Comprehensive biochemistry experiment,which is interlocked and has difficulties in a certain degree,requires considerable knowledge,multiple techniques and long time.In order to ensure the smooth progress of the experiment,biochemistry and molecular biology department of Hebei medical university has taken following measures in teaching preparation and teaching implementation:building a specialized laboratory;performing collective lesson preparation and pre-experiments;technical teaching;teaching with multimedia equipments;students submitting experimental preparatory reports before class and then completing the experiments in groups.These measures achieved the intended purpose of setting up a comprehensive experiment.

Objective To report the clinical and pathological characteristics of one patient with glycogen storage disease Ⅳ (Anderson disease). Methods The patient was received detailed clinical examinations, ultrasound, electromyography, head MRI and muscle biopsy. Results The onset of the 22 years old male patient was 7yrs. The main symptoms were intolerance and fatigue in proximal limbs muscular movement, cardiopalmus by chance. Abdominal ultrasound examinations showed cirrhosis, portal hypertension, splenomegaly. Echocardiogram showed left ventricular myohypertrophia, mild mitral and tricuspid valve insufficiency. Electrophysiology study revealed widespread myogenic changes. Cranial MRI, MRA and MRS were normal. Muscle biopsy showed basophilic intracytoplasmic material in a lot of fibers deposits, which was intensively PAS-positive material and partially resistant to diastase digestion. In the electron microscope, the storage material consisted of filamentous and finely granular material. Conclusions There was the first case of glycogen storage disease Ⅳ reported in our country, mainly involved skeletal muscle, liver, spleen and cardiac muscle.

Objective To observe the effect of T-2 toxin on growth and development of rat epiphyseal plate of left knee and metaphyseal bone of femur and tibia in normal and low nutritional status, to find out possible pathogenic factors of Kashin-Beck disease and provide experimental basis for early intervention. Methods Ninety 3-week-old Wistar rats, weighing 60 - 70 g, were randomly divided into three groups: control group(general feed), T-2 toxin + general feed group, T-2 toxin + low nutrition feed group, thirty rats in each group with equally sex ratio. T-2 toxin (1.0 mg/kg) was administered orally 5 times a week via a gavage needle for 4 weeks. The change of hair, activity and body weight was observed. After 1, 2, 4 weeks, the epiphyseal plate of left knee and metaphyseal bone of femur and tibia (including distal femur and proximal tibia) were collected. Specimens were processed with HE and Masson staining. The morphology of chondrocytes and matrix collagen content in epiphyseal plate was observed. Trabecular bone volume fraction in tibial metaphyseal bone was analyzed by Image-Pro Plus 6.0 software. Results In the control group, rats were in good movement and hair with light, but in T-2 toxin + general feed group and T-2 toxin + low nutrition feed group, rats were found with reduced activities and hair with dark color. Body weights(g) of the control group, the T-2 toxin + general feed group and the T-2 toxin + low nutrition feed group were 81.0 ± 6.2, 79.0 ±5.1, 77.0 ± 7.5, respectively, by the end of first week; 101.8 ± 6.7, 97.0 ± 6.8, 93.0 ± 5.3, respectively, by the end of second week; 151.1 ± 15.7, 126.5 ± 11.9, 106.5 ± 11.5, respectively, by the end of fourth week. There was significant difference in groups by second week and the fourth week (F = 9.72, 41.65, all P ＜ 0.05 ). There was significant difference among multi-groups by the fourth week(all P ＜ 0.01 ). Under light microscope, at the second weeks, coagulative necrosis of chondrocytes was found in hypertrophic zone in the two groups with T-2 toxin; at the fourth weeks, cell necrosis increased. Masson staining showed collagen staining in the two groups with T-2 toxin significantly turned to clear pale coloration, indicating that the collagen matrix was significantly reduced. Image analysis showed there was significant difference in groups at the second and fourth week(F= 9.72, 41.65, all P＜ 0.05)in tibial metaphyseal trabecular bone volume fraction. There was significant difference between T-2 toxin + low nutrition feed group[(0.55 ± 0.12)％, (0.21 ± 0.0)％] and control group[(0.67 ± 0.09)％, (0.51 ± 0.14)％] by the second and fourth week(all P ＜ 0.01 ). Conclusions Under normal nutritional status, T-2 toxin can induce hypertrophic epiphyseal cartilage necrosis, collagen content decreased in epiphyseal plate, metaphyseal trabecular bone formation disorders; in the low nutritional status, T-2 toxin can lead to rat epiphyseal necrosis and significant metaphyseal bone disorder, but whether the performance is related to Kaschin-Beck disease needs to be studied further.

OBJECTIVETo identify the anatomical variability of the left spermatic vein (LSV) and determine its effect on the induction of experimental left varicocele (ELV) in adolescent rats.

METHODSWe equally randomized 30 adolescent male SD rats to groups A (LSV collaterals fully ligated and the left renal vein constricted), B (only the left renal vein constricted), and C (sham operation), observed the courses of the LSVs and measured their diameters. At 30 days after operation, we analyzed the changes in the left kidneys and the diameters of the LSVs.

RESULTSIrregular collaterals were observed in 90% of the LSVs and no abnormal changes were found in the left kidneys after surgery. The postoperative LSV diameter was remarkably increased in group A as compared with the baseline ([1.47 +/- 0.15 ] vs [0.16 +/- 0.08] mm, P < 0.01), but showed no significant difference in group B ([0.31 +/- 0.49] vs [0.15 +/- 0.07] mm, P > 0.05) and C ([0.17 +/- 0.07] vs [0.16 +/- 0.06] mm, P > 0.05), and it was significantly longer in A than in B (P < 0.01). The success rate of ELV induction was 100% in group A and 10% in group B, but no varicocele was observed in group C.

CONCLUSIONCorrect identification of the anatomical course of the LSV and ligation of its irregular collaterals are essential for the establishment of a stable and consistent ELV model.

Animals ; Disease Models, Animal ; Kidney ; pathology ; Ligation ; Male ; Rats ; Rats, Sprague-Dawley ; Spermatic Cord ; blood supply ; Varicocele ; Veins ; abnormalities

Xylanase can hydrolyze xylans into xylooligosaccharides and D-xylose, and has great prospect for applications in feed industry, paper and pulp industry, food industry and environment science. The study of xylanase had been started in 1960's. With the development and application of the new technologies, such as molecular biology, structural biology and protein engineering, many progresses have been made in the research of structures and functions of xylanase. This paper reviews the research progress and trend in the structure correlating with the important properties of xylanase. Analyses of three-dimensional structures and properties of mutants have revealed that glutamine and aspartic acid residues are involved in the catalytic mechanism. The thermostability of xylanase correlated with many factors, such as disulfide bridges, salt bridges, aromatic interactions, cotent of arginine and proline, and some multidomain xylanase have thermostability domains in N or C terminal. But no single mechanism is responsible for the remarkable stability of xylanase. The isoelectic points and reaction pH of xylanase are influenced by hydrophobicity and content of electric charges. Many researches had demonstrated that aromatic amino acid, histidine, and tryptophan play an important role in improving enzyme-substrate affinity. The researches of structures and functions of xylanase are of great significance in understanding the catalytic mechanism and directing the improvement of xylanase properties to meet the application requirement.
Catalysis ; Endo-1,4-beta Xylanases ; chemistry ; metabolism ; Enzyme Stability ; Protein Engineering ; Substrate Specificity

Genomic DNA and cDNA sequences of lactase from Aspergillus candidus were cloned. Sequences analysis revealed that the genomic DNA was 3458 bp containing eight introns, cDNA was 3015 bp and encoding a polypeptide of 1005 amino acid residues. Signal peptide was 19 amino acid residues, eleven potential N-glycosylation sites were assumed. Comparing the gene cDNA and amino acid sequences with other lactase sequences from various sources, it showed a very low homology with most of other sequences. Although the gene had a higher homology to Aspergillus oryzae lactase sequence, characterization of both enzymes exhibited obvious difference. The gene from Aspergillus candidus was a promising new gene for food industry.
Amino Acid Sequence ; Aspergillus ; enzymology ; genetics ; Cloning, Molecular ; Lactase ; Molecular Sequence Data ; Sequence Homology ; beta-Galactosidase ; chemistry ; genetics

Adolescent ; Adult ; Aged ; Biopsy ; Delayed Graft Function ; diagnosis ; pathology ; Female ; Graft Rejection ; diagnosis ; pathology ; Humans ; Kidney ; pathology ; physiopathology ; Kidney Function Tests ; Kidney Transplantation ; pathology ; Male ; Middle Aged ; Young Adult

ObjectiveTo investigate the protective effect of paraoxonase 1 (PON1) gene against liver oxidative stress injury in mice due to dichlorvos poisoning.Methods Experiment 1: 12 male Balb/c mice were randomly divided into three groups, with 4 mice in each group: control group, green fluorescent protein lentivirus control group (Lv-GFP group), and recombinant PON1 lentivirus group (Lv-PON1 group). 2×107 TU of Lv-GFP or Lv-PON1 was transfected via tail vein, while normal saline was given to those in control group. Blood was collected on 0, 1, 3, 5, 7, 9 days via fundus venous plexus for the assay of serum PON1 activity. PON1 mRNA and protein expression levels were respectively determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot on the 3rd post-lentivirus transfection day. Experiment 2: according to the random number table method, another 96 male Balb/c mice were divided into four groups of 24 mice in each control group, dichlorvos group, Lv-GFP intervention group, and Lv-PON1 intervention group. Lv-GFP or Lv-PON1 was transfected via tail vein followed by intraperitoneal injection of dichlorvos 9 mg/kg, while those in control group were given normal saline. Six mice in each group were sacrificed respectively at 6, 12, 24, 48 hours, and liver tissue was collected. PON1 mRNA and nuclear factor E2-related factor 2 (Nrf2) mRNA expression levels were determined by RT-PCR, and PON1 protein level was determined by Western Blot. The content of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue were determined by chemical colorimetry. The activity of superoxide dismutase (SOD) and catalase (CAT) were measured by double antibody sandwich enzyme linked immunosorbent assay (ELISA).Results Experiment 1: after Lv-PON1 was transfected to normal mice, PON1 activity in serum gradually increased and maintained a high level on 3rd day, while that of the control group and Lv-GFP group showed a normal low level. On the 3rd post-lentivirus transfection day, PON1 mRNA and PON1 protein expressions in liver were significantly higher than those of control group and Lv-GFP group. Experiment 2: compared with control group, the mice in dichlorvos group showed significant decreases in PON1 mRNA, PON1 protein, Nrf2 mRNA as well as GSH, SOD, CAT levels at 6 hours [PON1 mRNA (gray value):0.237±0.075 vs. 0.674±0.011, PON1 protein (gray value): 0.602±0.086 vs. 0.998±0.124, Nrf2 mRNA (gray value): 0.089±0.012 vs. 0.126±0.010, GSH (mg/g): 3.84±0.33 vs. 5.52±0.40, SOD (μg/g): 0.383±0.040 vs. 0.564±0.052, CAT (ng/g): 7.32±1.28 vs. 12.46±1.54, allP< 0.05], and remarkable increase in MDA content (nmol/g: 7.78±0.41 vs. 2.34±0.25,P< 0.05). With the extension of time, PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels gradually increased, MDA content gradually decreased, Nrf2 mRNA expression level had risen to the level of control group at 24 hours (0.133±0.019 vs. 0.126±0.009,P> 0.05), and it was higher than that of the control group at 48 hours (0.206±0.028 vs. 0.124±0.010,P< 0.05). Compared with that of the dichlorvos group, Lv-PON1 intervention group showed a significant increase in PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels [PON1 mRNA (gray value): 0.726±0.021 vs. 0.237±0.075, PON1 protein (gray value): 0.739±0.050 vs. 0.602±0.086, Nrf2 mRNA (gray value): 0.158±0.007 vs. 0.089±0.012, GSH (mg/g): 4.30±0.22 vs. 3.84±0.33, SOD (μg/g): 0.454±0.062 vs. 0.383±0.040, CAT (ng/g): 8.98±1.02 vs. 7.32±1.28, allP< 0.05], and a decrease in MDA content (nmol/g: 6.56±0.44 vs. 7.78±0.41,P< 0.05).Conclusion Regulation of PON1 gene can reduce MDA content, enhance SOD and CAT activities, increase GSH content, and it may also up-regulate Nrf2 mRNA expression to play a protective effect against oxidative stress of liver injury induced by dichlorvos poisoning.

Depression is a serious mental illness with a high incidence. At present, we do not fully understand the specific pathological mechanisms of depression, and the efficacy of drug treatments is very limited. Recent studies have shown that epigenetic changes that occur in specific brain regions may be a key mechanism by which environmental factors to interact with individuals to influence the risk of depression. Therefore, drugs that target epigenetic regulation may become a new direction for the development of antidepressants. Histone deacetylase inhibitors (HDACi) are a class of compounds that inhibit histone deacetylase activity, which has been reported to be associated with depression; this article addresses the use of HDACi in preclinical studies, and their potential therapeutic role and limitations of use in depression.

Objective To study the toxicokinetics of Cymermethrin and its metabolites in dog bile and provide evidence for forensic cases of identification of Cymermethrin poisoning. Methods 1/4LD50 doses of Cymermethrin were given to 6 male dogs by oral perfusion after the gallbladder fistula surgery on them,and their bile were collected at different time, in which Cymermethrin and its metabolites were extracted by Liquid-liquid extraction with dichloromethane and detected by HPLC-MS-MS. The qualitative analysis was based on retention time and MRM ions. The quantitative analysis was based on an internal standard method and calibration curve. Toxicokinetics equations of Cymermethrin and its metabolites in the bile were established from the c-t curves which were fitted by the WinNonlin toxicokinetics software meanwhie toxicokinetics parameters were obtained. Results The toxicokinetics of Cymermethrin met first-order dynamic equation. The Tmax of Cymermethrin(CYM), 3-phenoxybenzoic acid (3-PBA), 3-(2,2-Dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate (DCVA) respectively were 1.52±0.30,1.29±0.04,0.93±0.41 h ; The Cmax of CYM, 3-PBA, DCVA respectively were 0.38±0.03,7.9±1.32,30.9±16.24 μg/mL ; The T1/2 of CYM, 3-PBA, DCVA respectively were3.93±0.71,1.36±0.11,4.49±2.81 h; Conclusion The toxicokinetics of Cymermethrin in dog bile met first-order dynamic equation ; The toxicokinetics model and parameters of Cymermethrin can provide evidence for forensic identification of Cymermethrin poisoning cases.