Genetic diversity analysis of sweet kernel apricot in China based on SSR and ISSR markers.

Simple sequence repeat (SSR) and inter-simple sequence repeat (ISSR) markers were used to evaluate genetic diversity among 22 sweet kernel apricot accessions and 12 cultivars in China to provide information on how to improve the utilization of kernel apricot germplasms. The results showed that 10 pairs of SSR primers screened from 40 primer pairs amplified 43 allelic variants, all of which were polymorphic (100%), and 9 ISSR primers selected from 100 primers amplified 67 allelic variants with 50 polymorphic bands (74.63%). There was a relatively distant genetic relationship between the 34 samples, where their genetic similarity coefficient was between 0.62 and 0.99. The UPGMA dendrogram constructed using combined data of the two marker systems separated the genotypes into three main clusters.

Alteration of coenzyme specificity of malate dehydrogenase from Streptomyces coelicolor A3(2) by site-directed mutagenesis.

We describe here for the first time the alteration of coenzyme specificity of malate dehydrogenase (MDH) from Streptomyces coelicolor A3(2) (ScMDH). In the present study, we replaced four amino acid residues in the Rossmann fold (ßB-αC) region of NADH-dependent ScMDH by site-directed mutagenesis with those of NADPH-dependent MDH (Glu42Gly, Ile43Ser, Pro45Arg, and Ala46Ser). The coenzyme specificity of the mutant enzyme (ScMDH-T4) was examined. Coenzyme specificity of ScMDH-T4 was shifted 2231.3-fold toward NADPH using kcat/Km(coenzyme) as the measurement of coenzyme specificity. Accordingly, the effect of the replacements on coenzyme specificity is discussed. Our work provides further insight into the coenzyme specificity of ScMDH.

Microsatellite markers for assessing genetic diversity of the medicinal plant Paris polyphylla var. chinensis (Trilliaceae).

Paris polyphylla var. chinensis is a perennial herb with medicinal properties that is widely used in traditional Chinese medicine. However, this plant has been on the edge of extinction during the last few decades because of excessive deforestation based on the intense ethnopharmaceutical interest. We isolated 12 microsatellite loci from a (CT)(n)-enriched genomic library of P. polyphylla var. chinensis. The polymorphism at each locus was analyzed by screening 30 individuals from a natural population. The number of alleles ranged from 2 to 5. The observed and expected heterozygosities ranged from 0.000 to 0.467, with a mean of 0.247, and from 0.383 to 0.662 with a mean of 0.537, respectively. Six loci (Pp1, Pp3, Pp6, Pp7, Pp9, and Pp12) were found to significantly deviate from Hardy-Weinberg equilibrium. This may be due to the small population size, inbreeding or null alleles. Five of the pairwise comparisons (Pp1 and Pp4, Pp2 and Pp5, Pp2 and Pp9, Pp2 and Pp12, Pp11 and Pp12) exhibited significant linkage disequilibrium (P < 0.05). We conclude that these microsatellite markers will be useful for population genetic studies of P. polyphylla var. chinensis.

Clinical effect of open reduction and internal fixation with a steel plate through the triceps approach in the treatment of fractures of the middle and lower 1/3 of the humerus.

OBJECTIVE: The stability of fractures of the middle and lower 1/3 of the displaced humeral shaft is poor, and surgery is currently the main treatment. The posterolateral approach to the upper arm has many advantages but it is not widely used in clinical applications. The aim of the study was to investigate the clinical effect of open reduction and internal fixation with a steel plate through the triceps approach in the treatment of fractures of the middle and lower 1/3 of the humerus. PATIENTS AND METHODS: A retrospective analysis was performed on 26 patients with fractures of the middle and lower 1/3 of the humerus who were admitted to our hospital from January 2018 to December 2021. According to the AO ASIF classification, 12 patients had type A, 8 patients had type B, and 6 patients had type C fractures. The posterior transtriceps approach was used for open reduction and internal fixation with a steel plate to evaluate its clinical efficacy. RESULTS: All patients were followed completely, and the follow-up time was 6.0-18.0 months. Fracture nonunion occurred in 1 patient after the operation, and the other 25 patients healed well; 2 patients showed symptoms of radial nerve injury and numbness in the tiger's mouth area, which improved after 2 weeks. The average healing time of the fracture was 12.3 weeks. There were no infections or complications such as plate fractures. Elbow joint function according to Mayo scoring standards was as follows: 19 cases were excellent, 3 cases were good, 2 cases were fair, and 2 cases were poor. The excellent and good rate was 84.6%. CONCLUSIONS: Open reduction through the triceps approach and internal fixation with a steel plate for the treatment of the middle and lower 1/3 of the humerus can directly expose and protect the radial nerve and its branches and reduce radial nerve damage, and plate fixation on the tension side is biomechanical and worthy of clinical application.

Polymorphic microsatellite loci for the genetic analysis of Lycoris radiata (Amaryllidaceae) and cross-amplification in other congeneric species.

Lycoris radiata is a perennial herb that has been used in traditional Chinese medicine for a long time and has two main medicinal components in its bulb, lycorine and galanthamine. However, the original microsatellite loci have not been developed for any species of Lycoris. Total genomic DNA was extracted from fresh bulbs using a modified CTAB protocol. We isolated 10 microsatellite loci from 21 L. radiata individuals of a natural population from Yellow Mountain in Anhui Province, China. The number of alleles ranged from two to nine. The observed and expected heterozygosities ranged from 0.238 to 0.952 and from 0.455 to 0.784, respectively. One locus significantly deviated from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these microsatellite loci was characterized in additional five species (L. sprengeri, L. anhuiensis, L. albiflora, L. longituba, and L. chinensis) of Lycoris. The results suggest that these microsatellite markers would contribute to the population genetic studies of L. radiata and other related species.

Tyrosinase immobilization on ZnO nanorods for phenol detection.

Directly using zinc powders as source material, ZnO nanorods were fabricated on gold wire by hydrothermal reaction without any other surfacant and stabilizing agent. The gold wire was skillfully treated to improve the nucleation for growth of ZnO nanostructures and to further improve the performance of the biosensor, which was construct by immobilizing tyrosinase (Tyr) on the ZnO nanorods for phenol detection. Electrochemical measurement, Fourier transform infrared and scanning electron microscopic analyses demonstrated that the Tyr was stably adsorbed on the ZnO nanorods surface with bioactivity for phenol oxidization. The biosensor reached 95% of steady-state current within 5s, and the sensitivity was as high as 103.08 microA/mM at C(phenol) > 20 microM and was 40.76 microA/mM at C(phenol) < 20 microM. The detection limit of 0.623 microM was obtained at a signal/noise ratio of 3.

Layer by layer immobilized horseradish peroxidase on zinc oxide nanorods for biosensing.

Using zinc powders as source material, ZnO nanorods (ZnONR) were fabricated on gold wire by a hydrothermal reaction without any other surfactant. The gold wire end was coated by a thin layer of Zn-Au alloy to improve the nucleation for growth of ZnO nanostructures and to further improve the performance of the biosensor, which was constructed by alternatively immobilizing poly(sodium 4-styrenesulfonate) (PSS) and horseradish peroxidase (HRP) on the ZnONR. Electrochemical measurement, ultraviolet-visible spectrum, zeta-potential, and scanning electron microscopic analysis demonstrated that PSS and HRP were stably adsorbed layer by layer on the ZnONR surface, and the HRP kept bioactivity for H(2)O(2) detection without an electron transfer mediator. The multilayered HRP sensors exhibited a wide linear range and low detection limit. The sensitivity of the biosensor increased with the immobilized HRP layers from the lowest value of 36.28 microA mM(-1) for a monolayer.

SELDI-TOF MS proteinchip technology for screening of serum markers of HBV-induced hepatocellular carcinoma.

The incidence of hepatocellular carcinoma (HCC) is highest among primary liver cancer. HBV and HBV-induced liver cirrhosis may lead to HCC (1). At present, it is difficult to diagnose HCC at early stage or to differentiate HCC. Therefore, it is much needed to explore and develop a simple, rapid diagnostic method, which has higher sensitivity and specificity for HCC at early stage (2, 3). Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel non-electrophoresis-based proteomic technology. SELDI offers the advantages of rapid and simple examination as well as high specificity and sensitivity (4, 5). To our knowledge,there has been little study reported using SELDI-TOF-MS technology to investigate HCC. In this study, 25 cases of HCC patients not receiving any therapy, 25 cases receiving the interposition chemotherapy and 50 cases of the healthy people were tested by Weak cationic exchange (WCX2) protein chip and SELDI-TOF-MS. The differentially expressed peptides or proteins were analyzed by BioMarker Wizard software. At the different M/Z value range, seven peptides/ proteins were obviously different among these three groups. Four peptides including 6489.48Da, 6662.34Da, 8593.75Da and 8720.23Da were up-regulated in healthy controls, two including 7777.27Da and 9250.00Da were up-regulated in the patients with HCC without receiving any therapy and one protein of 16200.17Da was up-regulated in the patients with HCC receiving the interposition chemotherapy. Using Biomarker Pattern software, one pattern including two peptides (7777.27Da, 9250.00Da) can separate HCC without receiving any therapy from normal controls. This diagnosis pattern gave the much-improved sensitivity of 92% and the specificity of 100%. Through searching protein database, these seven peptides or proteins were identified as possible Galanin related peptide, Pro-neuregulin-4 protein, small inducible cytokine A15 precursor, 9 kDa protein, CSL-zincfinger protein 1, mitochondrial hinge protein, actin related protein, respectively. Using SELDI-TOF MS, the method of sieving the tumor markers from HCC becomes quick and valid. These differentially expressed peptides or proteins could be biomarkers of HCC in the serum and drug targets for treating HCC.

Elevated serum retinol-binding protein 4 levels are correlated with blood pressure in prehypertensive Chinese.

Retinol-binding protein 4 (RBP4) has been reported involving in the occurrence and development of hypertension. However, to date, few data are available on the correlation between serum RBP4 level and blood pressure (BP) in prehypertension. Therefore, this association was investigated in prehypertensive Chinese. Overall, 160 subjects with prehypertension (Pre-HT group) and 160 subjects with normal BP (NBP group) were recruited in this study. The subjects were divided into the following four subgroups according to body mass index (BMI): obese Pre-HT subgroup; non-obese Pre-HT subgroup; obese NBP subgroup; and non-obese NBP subgroup (n=80 in each). Anthropometric parameters, systolic BP (SBP), diastolic BP (DBP) and several biochemical parameters were measured. Fasting insulin was evaluated by radioimmunoassay. Serum RBP4 level was measured by enzyme-linked immunosorbent assay. The Pre-HT group had higher levels of serum RBP4 level than did the NBP group (P<0.001). Moreover, higher RBP4 levels were identified in the obese Pre-HT subgroup relative to the non-obese Pre-HT subgroup (P=0.005). However, no difference in RBP4 level was identified between the obese and the non-obese NBP subgroups (P=0.317). RBP4 level was positively correlated with BMI (r=0.226, P=0.001), SBP (r=0.468, P<0.001) and DBP (r=0.358, P<0.001) after adjustment for age, sex, smoking status and alcohol consumption. The results of the multiple regression analyses demonstrated that RBP4 level was independently associated with SBP (ß=0.427, P<0.001) and DBP (ß=0.338, P<0.001). In conclusion, serum RBP4 level was significantly higher and closely associated with BP in prehypertensive Chinese.

The development of nao li shen and its clinical application.

The traditional Chinese medicine Nao Li Shen (containing ginseng, gastrodia tuber, chuanxiong rhizome and red sage root) is used in craniocerebral injury, cervical spondylosis and cerebrovascular diseases. The preparation, as an orally administered liquid, was tested in Mongolian gerbils and shown to increase tolerance to ischaemia and anoxia. Clinical use of the preparation resulted in improvement in 96% of 202 patients, as judged by right cerebral blood flow, TCD and CT examination. We conclude that Nao Li Shen has a positive curative effect upon craniocerebral injury and sequelae of cerebrovascular diseases.

[Glucose isomerase gene knock-out by denatured double-stranded DNA].

After Genetic background analysis of Streptomyces diastaticus No. 7 strain M1033, the modified conditions of M1033 protoplasts and transformation were established. Replacement plasmid for homologous recombination was also constructed by inserting tsr gene into glucose isomerase gene. The homologous recombination of GI gene in M1033 chromosomes was achieved by using denatured linearized DNA fragments and glucose isomerase deficient strain M1033LJ was obtained. It is basic for introducing mutation into M1033 chromosome and realizing site-directed molecular reformation.

[Comparison of chemical constituents of essential oils from Elsholtzia splendens and cultivated Mosla chinensis by GC-MS analysis].

Xiangru (Labiatae) is a well-known drug used in Chinese traditional medicine. Chemical constituents of essential oils from Elsholtzia splendens and cultivated Mosla chinensis were compared by GC-MS/DS combination on SE-54 capillary column (30 m x 0.32 mm). Forty-three components were identified in Elsholtzia splendens Nakai ex F.Maekawa. The total content of the components in the essential oil was found to be 90.61%. Among them the major constituents are elsholtzia ketone (80.81%). Fifty components were identified in cultivated Mosla chinensis, with a total content of 93.91% in the essential oil. The major constituents are carvacrol (51.11%), thymol (22.00%), and etc.

[Mutations of Q20L and G247D improved the specific-activity and optimum pH of glucose isomerase].

The mutants of Q20L and G247D of glucose isomerase (GI) were constructed by in vitro site-directed mutagenesis of GI gene with double-primersmethod. The recombinant plasmids pTKD-GIQ20L and pTKD-GIG247D were expressed in E. coli K38 strain. The comparison experiments of mutant enzymes with wild-type GI showed that: (1) the optimum temperature of GIQ20L was decreased by 5 degrees C. Its thermostability was only 78% half-time of the wild type. But its substrate affinity was enhanced. (2) The specific-activity of GIG247D was increased by 33%, and the optimum pH was lowered by 0.6 unit. However, the thermostability of GIG247D was decreased. We supposed, based on the above facts and 0.19 nm resolution crystal structure of SM33GI, that Gln20 locates between alpha 0-helix and alpha 1-helix, the substitution of hydrophobic side chain of Leu for hydrophilic side chain of Gln may enhance the hydrophobic interaction of the molecular surface, leading to the decrease of the stability and thermostability of GIQ20L. Gly247 which is the last amino acid of a beta-sheet from 242 to 247 residues locates in the active core of GI. After replacement, Asp247 which has strong negative electricity may change the electrostatic distribution and influence the charge transfer processes of the active core. So the specific-activity of GIG247D was increased. The introduced charge could alter the pKa of dissociable groups and make the optimum pH lower. In addition, the side chain of Asp247 seems to be very crowded in the surrounding space conformation and is easy to exclude with the other side chains, therefore influences the stability of beta-sheet. Furthermore, Asp247 is in the vicinity of the interface of subunits, so it could interfere with the stability of the interaction between subunits. Thus, the GIG247D decreased the thermostability of SM33GI. The higher enzyme activity and the lower optimum pH will be very useful for industrial production of GI.

Increasing the thermostability of D-xylose isomerase by introduction of a proline into the turn of a random coil.

Thermostability can be increased by introducing prolines at suitable sites in target proteins. Two single (G138P, G247D) mutants and one double (G138P/G247D) mutant of xylose isomerase from Streptomyces diastaticus No.7, strain M1033 have been constructed by site-directed mutagenesis. With respect to the wild-type enzyme, G138P showed about a 100% increase in thermostability, and G247D showed an increased catalytic activity. Significantly, the double mutant, G138P/G247D displayed even higher activity than G247D and better heat stability than G138P. Its half life was about 2.5-fold greater than the wild-type enzyme, using xylose as a substrate. Molecular modelling suggested that the introduction of a proline residue in the turn of a random coil may cause the surrounding conformation to be tightened by reducing the backbone flexibility. The change in thermostability can, therefore, be explained based on changes in the molecular rigidity. Furthermore, the improvements in the properties of the double mutant indicated that the advantages of two single mutants can be combined effectively.