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 K_m and k_cat 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.
Galactose/metabolism* ; Limosilactobacillus fermentum/genetics* ; Lactose ; Hexoses/metabolism* ; Aldose-Ketose Isomerases/genetics* ; Hydrogen-Ion Concentration

OBJECTIVETo investigate the correlation between nonsyndromic deafness and mitochondrial 12s rRNA A839G mutation.

METHODSAccording to the clinical manifestations of mitochondrial DNA sequencing and analysis to find and determine family containing mitochondrial 12s rRNA A839G mutation. Harvested its family members blood and transferred their lymphocytes into lymphoblastoid cell lines, followed by cells cultured, cell doubling experiment, susceptibility testing, cellular oxygen consumption rate experiment, ROS and mitochondrial membrane potential experimental tests were progressed to explore the correlation between the A839G mutation and nonsyndromic deafness.

RESULTSThe mitochondrial 12s rRNA A839G mutation pedigrees were determined through the full sequence detections of the Mitochondrial DNA, further phylogenetic analysis showed that 839 point conservative index (CI) up to 78.6%; in RPMI-galactose medium containing A839G gene mutant cell line, the doubling time was significantly longer than the control group, and the difference was significant (P = 0.033). The effect to cell lines containing the A839G mutation of aminoglycoside drugs was not obvious. When compared with the control group, cell lines containing the A839G mutation significantly reduced cellular oxygen consumption rate(P = 0.033); compared with the control group, the ROS levels of cell lines containing the A839G mutation appeared more substantial elevated with significan difference (P < 0.01). The mitochondrial membrane potential of cells of experimental group was significantly reduced than the control group.

CONCLUSIONThe present study proved that the mitochondria 12s rRNA A839G mutations affect the function of the mitochondrial respiratory chain at the cell level, which might reduce the growth rate of the mutant cell lines, result in hearing.

Aminoglycosides ; Cell Line ; DNA, Mitochondrial ; Deafness ; genetics ; Galactose ; Hearing Tests ; Mitochondria ; Mutation ; Pedigree ; Phylogeny ; RNA, Ribosomal ; genetics

L-Arabinose isomerase (L-AI) is an intracellular enzyme that catalyzes the reversible isomerization of D-galactose and D-tagatose. Given the widespread use of D-tagatose in the food industry, food-grade microorganisms and the derivation of L-AI for the production of D-tagatose is gaining increased attention. In the current study, food-grade strains from different foods that can convert D-galactose to D-tagatose were screened. According to physiological, biochemical, and 16S rDNA gene analyses, the selected strain was found to share 99% identity with Pediococcus pentosaceus, and was named as Pediococcus pentosaceus PC-5. The araA gene encoding L-AI from Pediococcus pentosaceus PC-5 was cloned and overexpressed in E. coli BL21. The yield of D-tagatose using D-galactose as the substrate catalyzed by the crude enzyme in the presence of Mn2+ was found to be 33% at 40 degrees C.
Aldose-Ketose Isomerases ; biosynthesis ; genetics ; Biotransformation ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Galactose ; metabolism ; Genetic Vectors ; genetics ; Hexoses ; metabolism ; Pediococcus ; classification ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVE: To study the expression of the glycoprotein in atrioventricular cell membrane of the acute myocardial infarction. METHODS: The glycoprotein changes occurred at the atrioventricular cell membrane of the acute myocardial infarction of 8 cases were observed by using immunohistochemical methods. RESULTS: Positive staining of PNA could be observed in atrioventricular cell membrane. CONCLUSION This experiment proved that atrioventricular cell membrane expressed D-galactose as same as myocardial cell membrane in the acute myocardial infarction.
Atrioventricular Node/metabolism* ; Cell Membrane/metabolism* ; Forensic Medicine ; Galactose/genetics* ; Humans ; Male ; Membrane Glycoproteins/genetics* ; Middle Aged ; Myocardial Infarction/metabolism* ; Myocardium/metabolism*

The study was purposed to develop a novel cryopreserved agent (CPA) for platelets, to investigate the morphology of cryopreserved platelets in different CPA and the CD62P expression on membrane of platelets after stimulating by thrombin, as well as to compare the effect of adding UDP-Gal on preserved efficiency of preservation solutions. A novel cryopreserved agent consisting of 2% DMSO, thrombosol and UDP-Gal was developed on basis of using higher concentration of DMSO. The morphology of chilled platelets was observed by transmission electron microscope and compared with fresh platelets. The expression of CD62P on the membrane of platelets was detected at 0, l, 3 months. The results indicated that the significant effect of cryopreservation on morphology of platelets was found according to percentages of round, dendritic and irregular shapes of cryopreserved platelets. The protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal were better than that of 5% DMSO. Compared with fresh platelets, the expression of CD62P on platelet membrane decreased obviously after cryopreservation, but not observed difference at preservation for 1 month and 3 months, as well as among 3 kinds of different CPA. It is concluded that the protective effects of 2% DMSO + thrombosol and 2% DMSO + thrombosol + UDP-Gal on morphology of platelets are similar, but better than that 5% DMSO. The reaction of cryopreserved platelets to thrombin decreases, while the significant difference is not found among these 3 kinds of CPA. The addition of UDP-Gal to cryopreserved agents not show the protective effect on platelets.
Blood Platelets ; Blood Preservation ; methods ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Dimethyl Sulfoxide ; pharmacology ; Humans ; P-Selectin ; biosynthesis ; genetics ; Uridine Diphosphate Galactose ; pharmacology

OBJECTIVETo research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of kanamycin.

METHODSFifty wistar rats were randomly divided into four groups: group A (D-galactose group, n = 14) were treated with hypodermic 5% D-galactose (150 mg x kg(-1) x d(-1)) for 8 weeks and then with intraperitoneal saline for 10 days; group B (D-galactose and kanamycin group, n = 14) were given the same dose of D-galactose but kanamycin (500 mg x kg(-1) x d(-1)) instead of saline; group C (kanamycin group, n = 12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n = 10) were given saline only. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and the mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique.

RESULTSThe incidence of mitochondrial DNA 4834 bp deletion mutation was 100% (28/28) in group A, 92.86% (26/28) in group B and 0% in group C or group D. The activity of GSHPX in group A was (59.07 +/- 8.70)U, (63.29 +/- 12. 40)U in group B, (136.67 +/- 9.53)U in group C and (142.10 +/- 7.02)U in group D. The difference between group A and D was significant (P = 0.000) while the difference between group A and B was not significant (P = 0.307), which was similarly as between group C and group D (P = 0.151). ABR threshold was (5.36 +/- 3.08) dB peSPL in group A, (61.79 +/- 11.20) dB peSPL in group B, (34.17 +/- 4.69) dB peSPL in group C and (6.50 +/- 3.37) dB peSPL in group D. No difference was found between group A and D (P = 0.398) while the difference in shift of ABR threshold between group B and group C (or group D) was significant (P = 0.000).

CONCLUSIONSThe mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats present high incidence of mtDNA4834 deletion which can greatly enhance the sensitivity of the inner ear to the kanamycin.

Aging, Premature ; chemically induced ; Animals ; DNA, Mitochondrial ; genetics ; Disease Models, Animal ; Disease Susceptibility ; Ear, Inner ; drug effects ; Galactose ; toxicity ; Kanamycin ; toxicity ; Rats ; Rats, Wistar ; Sequence Deletion

The purpose of this study was to investigate the molecular genetic basis of A2 subgroup and identify the novel alleles at ABO locus in Chinese Han population. All seven exons and their flanking sequences, enhancer and promoter in the ABO gene of five samples from individuals with serological discrepancies were amplified by polymerase chain reaction (PCR); the PCR products were screened by directly sequencing; the haplotypes of exon 6 and 7 were analyzed by TOPO cloning sequencing. The results showed that five samples were identified as A2 or A2B subgroup by serological technology. The A201 and A205 alleles were confirmed in one A2B individual and one A2 individual, respectively. A novel A2 variant allele was identified in three A2B individuals. The two nucleotide acid alterations (467C > T and 539G > C) at the exon 7 resulting in two amino acid substitutions (P156L and R180P) in this novel allele were observed, when compared with A101 allele. It is concluded that the polymorphism of A2 allele is found to be relatively variable in Chinese population, and a novel A208 allele responsible for A2 subgroup is firstly reported.
ABO Blood-Group System ; genetics ; Alleles ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; ethnology ; Female ; Fucosyl Galactose alpha-N-Acetylgalactosaminyltransferase ; genetics ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Point Mutation

OBJECTIVETo investigate effects of total paeony glucosides (TPGs) on the expressions of Toll receptors (TLR4) and interleukin-33 (IL-33) in the brain tissue of D-galactose-induced aging rats. METHODS; Fifty SD rats were randomly divided into 5 groups, i.e., the blank control group, the model group, the high dose TPG group, the middle dose TPG group, and the low dose TPG group, 10 in each group. Equal volume of normal saline was subcutaneously injected to rats in the blank control group, while 10% D-galactose was subcutaneously injected to rats in the rest groups at 0.125 mL/g, once a day for 8 successive weeks to induce the aging rat model. TPG was administered at 300 mg/kg, 150 mg/kg, and 75 mg/kg to rats in the high, middle, and low dose TPG groups while injecting D-galactose from the 5th week of model preparation, once daily for 4 successive weeks. Equal volume of normal saline was administered to rats in the blank control group and the model group, once daily. The capability for learning and memory was detected using Morris water. The mRNA expressions of TLR4 and IL-33 in the brain tissue were detected using ELISA.

RESULTSCompared with the blank control group, the capability for learning and memory decreased in the model group with statistical difference (P < 0.05). Compared with the model group, the capability for learning and memory was obviously improved in all the medicated groups in a dose-dependent manner, showing statistical difference (P < 0.05). Compared with the blank control group, mRNA expressions of TLR4 and IL-33 in the brain tissue obviously increased after medication in the model group, showing statistical difference (P < 0.05). Compared with the model group, mRNA expressions of TLR4 and IL-33 in the brain tissue obviously decreased after medication in all the medicated groups in a dose-dependent manner, showing statistical difference (P < 0.05).

CONCLUSIONTPGs improved D-galactose induced aging rats' capability for learning and memory through regulating changes of TLR4 and IL-33 expressions.

Aging ; drug effects ; Animals ; Brain ; drug effects ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Galactose ; adverse effects ; Interleukins ; metabolism ; Learning ; drug effects ; Male ; Memory ; drug effects ; Paeonia ; chemistry ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo evaluate the efficacy of gene delivery to the right lateral lobe of the rat liver via a branch of the bile duct using naked DNA.

METHODSWe evaluated regional gene delivery to the right lateral lobe of the rat liver via a branch of the bile duct, using naked DNA, including pGL3, pCMV beta and Cy3 labeled CMV beta.

RESULTSGene expression was observed in right lateral lobe of both the damaged and the normal rats liver. The gene delivery efficiency was similar in two groups (P > 0.05). Gene expression was found in the right lateral lobe of damaged and normal livers. Fluorescence was observed in the region of the portal triads, and occasionally, in the lobule.

CONCLUSIONRetrograde infusion of naked DNA via the bile duct is an effective way to deliver genes to in both damaged and normal rat liver.

Animals ; Bile Ducts ; metabolism ; DNA ; genetics ; Disease Models, Animal ; Female ; Galactose ; Gene Expression ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; Liver ; metabolism ; pathology ; Liver Diseases ; genetics ; pathology ; therapy ; Luciferases ; genetics ; metabolism ; Plasmids ; genetics ; Rats ; Rats, Inbred Lew ; beta-Galactosidase ; genetics ; metabolism

AIMTo investigate the mechanisms of Naoyikang (Traditional Chinese Medicine) on the Alzheimer's Disease (AD) model mice induced by D-galactose (D-gal) and NaNO2.

METHODSThe mouse model was established by intraperitoneal injection of D-gal and NaNO2. The capacity of learning and memory was tested on mice with electrical maze; the content of nitric oxide (NO) and the activity of monoamine oxidase-B (MAO-B), glutathione peroxidase (GSH-PX), Na(+) -K(+) -ATP enzyme and Ca(2+) -ATP enzyme in cerebral cortex and hippocampus were assayed by biochemical methods; expression of Bax and Bcl-2 mRNA was detested by RT-PCR.

RESULTSNaoyikang could ameliorate the capacity of learning and memory of AD model mice and reduce MAO-B activity in the brain tissue and activate the activity of Na(+) -K(+) -ATP enzyme and Ca(2+) -ATP enzyme in the brain tissue and decrease the expression of Bax mRNA, but increase the expression of Bcl-2 mRNA in the model brain tissue.

CONCLUSIONNaoyikang could protect AD model mice induced by D-gal and NaNO2. It could modify the metabolism of monoamine neurotransmitter in brain through reducing MAO-B activity and protect neurons by activating the activity of Na(+) -K(+) -ATP enzyme and Ca(2+) -ATP enzyme and decrease Bax expression and increase Bcl-2 expression in the model brain tissue.

Alzheimer Disease ; chemically induced ; drug therapy ; Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Galactose ; Male ; Maze Learning ; Mice ; Mice, Inbred ICR ; Neuroprotective Agents ; therapeutic use ; Phytotherapy ; RNA, Messenger ; genetics ; metabolism ; Random Allocation ; Sodium Nitrite ; Sodium-Potassium-Exchanging ATPase ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism