Rational design of L-arabinose isomerase from Lactobacillus fermentum and its application in D-tagatose production / 生物工程学报

L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The Km and kcat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.

Ershiwuwei Shanhu Pills regulate Akt/mTOR/GSK-3β signaling pathway to alleviate Alzheimer's disease mice / 中国中药杂志

The present study investigated the mechanism of the Tibetan patent medicine Ershiwuwei Shanhu Pills(ESP) in alleviating Alzheimer's disease in mice via Akt/mTOR/GSK-3β signaling pathway. BALB/c mice were randomly assigned into a blank control group, a model group, low(200 mg·kg~(-1)), medium(400 mg·kg~(-1)) and high(800 mg·kg~(-1)) dose groups of ESP, and donepezil hydrochloride group. Except the blank control group, the other groups were given 20 mg·kg~(-1) aluminum chloride by gavage and 120 mg·kg~(-1) D-galactose by intraperitoneal injection for 56 days to establish Alzheimer's disease model. Morris water maze was used to detect the learning and memory ability of mice. The level of p-tau protein in mouse hippocampus and the levels of superoxide dismutase(SOD), malondialdehyde(MDA), catalase(CAT), and total antioxidant capacity(T-AOC) in hippocampus and serum were detected. Hematoxylin-eosin staining and Nissl staining were performed for the pathological observation of whole brain in mice. TdT-mediated dUTP nick-end labeling(TUNEL) staining was employed for the observation of apoptosis in mouse cortex. Western blot was adopted to detect the protein levels of p-mTOR, p-Akt, and GSK-3β in the hippocampus. Compared with the model group, the ESP groups showcased alleviated pathological damage of the whole brain, decreased TUNEL positive cells, reduced level of p-tau protein in hippocampus, and risen SOD, CAT, and T-AOC levels and declined MDA level in hippocampus and serum. Furthermore, the ESP groups had up-regulated protein levels of p-mTOR and p-Akt while down-regulated protein level of GSK-3β in hippocampus. Therefore, ESP can alleviate the learning and memory decline and oxidative damage in mice with Alzheimer's disease induced by D-galactose combined with aluminum chloride, which may be related to Akt/mTOR/GSK-3β signaling pathway.

Modeling of lactose enzymatic hydrolysis using Monte Carlo method

Background: Mathematical modeling is useful in the analysis, prediction, and optimization of an enzymatic process. Unlike the conventional modeling methods, Monte Carlo method has special advantages in providing representations of the molecule's spatial distribution. However, thus far, Monte Carlo modeling of enzymatic system is namely based on unimolecular basis, not suitable for practical applications. In this research, Monte Carlo modeling is performed for enzymatic hydrolysis of lactose for the purpose of real-time applications. Results: The enzyme hydrolysis of lactose, which is conformed to Michaelis­Menten kinetics, is modeled using the Monte Carlo modeling method, and the simulation results prove that the model predicts the reaction kinetics very well. Conclusions: Monte Carlo modeling method can be used to model enzymatic reactions in a simple way for real-time applications.

Effect of chinese traditional herb Epimedium grandiflorum C.Morren and its extract Icariin on osteoarthritis via suppressing NF-kB pathway.

Osteoarthritis (OA), which is also called degenerative arthritis, is the leading cause of disabilities in the old people. The Chinese traditional herb Epimedium grandiflorum had long been found to attenuate osteoarthritis process, but the detailed mechanism was not clear. To study the mechanisms of E. grandiflorum in the treatment of osteoarthritis, rabbit osteoarthritis model combined with D-galactose was used. After different treatments for 10 weeks, cartilage sections were analyzed by immunohistochemistry for uPA, uPAR and PAI expression level. E. grandiflorum could significantly attenuate OA condition and decrease uPA, uPAR and PAI expression. The extract of E. grandiflorum, icariin also had a similar effect when compared with E. grandiflorum treatment alone. Rabbit chondrocytes were further isolated to be stimulated by TNFα combined with different reagents treatment. Here, icariin treatment significantly reduced nuclear factor kappa B NF-B (P65) activity, decreased uPA expression level and increased IBα protein level. The results indicated that E. grandiflorum and its extract icariin could attenuate OA condition, reduce the expression of uPA and uPAR and increase PAI in experimental rabbit model and this effect may be conducted by suppressing NF-kB activity by increasing IkBα level.

Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds.

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B.variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

Characterization of 2-deoxy-D-glucose uptake in fibroblast cultures derived from patients with A3243G mitochondrial DNA mutation.

We investigated cellular glucose uptake of fibroblast cultures derived from seven patients with mitochondrial DNA (mtDNA) A3243G mutation and from six healthy controls with no mtDNA mutations. Heteroplasmy of fibroblast cultures were shifted by culturing for 5 days in galactose-containing medium. The proportion of mutant mtDNA decreased by 7.7% to 10% in three patient fibroblast cultures, whereas 2-deoxy-D-glucose uptake increased 1.8-2.1-fold at basal state, 1.9-2.3-fold in the presence of 60 ng/ml of insulin, and 1.8-2.1-fold in 100 ng/ml of insulin. No significant changes in level of heteroplasmy or glucose uptake were observed in the other patients samples and control samples. This study showed that alteration in the proportion of fibroblast mtDNA A3243G mutation content directly affected basal and insulin-stimulated glucose uptake.

Congenital glucose galactose malabsorption complicated with rickets and nephrogenic diabetes insipidus

Congenital Glucose Galactose malabsorption [CGGM] is a rare disorder with limited data from the Arab world. We report the first case of CGGM in Oman. B.S.A two years old female who presented with chronic osmotic diarrhea since birth with hypernatraemic dehydration. B.S was found to have Glucose Galactose Malabsorption based on clinical trial of ORS and elemental formula. Symptoms resolved on introduction of Carbohydrate free formula. The patient developed many complications while on TPN including rickets and nephrogenic diabetes insipidus. These complications have not been reported earlier in CGGM

Alteración del metabolismo de la galactosa / Galactose metabolism defect

Las alteraciones del metabolismo de la galactosa se producen por el defecto de las enzimas: galactoquinasa (GALK), galactosa-1-fosfato-uridil transferasa (GAL1-PUT) y uridin difosfato galactosa 4' epimerasa (UDPGAL); de ellas la más frecuente es la galactosemia clásica producida por la deficiencia de GAL1PUT. Producto de este defecto se acumula galactosa-1-fosfato, galactosa libre y galactitol en sangre y tejidos, los que producen alteraciones hepáticas, renales y cerebrales. Su herencia es autosómica recesiva y la incidencia estimada a nivel mundial fluctúa entre 1:60.000 a 1:33.000 recién nacidos. Los síntomas y signos más característicos son vómito, diarrea, ictericia, hepatomegalia, cataratas. Si la enfermedad no es tratada oportunamente ocasiona la muerte del niño. El tratamiento consiste en eliminar la lactosa y galactosa de la alimentación, lo que incluye alimentos tales como la leche de todo tipo y sus derivados, la galactosa y alimentos o medicamentos que contenga alguno de estos productos. Se entrega leche de soya y los requerimientos de macro y micro nutrientes se indican según las recomendaciones para edad y sexo. La dieta dura toda la vida ya que la galactosa se transforma en galactitol, existiendo riesgo de producir catarata y daño renal en cualquier momento de la vida. Un buen control se obtiene al mantener el nivel sanguíneo de galactosa-1-fosfato igual o menor a 3.0 mg/dL y urinario de galactitol bajo 0.8 mmol/mol de creatinina.

Antioxidant enzymes and peroxidation in galactogenic cataract.

The antioxidant enzymes like catalase and superoxide dismutase (SOD) were studied in erythrocytes and lens at various stages of cataractogenesis in albino rats. The rate of peroxidation was measured by assessing the malondiadehyde (MDA) in lens and plasma. The insoluble and soluble protein fractions were measured in lens to study the protein crosslinkings in relation to the above said parameters. Cataract was induced in albino rats by feeding it with 30% galactose as part of the normal diet (w/w) for 30 days. The results show a decrease of SOD and catalase with concomitant increase of MDA and insoluble protein with the advancement of cataract.

Hemagglutination pattern of galactose specific lectin from Pedilanthus tithymaloides in diabetes mellitus.

The usefulness of a galactose specific lectin from P. tithymaloides was examined to study the hemagglutination pattern in 193 patients with diabetes mellitus out of which 34 cases were of insulin dependent. A control of 72 normal subjects was also included. The hemagglutination titre against a partially purified lectin from P. tithymaloides of control group ranged from 9.1 to 170 units per mg protein with a mean value of 54 units per mg protein. Significantly low titre was observed in the cases with insulin dependent diabetes mellitus, while non-insulin dependent diabetes mellitus cases did not show any significant change. Further significant reduction in the titre in insulin dependent diabetes mellitus group was shown to occur along with the increased duration of the diabetic condition, reflecting measurable erythrocyte surface alterations.

Free lysine, glycine, alanine, glutamic acid and aspartic acid reduce the glycation of human lens proteins by galactose.

The amino acids lysine, glycine, alanine, glutamate and aspartate formed adducts with galactose at physiological pH and temperature as shown by incorporation of U[14C] galactose. The percentage of galactose reacting with lysine, glycine, alanine, glutamate and aspartate was 4.5 to 7.8, 7.9 to 10.8, 3.2 to 4.6, 2.8 to 4.8 and 3 to 5.2, respectively. Studies with lysine showed that the extent of glycation of the free amino acid increased with time. Incubation of lens homogenate with galactose, effected glycation of proteins. Addition of lysine in concentrations of 5 and 10 mM to equimolar concentrations of galactose decreased the glycation of lens proteins by 64% to 71%; glycine, alanine, glutamate and aspartate decreased glycation by 23 to 68%, 32 to 61%, 35 to 56% and 26 to 61% respectively. Under similar conditions, glycine reacts to a greater extent than lysine, alanine, glutamic and aspartic acids. However, lysine was more effective than glycine, alanine, aspartic and glutamic acids in decreasing glycation of lens proteins by galactose. The decrease of glycation with added lysine increased with time. In general increase of amino acid concentration rather than that of sugar augmented the decrease of glycation of lens proteins.

Differential binding properties of Gal/GalNAc specific lectins available for characterization of glycoreceptors.

Differentiating the binding properties of applied lectins should facilitate the selection of lectins for characterization of glycoreceptors on the cell surface. Based on the binding specificities studied by inhibition assays of lectin-glycan interactions, over twenty Gal and/or GalNAc specific lectins have been divided into eight groups according to their specificity for structural units (lectin determinants), which are the disaccharide as all or part of the determinants and of GalNAc alpha 1-->Ser (Thr) of the peptide chain. A scheme of codes for lectin determinants is illustrated as follows: (1) F (GalNAc alpha 1-->3GalNAc), Forssman specific disaccharide--Dolichos biflorus (DBL), Helix pomatia (HPL) and Wistaria floribunda (WFL) lectins. (2) A (GalNAc alpha 1-->3 Gal), blood group A specific disaccharide--Codium fragile subspecies tomentosoides (CFT), Soy bean (SBL), Vicia villosa-A4 (VVL-A4), and Wistaria floribunda (WFL) lectins. (3) Tn (GalNAc alpha 1-->Ser (Thr) of the protein core)--Vicia villosa B4 (VVL-B4), Salvia sclarea (SSL), Maclura pomifera (MPL), Bauhinia purpurea alba (BPL) and Artocarpus integrifolia (Jacalin, AIL). (4) T (Gal beta 1-->3GalNAc), the mucin type sugar sequences on the human erythrocyte membrane(T alpha), T antigen or the disaccharides at the terminal nonreducing end of gangliosides (T beta)--Peanut (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera (MPL), Sophora japonica (SJL), Artocarpus lakoocha (Artocarpin) lectins and Abrus precatorius agglutinin (APA).(5) I and II (Gal beta 1-->3(4)GlcNAc)--the disaccharide residue at the nonreducing end of the carbohydrate chains derived from either N- or O-glycosidic linkage--Ricinus communis agglutinin (RCA1), Datura stramonium (TAL, Thorn apple), Erythrina cristagalli (ECL, Coral tree), and Geodia cydonium (GCL). (6) B (Gal alpha 1-->3Gal), human blood group B specific disaccharide--Griffonia(Banderiaea) simplicifolia B4 (GSI-B4). (7) E (Gal alpha 1-->4Gal), receptors for pathogenic E. coli agglutinin, Shiga toxin and Mistletoe toxic lectin-I (ML-I) and abrin-a.

Padronizaçäo do "spot-test" de Beutler e Baluda para o diagnóstico da galactosemia / Standardisation of Beutler-Baluda spot-test for the galactosemia diagnostic

A galactosemia é uma doença metabólica, causada por um déficit da enzima hexose 1-fosfato uridiltransferase (E.C.2.2.7.12). Seus principais sintomas clínicos säo vömitos e diarréias, que aparecem desde as primeiras ingestöes de leite, alteraçäo hepática e alteraçäo do cristalino com catarata bilateral. Com o objetivo de diagnosticar a galactosemia, foi padronizado um método qualitativo, o "spot-test" de Beutler-Baluda, que permite evidenciar a atividade da enzima, através de uma cascata de reaçöes enzimáticas que leva a formaçäo de NADP reduzido, cuja fluorescência é observada sob luz ultravioleta. A partir desta avaliaçäo 7 pacientes tiveram a confirmaçäo laboratorial do diagnóstico de galactosemia

Effects of monensin on glycosphingolipid metabolism in cultured human proximal tubular cells.

Effects of monensin, a monovalent cationic ionophore which disrupts Golgi apparatus and its related functions, on glycosphingolipid (GSL) metabolism were investigated in cultured human proximal tubular (PT) cells. Monensin (10(-6) M) stimulated [3H]Gal incorporation into GlcCer, GalCer and LacCer by 8.5-fold and 15-fold, respectively, in PT cells as compared to control. In contrast, [3H]Gal incorporation into GbOse3Cer and GM3 remained unchanged and that into GbOse4Cer was decreased 2-fold as compared to control. GSL measured by HPLC revealed that in cells incubated with monensin, GlcCer, GalCer and LacCer levels were increased 1.6-fold and 7-fold, respectively, whereas GbOse3Cer and GbOse4Cer levels were decreased several folds. Cells incubated with monensin contained 2.5- to 3-fold higher activity of alpha-galactosidase, beta-galactosidase and beta-glucosidase than control, whereas the activity of UDP-gal: glucosylceramide galactosyltransferase (beta-GalT-2) was 8-fold lower than control cells. Cells incubated with monensin took up and degraded one-half as much 125I-LDL as that of control cells. In control cells, exogenously derived [3H]LacCer on LDL was rapidly taken up and catabolized to monoglycosylceramide, or it was used for the endogenous synthesis of globotriosylceramide (trihexosylceramide), globotetraosylceramide (tetrahexosylceramide) and a ganglioside, GM3. In contrast, cells incubated with monensin accumulated most of the [3H]LacCer-LDL. Exogenously derived [3H]LacCer on LDL was catabolized to GlcCer, but was not utilized, for the synthesis of globotriosylceramide, globotetraosylceramide and GM3 in cells incubated with monensin.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic analysis of galactose toxicity in Escherichia coli with 2-deoxygalactose as the probe.

Biochemical basis of galactose toxicity has been studied in gal T mutants (CGSC 4974) using 2-deoxygalactose, a non-metabolizable analogue of galactose, as the probe. It is found that biochemical features of toxicity in wild type cells either with 2-deoxygalactose or with 2-deoxyglucose are very similar to the picture obtained with gal T mutants and the observed bacteriostasis is probably due to futile phosphorylation and not due to any specific inhibitory effect of phosphorylated galactose.