diosgenin rich food can be recommended to diabetic subjects as dietary management to postpone the occurrence of sugar cataract, and diosgenin deserves further investigation for chronic diabetic complications.

A strictly anaerobic, Gram-stain-positive, non-spore-forming, rod-shaped bacterial strain, designated BS-1T, was isolated from an anaerobic digestion reactor during a study of bacteria utilizing galactitol as the carbon source. Its cells were 0.3-0.5 μm × 2-4 μm, and they grew at 35-45 °C and at pH 6.0-8.0. Strain BS-1T produced H2, CO2, ethanol, acetic acid, butyric acid and caproic acid as metabolic end products of anaerobic fermentation. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain BS-1T represented a novel bacterial genus within the family Ruminococcaceae, Clostridium Cluster IV. The type strains that were most closely related to strain BS-1T were Clostridium sporosphaeroides KCTC 5598T (94.5 %), Clostridium leptum KCTC 5155T (94.3 %), Ruminococcus bromii ATCC 27255T (92.1 %) and Ethanoligenens harbinense YUAN-3T (91.9 %). Strain BS-1T had 17.6 % and 20.9 % DNA-DNA relatedness values with C. sporosphaeroides DSM 1294T and C. leptum DSM 753T, respectively. The major components of the cellular fatty acids were C16 : 0 dimethyl aldehyde (DMA) (22.1 %), C16 : 0 aldehyde (14.1 %) and summed feature 11 (iso-C17 : 0 3-OH and/or C18 : 2 DMA; 10.0 %). The genomic DNA G+C content was 50.0 mol%. Phenotypic and phylogenetic characteristics allowed strain BS-1T to be clearly distinguished from other taxa of the genus Clostridium Cluster IV. On the basis of these data, the isolate is considered to represent a novel genus and novel species within Clostridium Cluster IV, for which the name Caproiciproducens galactitolivorans gen. nov., sp. nov. is proposed. The type species is BS-1T ( = JCM 30532T and KCCM 43048T).

Innovations in the discovery of the functions of uncharacterized proteins/enzymes have become increasingly important as advances in sequencing technology flood protein databases with an exponentially growing number of open reading frames. This study documents one such innovation developed by the Enzyme Function Initiative (EFI; U54GM093342), the use of solute-binding proteins for transport systems to identify novel metabolic pathways. In a previous study, this strategy was applied to the tripartite ATP-independent periplasmic transporters. Here, we apply this strategy to the ATP-binding cassette transporters and report the discovery of novel catabolic pathways for d-altritol and galactitol in Agrobacterium tumefaciens C58. These efforts resulted in the description of three novel enzymatic reactions as follows: 1) oxidation of d-altritol to d-tagatose via a dehydrogenase in Pfam family PF00107, a previously unknown reaction; 2) phosphorylation of d-tagatose to d-tagatose 6-phosphate via a kinase in Pfam family PF00294, a previously orphan EC number; and 3) epimerization of d-tagatose 6-phosphate C-4 to d-fructose 6-phosphate via a member of Pfam family PF08013, another previously unknown reaction. The epimerization reaction catalyzed by a member of PF08013 is especially noteworthy, because the functions of members of PF08013 have been unknown. These discoveries were assisted by the following two synergistic bioinformatics web tools made available by the Enzyme Function Initiative: the EFI-Enzyme Similarity Tool and the EFI-Genome Neighborhood Tool., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD(+)-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35°C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8mM, kcat of 835min(-1) and a kcat/Km of 94.9min(-1)mM(-1). Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Poly(lactate-co-3-hydroxybutyrate) (P(LA-co-3HB)) was previously produced from xylose in engineered Escherichia coli. The aim of this study was to increase the polymer productivity and LA fraction in P(LA-co-3HB) using two metabolic engineering approaches: (1) deletions of competing pathways to lactate production and (2) overexpression of a galactitol transporter (GatC), which contributes to the ATP-independent xylose uptake. Engineered E. coli mutants (ΔpflA, Δpta, ΔackA, ΔpoxB, Δdld, and a dual mutant; ΔpflA + Δdld) and their parent strain, BW25113, were grown on 20 g l(-1) xylose for P(LA-co-3HB) production. The single deletions of ΔpflA, Δpta, and Δdld increased the LA fraction (58-66 mol%) compared to BW25113 (56 mol%). In particular, the ΔpflA + Δdld strain produced P(LA-co-3HB) containing 73 mol% LA. Furthermore, GatC overexpression increased both polymer yields and LA fractions in ΔpflA, Δpta, and Δdld mutants, and BW25113. The ΔpflA + gatC strain achieved a productivity of 8.3 g l(-1), which was 72 % of the theoretical maximum yield. Thus, to eliminate limitation of the carbon source, higher concentration of xylose was fed. As a result, BW25113 harboring gatC grown on 40 g l(-1) xylose reached the highest P(LA-co-3HB) productivity of 14.4 g l(-1). On the other hand, the ΔpflA + Δdld strain grown on 30 g l(-1) xylose synthesized 6.4 g l(-1) P(LA-co-3HB) while maintaining the highest LA fraction (73 mol%). The results indicated the usefulness of GatC for enhanced production of P(LA-co-3HB) from xylose, and the gene deletions to upregulate the LA fraction in P(LA-co-3HB). The polymers obtained had weight-averaged molecular weights in the range of 34,000-114,000.

Diabetic complications are attributed to hyperglycaemic condition which is in turn associated with the polyol pathway and advanced glycation end products. Aldose reductase (AR) is the principal enzyme of polyol pathway which plays a vital role in the development of diabetic complications. AR inhibitory activity can be screened by both in vitro and in vivo methods. In vitro assays for AR enzyme are further classified on the basis of the source of enzyme such as rat lens, rat kidney, cataracted human eye lens, bovine eyes and human recombinant AR enzymes, whereas the in vivo model is based on the determination of lens galactitol levels. A number of synthetic AR inhibitors (ARIs) including tolrestat and sorbinil have been developed, but all of these suffer from drawbacks such as poor permeation and safety issues. Therefore, pharmaceutical companies and many researchers have been carrying out research to find new, potent and safe ARIs from natural sources. Thus, many naturally occurring compounds have been reported to have AR inhibitory activity. The present review attempts to highlight phytochemicals and plant extracts with potential AR inhibitory activity. It also summarizes the classes of compounds which have proven AR inhibitory activity. Phytochemicals such as quercetin, kaempferol and ellagic acid are found to be the most promising ARIs. The exhaustive literature presented in this article clearly indicates the role of plant extracts and phytochemicals as potential ARIs., (Copyright © 2013 John Wiley & Sons, Ltd.)

Purpose: Recent studies report that growth factor and signaling changes in rat lenses do not directly result from the presence of diabetes or sorbitol/galactitol (polyol) formation/accumulation, but from secondary osmotic changes associated with the aldose reductase (AR) catalyzed polyol formation. AR is also present in rat retinal pericyte and endothelial cells; however, significant polyol formation only occurs in pericytes and this does not appear to be linked to osmotic changes. The purpose of this study was to determine whether polyol formation and AR activity are similarly linked to growth factor and signaling changes in the rat capillary cells despite the apparent absence of osmotic stress., Methods: Conditionally immortalized rat retinal pericyte (TR-rPCT) and endothelial (TR-iBRB) cell lines were cultured on collagen type 1-coated dishes in the DMEM containing 5.5 mM glucose. After 24 h of initial culture, the medium was replaced with a serum-free medium containing 5.5, 25, or 50 mM glucose or galactose with/without the aldose reductase inhibitors (ARIs) AL1576 or tolrestat for periods of up to 48 h. Growth factors and transduction pathways were measured by Western blots using the antibodies against basic FGF, IGF-1, TGF-β, P-ERK1/2, P-SAPK/JNK, and P-Akt., Results: Sorbitol accumulation was only observed in pericytes, while galactitol was present in both pericytes and endothelial cells. Pericytes cultured in high glucose showed increased expression of the growth factors basic FGF, IGF-1, TGF-β, and signaling in P-Akt, P-ERK1/2, and P-SAPK/JNK compared with those cultured in 5.5 mM glucose and these expressions were normalized by the presence of ARIs. Similar results were observed with galactose media. In contrast, endothelial cells cultured in high glucose media showed neither growth factor or signaling changes. In galactose media, endothelial cells showed increased expression of basic FGF, IGF-1, TGF-β, P-ERK1/2, and P-SAPK/JNK, which were only partially reduced by ARIs., Conclusion: Growth factor and MAPK signaling expression in pericytes are linked to the presence of polyols. Pericytes, which readily accumulate sorbitol/galactitol that is inhibited by ARIs, show expression changes similar to those observed in rat lenses. In contrast, endothelial cells only show partial expression changes that are linked to galactitol accumulation.

Purpose: We investigated the effect of an aldose reductase inhibitor (ARI) and the role of matrix metalloproteinase (MMP)-10 on recovery after corneal epithelium removal in a rat diabetic keratopathy model., Methods: Three-week-old Sprague-Dawley rats were fed the following diets for 6 weeks: normal MF chow (MF), 50% galactose (Gal), and 50% Gal containing 0.01% ARI (Gal +ARI). The corneal epithelium was removed using n-heptanol, and the area of epithelial defects was photographed and measured every 24 h. Real-time reverse transcriptase PCR, western blotting, and immunohistochemistry were used to determine the expression profile of MMP-10 and integrin α3., Results: Compared to the MF control group, the amount of galactitol in the Gal group increased approximately 200-fold, which was reduced to sevenfold by ARI treatment. The area of corneal erosion in the Gal group was significantly larger than in the MF group at 72 h and thereafter (p<0.01, unpaired t test). The expression level of MMP-10 was enhanced at both the protein and mRNA levels by exposure to a high concentration of Gal, while integrin α3 expression decreased at the protein level but remained unchanged at the mRNA level. Delayed epithelial wound healing and alterations in the expression levels of MMP-10 and integrin α3 were normalized by ARI. The corneal erosion closure rate was significantly decreased with topical recombinant MMP-10., Conclusions: These studies confirm that the increased expression of MMP-10 induced by Gal feeding is counteracted by ARI treatment and suggest a role of MMP-10 in modulating corneal epithelial wound healing.

As part of our ongoing search for natural sources of therapeutic and preventive agents for diabetic complications, we evaluated the inhibitory effects of components of the fruit of Xanthium strumarium (X. strumarium) on aldose reductase (AR) and galactitol formation in rat lenses with high levels of glucose. To identify the bioactive components of X. strumarium, 7 caffeoylquinic acids and 3 phenolic compounds were isolated and their chemical structures were elucidated on the basis of spectroscopic evidence and comparison with published data. The abilities of 10 X. strumarium-derived components to counteract diabetic complications were investigated by means of inhibitory assays with rat lens AR (rAR) and recombinant human AR (rhAR). From the 10 isolated compounds, methyl-3,5-di-O-caffeoylquinate showed the most potent inhibition, with IC₅₀ values of 0.30 and 0.67 μM for rAR and rhAR, respectively. In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate, methyl-3,5-di-O-caffeoylquinate showed competitive inhibition of rhAR. Furthermore, methyl-3,5-di-O-caffeoylquinate inhibited galactitol formation in the rat lens and in erythrocytes incubated with a high concentration of glucose, indicating that this compound may be effective in preventing diabetic complications.

Clostridium sp. BS-1 produces hexanoic acid as a metabolite using galactitol and enhanced hexanoic acid production was obtained by in situ extractive fermentation with Clostridium sp. BS-1 under an optimized medium composition. For medium optimization, five ingredients were selected as variables, and among them yeast extract, tryptone, and sodium butyrate were selected as significant variables according to a fractional factorial experimental design, a steepest ascent experimental design, and a Box-Behnken experimental design. The optimized medium had the following compositions in modified Clostridium acetobutyricum (mCAB) medium: 15.5gL(-1) of yeast extract, 10.13gL(-1) of tryptone, 0.04gL(-1) of FeSO4·7H2O, 0.85gL(-1) of sodium acetate, and 6.47gL(-1) of sodium butyrate. The predicted concentration of hexanoic acid with the optimized medium was 6.98gL(-1), and this was validated experimentally by producing 6.96gL(-1) of hexanoic acid with Clostridium sp. BS-1 under the optimized conditions. In situ extractive fermentation for hexanoic acid removal was then applied in a batch culture system with the optimized medium and 10% (v/v) alamine 336 in oleyl alcohol as an extractive solvent. The pH of the culture in the extractive fermentation was maintained at 5.4-5.6 by an acid balance between production and retrieval by extraction. During a 16 day culture, the hexanoic acid concentration in the solvent increased to 32gL(-1) while it was maintained in a range of 1-2gL(-1) in the medium. The maximum rate of hexanoic acid production was 0.34gL(-1)h(-1) in in situ extractive fermentation., (Copyright © 2013. Published by Elsevier Inc.)

Aldose reductase is the first enzyme in the polyol pathway and catalyzes the reduction of glucose to sorbitol by coupling with the oxidation of NADPH to NADP(+) . This sorbitol accumulation leads to various diabetic complications, including neuropathy, nephropathy, cataracts, and retinopathy. In the present study, aldose reductase inhibitory (ARI) activity of the methanolic as well as standardized extracts of Andrographis paniculata (Burm. f.) Wall. ex Nees (Acanthaceae) and its chief constituent, andrographolide, were studied using in vitro and in vivo methods. In the in vitro method, rat lens as well as kidney homogenates were used for the preparation of enzyme, whereas the effect of these test samples on the galactitol level in the eye lens was studied in a galactosemic rat model in vivo. The results of the study revealed that both extracts of the plant and its major compound, andrographolide, possess ARI activity in vitro. They were also found to significantly decrease galactitol accumulation in vivo., (Copyright © 2012 John Wiley & Sons, Ltd.)

Aldose reductase (AR) inhibitors have a considerable therapeutic potential against diabetes complications and do not increase the risk of hypoglycemia. Through bioassay-guided fractionation of an EtOH extract of the kernel from purple corn (Zea mays L.), 7 nonanthocyanin phenolic compounds (compound 1-7) and 5 anthocyanins (compound 8-12) were isolated. These compounds were investigated by rat lens aldose reductase (RLAR) inhibitory assays. Kinetic analyses of recombinant human aldose reductase (rhAR) were performed, and intracellular galactitol levels were measured. Hirsutrin, one of 12 isolated compounds, showed the most potent RLAR inhibitory activity (IC(50), 4.78 μ M). In the kinetic analyses using Lineweaver-Burk plots of 1/velocity and 1/substrate concentration, hirsutrin showed competitive inhibition against rhAR. Furthermore, hirsutrin inhibited galactitol formation in rat lens and erythrocytes sample incubated with a high concentration of galactose; this finding indicates that hirsutrin may effectively prevent osmotic stress in hyperglycemia. Therefore, hirsutrin derived from Zea mays L. may be a potential therapeutic agent against diabetes complications.

A series of UDP-galactitols were designed as analogues of high-energy intermediates of the UDP-galactopyranose mutase (UGM) catalyzed furanose/pyranose interconversion, an essential step of Mycobacterium tuberculosis cell wall biosynthesis. The final compounds structurally share the UDP and the galactitol substructures that were connected by four distinct electrophilic connections (epoxide, lactone and Michael acceptors). All molecules were synthesized from a common perbenzylated acyclic galactose precursor that was derivatized by alkenylation, alkynylation and cyclopropanation. The inhibition study against UGM could clearly show that slight changes in the relative orientation of the UDP and the galactitol moieties resulted in dramatic variations of binding properties. Compared to known inhibitors, the epoxide derivative displayed a very tight, reversible, inhibition profile. Moreover, a time-dependent inactivation study showed that none of these electrophilic structures could react with UGM, or its FAD cofactor, the catalytic nucleophile of this still intriguing reaction., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

For the catabolism of D-galactose three different metabolic pathways have been described in filamentous fungi. Apart from the Leloir pathway and the oxidative pathway, there is an alternative oxido-reductive pathway. This oxido-reductive pathway has similarities to the metabolic pathway of L-arabinose, and in Trichoderma reesei (Hypocrea jecorina) and Aspergillus nidulans the same enzyme is employed for the oxidation of L-arabitol and galactitol. Here we show evidence that in Aspergillus niger L-arabitol dehydrogenase (LadA) is not involved in the D-galactose metabolism; instead another dehydrogenase encoding gene, ladB, is induced in response to D-galactose and galactitol and functions as a galactitol dehydrogenase. Deletion of ladB in A. niger results in growth arrest on galactitol and significantly slower growth on D-galactose supplemented with a small amount of D-xylose. D-galactose alone cannot be utilised by A. niger and the addition of D-xylose stimulates growth on D-galactose via transcriptional activation of the D-xylose-inducible reductase gene, xyrA. XyrA catalyses the first step of the D-galactose oxido-reductive pathway, the reduction to galactitol, which in turn seems to be an inducer of the downstream genes such as LadB. The deletion of xyrA results in reduced growth on D-galactose. The ladB gene was expressed in the heterologous host Saccharomyces cerevisiae and the tagged and purified enzyme characterised. LadB and LadA have similar in vitro activity with galactitol. It was confirmed that the reaction product of the LadB reaction from galactitol is L-xylo-3-hexulose as in the case of the T. reesei Lad1., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Galactokinase deficiency (GALK-D), an autosomal recessive disorder in the Leloir pathway, results in accumulation of galactose, galactitol, and galactonate and leads to early onset of juvenile bilateral cataract. Highest incidence of GALK-D is found in Romani populations. The migration wave due to the Yugoslavian civil war has changed the spectrum of inborn errors of metabolism within Europe. Hence, newborn screening (NBS) in the Berlin region, performed from 1991 until 2010 in 683,675 neonates, revealed an increased incidence of GALK-D of 1:40,000, comparable to that of galactose-1-phosphate-uridyltransferase deficiency. A total of 44% of GALK-D patients were of Romani origin. All patients of Bosnian or Serbian origin were homozygous for the Romani founder mutation p.P28T. Detection of GALK-D by NBS and early start of galactose-restricted diet resulted in regression or prevention of cataracts. Slight cataracts without visual impairment occurred in 50% of the patients, 56% of whom were noncompliant. Further clinical symptoms, e.g., hypoglycemia, mental retardation, microcephaly, and failure to thrive, were associated with noncompliance. With treatment, galactose in blood decreased from 8,892 ± 5,243 to 36.5 ± 49.3 μmol/l, galactose in urine from 31,820 ± 32,103 to 30.0 ± 36.1 μmol/mmol creatinine, galactitol in RBC from 1,584 ± 584 to 12.3 ± 9.4 μmol/l, and galactitol in urine from 11,724 ± 4,496 to 236 ± 116 μmol/mmol creatinine. This is the first presentation of outcome and clinical features in GALK-D patients diagnosed by NBS. As our data suggest, GALK-D should be considered for inclusion in NBS in populations expected to have substantial numbers of GALK-D carriers, e.g., Yugoslavian immigrants.

In a study screening anaerobic microbes utilizing D: -galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with D: -galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294(T), with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from D-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from D-galactitol and D: -glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.

We report the structure-activity relationship of a series of coumarins as aldose reductase 2 (ALR2) inhibitors and their suppressive effect on the accumulation of galactitol in the rat lens. We evaluated their ALR2 selectivity profile against sorbitol dehydrogenase and aldehyde reductase (ALR1). Our study revealed that substitutions in the C7 OH group enhanced the potency toward ALR2, while the C6 OH group interferes with ALR1 inhibition activity. Having the phenyl moiety at C4 leads to improved potency and improved selectivity. A molecular docking study suggested that 6,7-dihydroxy-4-phenylcoumarin (15) binds to ALR2 in a different manner from epalrestat. Furthermore, compound 15 clearly suppressed galactitol accumulation in a dose-dependent manner. These results provide an insight into the structural requirements of coumarins for developing a new-type of selective ALR2 inhibitor., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: To explore the metabolic profile of vitreous fluid of patients with proliferative diabetic retinopathy (PDR) using 1H-NMR-based metabonomic analysis., Methods: 1H-NMR spectra were acquired from vitreous samples obtained during vitrectomy from 22 patients with type 1 diabetes with PDR and from 22 nondiabetic patients with macular hole (control group). Data analysis included a principal component analysis and partial least squares discriminant analysis (PLS-DA). In addition, 1H-(1)H and 1H-(13)C HMQC correlation spectra were acquired for the identification of metabolites. Furthermore, the main metabolites accounting for the differences in metabolic profile were assessed by current biochemical methods., Results: Lactate was the most abundant metabolite, and it was present at higher levels in samples from PDR patients than from nondiabetic patients (P=0.02). Glucose was significantly higher in samples from PDR patients than nondiabetic patients (P=0.03). After removing the lactate peak at 1.35 ppm and with the use of PLS-DA, a model was obtained that was able to correctly classify 19 of 22 patients with PDR and 18 of 22 controls, resulting in a sensitivity of 86% and a specificity of 81%. The main metabolites involved in this specific pattern recognition were galactitol and ascorbic acid (AA); levels of both were significantly lower in PDR patients., Conclusions: 1H-NMR-based metabonomic analysis of vitreous fluid permits the obtainment of a metabolic signature of PDR. Apart from the higher abundance of lactate and glucose, significant deficits of galactitol and AA are the main metabolic fingerprints of vitreous fluid from PDR patients.

Purpose: Experiments have been conducted to study the hypothesis that caffeine would inhibit reactive oxygen species induced oxidative stress in the lens in vivo, with implications of attenuating or preventing cataract formation., Methods: Oxidative stress was directly induced by administering 24% galactose diet to young adult rats. The treated group was fed a diet containing 24% galactose + 1% caffeine. Oxidative stress inflicted to the lens was assessed by measurement of glutathione (GSH) depletion and observing the status of lens clarity., Results: Caffeine administration was found to minimize the loss of GSH. This was also associated with a better maintenance of lens transparency as compared to the untreated galactosemic group., Conclusions: The studies demonstrate that caffeine could be helpful in inhibiting oxidative stress in the lens with the consequence of attenuating cataract formation.

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) isolates derived from diseased pigs in Japan during 2001 and 2005 were analyzed for biotype, based on H(2)S production and dulcitol fermentation, pulsed-field gel electrophoresis (PFGE) profile, and antimicrobial resistance profile. S. Choleraesuis biotype Choleraesuis (biotype Choleraesuis) was classified into one genotype, while varietas Kunzendorf (var. Kunzendorf) was classified into two genotypes. The isolates of var. Kunzendorf belonging to one genotype were isolated in a limited area of Japan. Variation in the antimicrobial resistance pattern was observed in isolates of both biotypes Choleraesuis and var. Kunzendorf. We have also shown that the PFGE profile was associated with the biotype and isolation region of each isolate., (2008 Elsevier Ltd. All rights reserved.)

Both carbohydrate monomers L-gulose and L-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of L-gulose and L-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing L-galactose at 4.6 g L(-1)d(-1) and L-gulose at 0.90 g L(-1)d(-1)., ((c) 2009 Elsevier Ltd. All rights reserved.)

Candida antarctica lipase B (CALB) has been used to polymerize and degrade polyesters. We developed a convenient method for investigating the biodegradability of plastics that involves the use of CALB-displaying "arming yeast." Polyurethane containing dulcitol units was prepared and used as the model material. Additionally, standard polyurethane with no dulcitol units was prepared by reacting 2,4-toluene diisocyanate with ethylene glycol. These polymers were incubated with CALB-displaying yeast cells. The polyurethane containing dulcitol was degraded, while the standard polyurethane was relatively unaffected. Arming yeast displaying appropriate enzymes can be used to investigate the biodegradability of synthetic plastics. It was also revealed that arming yeasts were applicable to evaluate the degradation of the film state of polyurethane.

In most organisms, productive utilization of galactose requires the highly conserved Leloir pathway of galactose metabolism. Yet, if this metabolic pathway is perturbed due to congenital deficiencies of the three associated enzymes, or an overwhelming presence of galactose, this monosaccharide which is abundantly present in milk and many non-dairy foodstuffs, will become highly toxic to humans and animals. Despite more than four decades of intense research, little is known about the molecular mechanisms of galactose toxicity in human patients and animal models. In this contemporary review, we take a unique approach to present an overview of galactose toxicity resulting from the three known congenital disorders of galactose metabolism and from experimental hypergalactosemia. Additionally, we update the reader about research progress on animal models, as well as advances in clinical management and therapies of these disorders.

Purpose: The two most widely investigated animal models for diabetic retinopathy (DR) are the rat and dog. In dogs, aldose reductase (AR) is present only in retinal capillary pericytes and their destruction has been linked to polyol accumulation and resulting apoptosis. Since both rat capillary pericytes and endothelial cells have been reported to contain AR, the role of polyol pathway activity in capillary cell destruction has been investigated in rat retinal capillary pericyte (TR-rPCT) and endothelial (TR-iBRB) cells., Methods: TR-rPCT and TR-iBRB cell lines were recloned and their identities were reconfirmed by characteristic immunostaining. Cells were cultured up to 72 h in media containing 50 mM glucose or galactose with/without the AR inhibitors or a sorbitol dehydrogenase inhibitor (SDI) or with 30 mM 3-fluoro-3-deoxyglucose. Polyol levels were determined by HPLC or (19)F-NMR. Apoptosis was detected with TUNEL/DAPI staining., Results: Smooth muscle actin is present only in pericytes while only endothelial cells stain for von Willebrand factor and accumulate acetylated low-density lipoprotein. AR is present in both cells but AR levels are lower in endothelial cells. Aldehyde reductase is also present in both cells. Cells cultured in 50 mM glucose or galactose show significant polyol accumulation in pericytes but endothelial cells show little accumulation of galactitol and no accumulation of sorbitol. Sorbitol accumulation in pericytes resulted in increased cellular permeability and increased TUNEL staining, which was reduced by AR inhibition., Conclusions: Although both rat retinal pericytes and endothelial cells contain AR, sorbitol accumulation and TUNEL staining primarily occur in pericytes and are inhibited by AR inhibitors.

Pregnancy, delivery, and postpartal metabolic control was monitored biochemically in five patients (22-38 years of age) with clinically, enzymatically, and genotypically established classical galactosaemia and good dietary compliance. Three of the patients performed breast feeding of their newborns. Monitoring parameters were galactose-1-phosphate and galactitol concentrations in erythrocytes and urinary excretion of galactose, galactitol, galactonate, and lactose. During pregnancy, a small but steady increase of renal metabolite excretion rates was observed. After delivery, a moderate transient increase of metabolite concentrations with peak values within the first week post partum occurred, irrespective of breast feeding. Altogether, there was no evidence for clinically or subclinically significant changes of metabolic control during pregnancy, delivery, or lactation. In conclusion, a specific metabolic monitoring is apparently not required in pregnant galactosemic women, and breast feeding of the nongalactosemic offspring can be recommended.

The human aldose reductase inhibitory effects of the methanol extracts of 17 medicinal and edible mushrooms were examined. Ganoderma lucidum showed the highest aldose reductase inhibitory activity compared with the other mushrooms. The effect of an ethanol extract of G. lucidum on the galactitol level in the eye lens was studied in a galactosemic rat model in vivo. This mushroom significantly decreased the galactitol accumulation., (Copyright 2008 John Wiley & Sons, Ltd.)

The uptake and catabolism of galactose by the yeast Saccharomyces cerevisiae is much lower than for glucose and fructose, and in applications of this yeast for utilization of complex substrates that contain galactose, for example, lignocellulose and raffinose, this causes prolonged fermentations. Galactose is metabolized via the Leloir pathway, and besides the industrial interest in improving the flux through this pathway it is also of medical relevance to study the Leloir pathway. Thus, genetic disorders in the genes encoding galactose-1-phosphate uridylyltransferase or galactokinase result in galactose toxicity both in patients with galactosemia and in yeast. In order to elucidate galactose related toxicity, which may explain the low uptake and catabolic rates of S. cerevisiae, we have studied the physiological characteristics and intracellular metabolite profiles of recombinant S. cerevisiae strains with improved or impaired growth on galactose. Aerobic batch cultivations on galactose of strains with different combinations of overexpression of the genes GAL1, GAL2, GAL7, and GAL10, which encode proteins that together convert extracellular galactose into glucose-1-phosphate, revealed a decrease in the maximum specific growth rate when compared to the reference strain. The hypothesized toxic intermediate galactose-1-phosphate cannot be the sole cause of galactose related toxicity, but indications were found that galactose-1-phosphate might cause a negative effect through inhibition of phosphoglucomutase. Furthermore, we show that galactitol is formed in S. cerevisiae, and that the combination of elevated intracellular galactitol concentration, and the ratio between galactose-1-phosphate concentration and phosphoglucomutase activity seems to be important for galactose related toxicity causing decreased growth rates.

This study reports the vital regulatory influence of Xyr1 (xylanase regulator 1) on the transcription of hydrolytic enzyme-encoding genes and hydrolase formation on lactose in Hypocrea jecorina. While the transcription of the xyr1 gene itself is achieved by release of carbon catabolite repression, the transcript formation of xyn1 (xylanase 1) is regulated by an additional induction mechanism mediated by lactose. Xyr1 has an important impact on lactose metabolism by directly activating xyl1 (xylose reductase 1) transcription and indirectly influencing transcription of bga1 (beta-galactosidase 1). The latter is achieved by regulating the conversion of D-galactose to the inducing carbon source galactitol.

The ability of Hypocrea jecorina (Trichoderma reesei) to grow on lactose strongly depends on the formation of an extracellular glycoside hydrolase (GH) family 35 beta-galactosidase, encoded by the bga1 gene. Previous studies, using batch or transfer cultures of pregrown cells, had shown that bga1 is induced by lactose and d-galactose, but to a lesser extent by galactitol. To test whether the induction level is influenced by the different growth rates attainable on these carbon sources, bga1 expression was compared in carbon-limited chemostat cultivations at defined dilution (=specific growth) rates. The data showed that bga1 expression by lactose, d-galactose and galactitol positively correlated with the dilution rate, and that galactitol and d-galactose induced the highest activities of beta-galactosidase at comparable growth rates. To know more about the actual inducer for beta-galactosidase formation, its expression in H. jecorina strains impaired in the first steps of the two d-galactose-degrading pathways was compared. Induction by d-galactose and galactitol was still found in strains deleted in the galactokinase-encoding gene gal1, which is responsible for the first step of the Leloir pathway of d-galactose catabolism. However, in a strain deleted in the aldose/d-xylose reductase gene xyl1, which performs the reduction of d-galactose to galactitol in a recently identified second pathway, induction by d-galactose, but not by galactitol, was impaired. On the other hand, induction by d-galactose and galactitol was not affected in an l-arabinitol 4-dehydrogenase (lad1)-deleted strain which is impaired in the subsequent step of galactitol degradation. These results indicate that galactitol is the actual inducer of Bga1 formation during growth on d-galactose in H. jecorina.

The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate-specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol-specific transporter (II(gat)) belongs to the glucitol family and is structurally the least well-characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three-dimensional structure of the IIB subunit (GatB). GatB consists of a central four-stranded parallel beta-sheet flanked by alpha-helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between beta1 and alpha1 that folds into a P-loop-like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA-interacting residues.

Ginger (Zingiber officinale Roscoe) continues to be used as an important cooking spice and herbal medicine around the world. Scientific research has gradually verified the antidiabetic effects of ginger. Especially gingerols, which are the major components of ginger, are known to improve diabetes including the effect of enhancement against insulin-sensitivity. Aldose reductase inhibitors have considerable potential for the treatment of diabetes, without increased risk of hypoglycemia. The assay for aldose reductase inhibitors in ginger led to the isolation of five active compounds including 2-(4-hydroxy-3-methoxyphenyl)ethanol (2) and 2-(4-hydroxy-3-methoxyphenyl)ethanoic acid (3). Compounds 2 and 3 were good inhibitors of recombinant human aldose reductase, with IC50 values of 19.2 +/- 1.9 and 18.5 +/- 1.1 microM, respectively. Furthermore, these compounds significantly suppressed not only sorbitol accumulation in human erythrocytes but also lens galactitol accumulation in 30% of galactose-fed cataract rat model. A structure-activity relationship study revealed that the applicable side alkyl chain length and the presence of a C3 OCH3 group in the aromatic ring are essential features for enzyme recognition and binding. These results suggested that it would contribute to the protection against or improvement of diabetic complications for a dietary supplement of ginger or its extract containing aldose reductase inhibitors.

Lactose (1,4-O-beta-d-galactopyranosyl-d-glucose) is a soluble and economic carbon source for the industrial production of cellulases or recombinant proteins by Hypocrea jecorina (anamorph Trichoderma reesei). The mechanism by which lactose induces cellulase formation is not understood. Recent data showed that the galactokinase step is essential for cellulase induction by lactose, but growth on d-galactose alone does not induce cellulases. Consequently, the hypothesis was tested that d-galactose may be an inducer only at a low growth rate, which is typically observed when growing on lactose. Carbon-limited chemostat cultivations of H. jecorina were therefore performed at different dilution rates with d-galactose, lactose, galactitol and d-glucose. Cellulase gene expression was monitored by using a strain carrying a fusion between the cbh2 (encoding cellobiohydrolase 2, Cel6A) promoter region and the Aspergillus niger glucose oxidase gene and by identification of the two major cellobiohydrolases Cel7A and Cel6A. The results show that d-galactose indeed induces cbh2 gene transcription and leads to Cel7A and Cel6A accumulation at a low (D=0.015 h(-1)) but not at higher dilution rates. At the same dilution rate, growth on d-glucose did not lead to cbh2 promoter activation or Cel6A formation but a basal level, lower than that observed on d-galactose, was detected for the carbon-catabolite-derepressible Cel7A. Lactose induced significantly higher cellulase levels at 0.015 h(-1) than d-galactose and induced cellulases even at growth rates up to 0.042 h(-1). Results of chemostats with an equimolar mixture of d-galactose and d-glucose essentially mimicked the behaviour on d-galactose alone, whereas an equimolar mixture of d-galactose and galactitol, the first intermediate of a recently described second pathway of d-galactose catabolism, led to cellulase induction at D=0.030 h(-1). It is concluded that d-galactose indeed induces cellulases at low growth rate and that the operation of the alternative pathway further increases this induction. However, under those conditions lactose is still a superior inducer for which the mechanism remains to be clarified.

Inhibition of Na+,K+-ATPase activity causes edema and cell death in central nervous system. We determined the in vitro effects of galactose-l-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) (mix A = classical galactosaemia) or Galtol and Gal (mix B = galactokinase deficiency galactosaemia), on Na+,K+-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P or Galtol alone at different concentrations, significantly inhibited Na+,K+-ATPase whereas Gal activated the enzyme in all investigated brain regions. Both mix A and mix B inactivated the enzyme by 20-30% (p < 0.001) in all studied areas. L-Cysteine (Cys) and glutathione (GSH) supplementation in mix B not only reversed the enzyme inhibition but also resulted in an activation of 50-60%, (p < 0.001) in all brain areas. Their presence in mix A also activated the inhibited Na+,K+-ATPase in hippocampus and hypothalamus to a lower degree, whereas Cys reversed the frontal cortex enzyme activity to control value only. These findings indicate that oxidation of the enzyme critical groups may be involved in galactosaemia, producing inhibitory effect. This phenomenon is reversed by antioxidants Cys and GSH, implying that free radicals may be implicated in the observed enzyme inactivation.

Genetic diversity among 86 Pasteurella multocida isolates was investigated by comparative sequence analysis of a 1468 bp fragment of the 16S rRNA gene. The strains included 79 field isolates recovered from birds (poultry) (22), cattle (21), pigs (26) and sheep (10) within England and Wales, four Asian isolates associated with bovine haemorrhagic septicaemia, and the type strains of the three subspecies of P. multocida. Dulcitol and sorbitol fermentation patterns were also determined to establish correlations between subspecies status and phylogenetic relatedness. Nineteen 16S rRNA types were identified, but these were clustered into two distinct phylogenetic lineages, A and B. Sequences within lineages A and B had a mean number of nucleotide differences of 21.12+/-3.90. Isolates within lineage A were associated with birds, cattle, pigs and sheep, whereas those belonging to lineage B were recovered from birds and a cat. Eighty-seven per cent of the isolates were classified as P. multocida subsp. multocida by dulcitol and sorbitol fermentation patterns, but these have diverse 16S rRNA gene sequences that were represented in both lineages A and B. Avian P. multocida subsp. septica isolates were associated exclusively with lineage B, but bovine P. multocida subsp. septica isolates were present in lineage A. P. multocida subsp. gallicida isolates of avian, bovine and porcine origin represent a homogeneous group within lineage A, but they have the same 16S rRNA type as certain P. multocida subsp. multocida isolates. These findings provide strong support for the view that dulcitol and sorbitol fermentation patterns are inaccurate indicators of genetic relatedness among P. multocida strains. Avian capsular type B isolates and capsular type B and E isolates associated with haemorrhagic septicaemia of cattle and water buffaloes are closely related and form a distinct cluster within lineage A. The current subspecies nomenclature of P. multocida neither accurately reflects the 16S rRNA-based phylogenetic relationships among isolates nor does it adequately encompass the full range of diversity within the species. The study provides a 16S rRNA-based evolutionary framework that will form the basis of further studies into the genetic diversity of P. multocida and will also help in the reclassification of the species.

Children and adolescents (n = 25) with galactosemia homozygous for the common Q188R mutation (substitution of glutamine codon 188 with arginine) were group matched for sex and age with healthy control participants (n = 20). Participants were administered an abbreviated neuropsychological battery by a doctoral-level psychologist. Results indicate that children and adolescents with galactosemia function generally within the low average IQ range, with a small standard deviation (indicating a relatively homogeneous IQ profile), and have many features suggestive of left-hemisphere dysfunction. Word retrieval difficulties are a primary component of the galactosemia profile. In addition, participants with galactosemia have less well-developed executive functions. Child and parental reports of behavioral symptoms differ; parents reported that their children had more internalizing symptoms than the children with galactosemia self-reported. Cognitive complications in galactosemia appear to emerge even in well-treated children.

Enteric bacteria (Enteriobacteriaceae) carry on their single chromosome about 4000 genes that all strains have in common (referred to here as "obligatory genes"), and up to 1300 "facultative" genes that vary from strain to strain and from species to species. In closely related species, obligatory and facultative genes are orthologous genes that are found at similar loci. We have analyzed a set of facultative genes involved in the degradation of the carbohydrates galactitol, D-tagatose, D-galactosamine and N-acetyl-galactosamine in various pathogenic and non-pathogenic strains of these bacteria. The four carbohydrates are transported into the cell by phosphotransferase (PTS) uptake systems, and are metabolized by closely related or even identical catabolic enzymes via pathways that share several intermediates. In about 60% of Escherichia coli strains the genes for galactitol degradation map to a gat operon at 46.8 min. In strains of Salmonella enterica, Klebsiella pneumoniae and K. oxytoca, the corresponding gat genes, although orthologous to their E. coli counterparts, are found at 70.7 min, clustered in a regulon together with three tag genes for the degradation of D-tagatose, an isomer of D-fructose. In contrast, in all the E. coli strains tested, this chromosomal site was found to be occupied by an aga/kba gene cluster for the degradation of D-galactosamine and N-acetyl-galactosamine. The aga/kba and the tag genes were paralogous either to the gat cluster or to the fru genes for degradation of D-fructose. Finally, in more then 90% of strains of both Klebsiella species, and in about 5% of the E. coli strains, two operons were found at 46.8 min that comprise paralogous genes for catabolism of the isomers D-arabinitol (genes atl or dal) and ribitol (genes rtl or rbt). In these strains gat genes were invariably absent from this location, and they were totally absent in S. enterica. These results strongly indicate that these various gene clusters and metabolic pathways have been subject to convergent evolution among the Enterobacteriaceae. This apparently involved recent horizontal gene transfer and recombination events, as indicated by major chromosomal rearrangements found in their immediate vicinity.

Unlabelled: Galactosaemia is an inborn error of galactose (Gal) metabolism characterized by irreversible brain damage. The aim of this study was to evaluate whether the antioxidants L-cysteine (Cys) and the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes Na+,K+ -ATPase and Mg2+ -ATPase in in vitro galactosaemia. Mixture A (mix. A: galactose-1-phosphate (Gal-1-P, 2mM) plus galactitol (Galtol, 2mM) plus Gal (4mM) = classical galactosaemia) or Mixture B (mix. B: Galtol (2mM) plus Gal (1mM) = galactokinase deficiency galactosaemia) were preincubated in the presence or absence of Cys (0.83mM) or GSH (0.83 mM) with whole brain homogenates of suckling rats at 37 degrees C for 1h. TAS and the enzyme activities were determined spectrophotometrically. The preincubation of brain homogenates with mix. A or mix. B resulted in a decrease of TAS to 30% (P < 0.01), while the presence of Cys or GSH increased TAS to 20% (P < 0.01) and 60% ( P < 0.001), respectively. The antioxidants reversed the inhibited Na+,K+ -ATPase by mix. A or mix. B and the stimulated Mg2+ -ATPase by mix. B to control values, whereas no effect was observed on the enormously activated Mg2+ -ATPase by mix. A., Conclusions: (a) Gal and its derivatives may produce free radicals in the suckling rat brain, reported for first time, (b) Na+,K+ -ATPase inhibition and Mg2+ -ATPase activation are probably due to the oxidative stress from the above compounds, (c) Cys or GSH could play a protective role reversing the inhibited Na+,K+ -ATPase toward normal in in vitro galactosaemia and (d) the addition of the above antioxidants may reduce the consequences of brain Mg2+ -ATPase activation by Gal and Galtol in galactokinase deficiency galactosaemia.

Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.