[Construction and culture condition optimization of a Saccharomyces cerevisiae strain for production of galactitol].

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 transport of mannitol in Sinorhizobium meliloti is carried out by a broad-substrate polyol transporter SmoEFGK and is affected by the ability to transport and metabolize fructose.

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.

Membrane-bound sorbitol dehydrogenase is responsible for the unique oxidation of D-galactitol to L-xylo-3-hexulose and D-tagatose in Gluconobacter oxydans.

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 Ca2+-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.

Characterization of the sorbitol dehydrogenase SmoS from Sinorhizobium meliloti 1021.

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 Šresolution and those of a complex with sorbitol diffracted to 2.0 Šresolution. 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.

L-Xylo-3-hexulose, a new rare sugar produced by the action of acetic acid bacteria on galactitol, an exception to Bertrand Hudson's rule.

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.

Sugar Alcohols of Polyol Pathway Serve as Alarmins to Mediate Local-Systemic Innate Immune Communication in Drosophila.

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.

Galactitol catabolism in Sinorhizobium meliloti is dependent on a chromosomally encoded sorbitol dehydrogenase and a pSymB-encoded operon necessary for tagatose catabolism.

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.

Assessment of lactase activity in humans by measurement of galactitol and galactonate in serum and urine after milk intake.

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.

Diosgenin, a Novel Aldose Reductase Inhibitor, Attenuates the Galactosemic Cataract in Rats.

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 IC50 of 4.59 × 10-6 mol/L. Diosgenin (10 µM) 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.

Recurrent Reverse Evolution Maintains Polymorphism after Strong Bottlenecks in Commensal Gut Bacteria.

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.

Galactose and its Metabolites Deteriorate Metaphase II Mouse Oocyte Quality and Subsequent Embryo Development by Disrupting the Spindle Structure.

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.

Genetic Characterization of the Galactitol Utilization Pathway of Salmonella enterica Serovar Typhimurium.

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.

ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58.

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.

Caproiciproducens galactitolivorans gen. nov., sp. nov., a bacterium capable of producing caproic acid from galactitol, isolated from a wastewater treatment plant.

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).

Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in D-tagatose production.

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.

Aldose reductase inhibitors of plant origin.

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.

Polyol effects on growth factors and MAPK signaling in rat retinal capillary cells.

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-ß, 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-ß, 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-ß, 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.

Enhanced production of poly(lactate-co-3-hydroxybutyrate) from xylose in engineered Escherichia coli overexpressing a galactitol transporter.

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.

Aldose reductase inhibitor counteracts the enhanced expression of matrix metalloproteinase-10 and improves corneal wound healing in galactose-fed rats.

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.