Effects of two novel amino acid substitutions on the penicillin binding properties of the PBP5 C­terminal from Enterococcus faecium.

The low­affinity penicillin­binding protein (PBP)5 is responsible for resistance to ß­lactam antibiotics in Enterococcus faecium. (E. faecium). In order to evaluate more fully the potential of this species for the development of resistance to ß-lactam antibiotics, the present study aimed to examine the extent of penicillin-binding protein (PBP) variations in a collection of clinical E. faecium isolates. In the present study, the C­terminal domain of PBP5 (PBP5­CD) of 13 penicillin­resistant clinical isolates of E. faecium were sequenced and the correlation between penicillin resistance and particular amino acid changes were analyzed. The present study identified for the first time, to the best of our knowledge, two novel substitutions (Tyr460Phe and Ala462Thr or Val462Thr) of E. faecium PBP5­CD. The covalent interaction between penicillin and PBP5­CD was also investigated using homology modeling and molecular docking methods. The theoretical calculation revealed that Phe460 and Thr462 were involved in penicillin binding, suggesting that substitutions at these positions exert effects on the affinity for penicillin, and this increased affinity translates into lower resistance in vitro.

[Study on fluorescence sequencing typing technology identification of raw materials in liuwei dihuang pill].

In this paper, Liuwei Dihuang pill was used to study the identification of Chinese patent medicine by fluorescence sequencing typing technology. The DNA of Paeonia suffruticosa was used as template to amplify by five pair of FAM fluorescence labeling primers. Then, the amplified products were sequenced. The sequencing results were analyzed by GeneMarker V1.80 to screen the best fluorescence labeling primers. As a result, psbA-trnH fluorescence labeling primer was used to identify the raw materials of Liuwei Dihuang pill. The results showed that three kinds of raw plant medicinal materials in Liuwei Dihuang pill were able to be correctly identified by psbA-trnH fluorescence labeling primer. The fluorescence sequencing typing technology can stably and accurately distinguish raw medicinal materials in Chinese patent medicine.

[Study on molecular identification of water extracts from Gelsemium elegans and Lonicera japonica and its close species by specific PCR amplification].

OBJECTIVE: To explore the new method of discriminating Gelsemium elegans from Lonicera japonica and its close species by using specific PCR amplification. METHOD: Thirteen samples of the different G. elegans materials and 58 samples of L. japonica, L. macranthoides and L. dasystyla were collected. The total DNA of the samples were extracted, and the DNA of G. elegans, L. japonica and L. macranthoides water extracts were extracted. PsbA-rnnH sequence from G. elegans was amplified by PCR and sequenced unidirectionally, ClustulW was used to align psbA-trnH sequences of the G. elegans and L. japonica and its close species from GenBank database. RESULT: All samples were amplified by PCR with specific primer, DNA from G. elegans would be amplified 97 bp whereas PCR products from all of Lonicera samples had not bands. CONCLUSION: Specific PCR amplification can be used to identify G. elegans from L. japonica and its close species successfully and is an efficient molecular marker for authentication of G. elegans and L. japonica and its close species.

[Study on identification of Astragali Radix and Hedysari Radix by PCR amplification of specific alleles].

To explore the new method of discriminating Astragali Radix and Hedysari Radix by using PCR amplification of specific alleles, 30 samples of the different Astragali Radix materials and 28 samples of Hedysari Radix were collected. The total DNA of all samples were extracted, trnL-trnF sequence from Astragali Radix and Hedysari Radix was amplified by PCR and sequenced unidirectionally. These sequences were aligned by using Clustul W. Primer was designed and the PCR reaction systems including annealing temperature, dNTP, etc were optimized. All samples were amplified by PCR with specific primer, DNA from Astragali Radix would be amplified 136 bp, whereas PCR products from all of Hedysari Radix were 323 bp. This method can detect 10% of intentional Hedysari Radix DNA into Astragali Radix. PCR amplification of alleles can be used to identify Astragali Radix and Hedysari Radix successfully and is an efficient molecular marker for authentication of Astragali Radix and Hedysari Radix.

[Molecular identification of raw materials from lian qiao bai du wan].

Lian Qiao Bai Du Wan was used to study the identification of Chinese patent medicine by molecular marker technique. DNA was extracted through modified CTAB method. The psbA-trnH and rbcL sequences were gradient amplified, and PCR products were ligated with the pEASY-T5 vector and then transformed into Trans1-T1 cells, respectively. Clones were selected randomly and sequenced. All sequences were analyzed by BlastN and the neighbor-joining (NJ) phylogenetic tree was constructed by MEGA 4.0. The results showed that nine kinds of medicinal materials can be identified by psbA-trnH sequences, and six kinds of medicinal materials by rbcL sequences from Lian Qiao Bai Du Wan. Molecular marker technique can stably and accurately distinguish multi-origin medicinal materials in Chinese patent medicine.

Lack of association of common polymorphisms in MUC1 gene with H. pylori infection and non-cardia gastric cancer risk in a Chinese population.

Several lines of evidence support the notion that MUC1 is often aberrantly expressed in gastric cancer, and it is a ligand for Helicobacter pylori. Genetic variation in MUC1 gene may confer susceptibility to H. pylori infection and gastric cancer. We assessed the association of common polymorphisms in MUC1 gene with H. pylori infection and non-cardia gastric cancer using an LD-based tag SNP approach in north-western Chinese Han population. A total of four SNPs were successfully genotyped among 288 patients with non-cardia gastric cancer and 281 age- and sex-matched controls. None of the tested SNPs was associated with H. pylori infection. SNP rs9426886 was associated with a decreased risk of non-cardia gastric cancer, but lost significance after adjustment for multiple testing. Overall, our data indicated that common genetic variations in MUC1 gene might not make a major contribution to the risk of H. pylori infection and non-cardia gastric cancer in our studied population.

[Molecular identification of astragali radix and its adulterants by ITS sequences].

OBJECTIVE: To explore a new method for identification Astragali Radix from its adulterants by using ITS sequence. METHOD: Thirteen samples of the different Astragali Radix materials and 6 samples of the adulterants of the roots of Hedysarum polybotrys, Medicago sativa and Althaea rosea were collected. ITS sequence was amplified by PCR and sequenced unidirectionally. The interspecific K-2-P distances of Astragali Radix and its adulterants were calculated, and NJ tree and UPGMA tree were constructed by MEGA 4. RESULT: ITS sequences were obtained from 19 samples respectively, there were Astragali Radix 646-650 bp, H. polybotrys 664 bp, Medicago sativa 659 bp, Althaea rosea 728 bp, which were registered in the GenBank. Phylogeny trees reconstruction using NJ and UPGMA analysis based on ITS nucleotide sequences can effectively distinguish Astragali Radix from adulterants. CONCLUSION: ITS sequence can be used to identify Astragali Radix from its adulterants successfully and is an efficient molecular marker for authentication of Astragali Radix and its adulterants.

Angiotensin-I-converting enzyme inhibitory peptides: Chemical feature based pharmacophore generation.

A validated 3D pharmacophore model was generated for a series of ACE inhibitory peptides, which consisted of five features (two hydrophobic functions, two hydrogen bond acceptors, and a negative ionizable function). The built model was able to correctly predict the activity of known ACE inhibitors. The model was then used as query to search 3D databases of peptides. Three novel peptides (I, II and III) were synthesized and biologically evaluated in vitro. It appears that the in vitro activity of peptides I, II and III was consistent with their molecular modeling results. Our results provided confidence for the utility of the pharmacophore model to retrieve novel ACE inhibitory peptides with desired biological activity by virtual screening.

Computer-aided drug design for AMP-activated protein kinase activators.

AMP-activated protein kinase (AMPK) is an important therapeutic target for the potential treatment of metabolic disorders, cardiovascular disease and cancer. Recently, various classes of compounds that activate AMPK by direct or indirect interactions have been reported. The importance of computer-aided drug design approaches in the search for potent activators of AMPK is now established, including structure-based design, ligand-based design, fragment-based design, as well as structural analysis. This review article highlights the computer-aided drug design approaches utilized to discover of activators targeting AMPK. The principles, advantages or limitation of the different methods are also being discussed together with examples of applications taken from the literatures.

[Application of molecular pharmacognosy in research of Mongolian medicine].

Molecular pharmacognosy has developed as a new borderline discipline. Using the method and technology of molecular pharmacognosy, a wide range of challenging problems were resolved, such as the identification of Mongolian medicinal raw materials, etiology of endangerment and protection of endangered Mongolian medicinal plants and animals, biosynthesis and bioregulation of active components in Mongolian medicinal plants, and characteristics and the molecular bases of Dao-di Herbs. So molecular pharmacognosy will provide the new methods and insights for modernization of Mongolian medicine.

Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test.

To elucidate the protein-protein interactions of hemoglobin (Hb) variants A and A(2), HbA was first shown to bind with HbA(2) in live red blood cells (RBCs) by diagonal electrophoresis and then the interaction between HbA and HbA(2) outside the RBC was shown by cross electrophoresis. The starch-agarose gel electrophoresis of hemolysate, RBCs, freeze-thawed RBCs and the supernatant of freeze-thawed RBCs showed that the interaction between HbA and HbA(2) was affected by membrane integrity. To identify the proteins involved in the interaction, protein components located between HbA and HbA(2) in RBCs (RBC HbA-HbA(2)) and hemolysate (hemolysate HbA-HbA(2)) were isolated from the starch-agarose gel and separated by 5-12% SDS-PAGE. The results showed that there was a ≈22 kDa protein band located in the RBC HbA-HbA(2) but not in hemolysate HbA-HbA(2). Sequencing by LC/MS/MS showed that this band was a protein complex that included mainly thioredoxin peroxidase B, α-globin, δ-globin and ß-globin. Thus, using our unique in vivo whole blood cell electrophoresis release test, Hbs were proven for the first time to interact with other proteins in the live RBC.

RBC electrophoresis with discontinuous power supply - a newly established hemoglobin release test.

In this paper, we aimed to introduce a newly established red blood cells (RBCs) electrophoresis method hemoglobin release test (HRT) and tried to determine its significance. Human blood samples from beta-thalassemia patients and healthy controls were analyzed with HRT, which was carried out on starch-agarose mixed gel. First, the whole blood samples were electrophoresed for 2 h, then paused for 15 min and ran for 15 min by turns. This "pause-run-pause" experiment was performed for several turns and the total electrophoresis time lasted for about 6 h. The results showed that some other hemoglobin (Hb) components were released from the origin of each sample during the HRT, and the samples from beta-thalassemia patients released more Hb than the healthy controls. This finding demonstrates that Hb may exist differently associated in RBCs, and it may have an important theoretical and clinical significance in Hb and RBC research.

[Polymorphism of vitamin D receptor gene Fok I in Mongolian population of China].

OBJECTIVE: To investigate the polymorphism distribution of vitamin D receptor (VDR) gene Fok I in Mongolian population of China. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze three genotypes FF, Ff and ff in the start codon of VDR gene (Fok I) in unrelated normal healthy Mongolian individuals of China. RESULTS: In the population, we obtained the allelic frequencies of 57% and 43% for (F) and (f) allele and the percentage of genotypes FF, Ff and ff to be 31%, 52%, and 17% respectively. CONCLUSION: The polymorphism frequency and distribution of this VDR gene Fok I in Mongolian population of China exhibit its own characteristics.