Preparation and pharmacodynamic evaluation of naringenin lyophilized liposome / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prepare the lyophilized powder of naringenin liposome and investigate its pharmacodynamics in rat models of acute lung injury (ALI).</p><p><b>METHODS</b>Naringenin liposome was prepared by ethanol injection method and then its quality was evaluated. Also, the related characteristics was evaluated by adding mannitol (5%,W/V) as lyoprotectant to be freeze-dried. The rat ALI models were established by inhaling lipopolysaccharide (LPS) (5 mg/kg). Totally 48 female Sprague-Dawley rats were randomly divided into six groups:control group(A), LPS group(B), LPS+naringenin group(C), LPS+lyophilized liposome group(D), LPS+dexamethasone group(E), and LPS+blank liposome group(F), with 8 rats in each group. Lung wet/dry weight ratio was calculated, and the histopathological morphologies were observed under the light microscope.</p><p><b>RESULTS</b>The encapsulation efficiency of the prepared liposome was (82.44 ± 0.98)%, the average particle size was (133 ± 11)nm, and the Zeta potential was (-35.9 ± 5)mV. The angle of repose of lyophilized powder was 36℃ and the bulk density was 0.3 g/ml. Compared with the group A, the lung tissues from groups B to F showed different remarkable histopathological changes under a light microscope, including infiltration of inflammatory cells, capillary congestion, hemorrhage, and marked thickening of the alveolar wall,among which group B and F changed the most significant, followed by group C, whereas groups D and E were the lightest. The wet/dry weight ratios increased in groups B to F compared with group A in some degree, and the increase of the lung wet/dry weight ratio in group D and E was significantly lower than in group B(P=0.0012, P=0.0018).</p><p><b>CONCLUSION</b>The technology of preparing naringenin liposome by ethanol injection is simple and feasible, and lyophilized powder has an obvious therapeutic effect on ALI.</p>

Effect of traditional and integrative regimens on quality of life and early renal impairment in elderly patients with isolated systolic hypertension / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the effect of Chinese medical regimen and integrative medical regimen on quality of life and early renal impairment in elderly patients with isolated systolic hypertension (EISH).</p><p><b>METHODS</b>A multi-center, randomized, double-blinded controlled trail was adopted. A total of 270 cases of EISH were randomly divided into 3 groups: Chinese medicine group (CM), combination group and Western medicine group (WM). The course of treatment was 4 weeks. The clinical blood pressure, integral of quality of life (SF-36 scale), immunoglubin G (IgG), microalbumin (mALB), beta(2)-microglobulin (beta(2)-MG), transferrin (TRF) and N-acetyl-beta'-D-glucosa-minidase (NAG) in urine were determined before and after the treatment.</p><p><b>RESULTS</b>After treatment, systolic blood pressure depressed significantly in each group (P<0.05), and the combination group was superior to CM or WM group in depressing SBP (P<0.05); in each group, integral of quality of life improved in different degree, and combination group was superior to WM group in all 8 dimensions (P<0.05). The level of mALB and beta(2)-MG in urine decreased in all groups (P<0.05), and the combination group was superior to CM group or WM group in decreasing mALB (P<0.05).</p><p><b>CONCLUSIONS</b>Chinese medical regimen has affirmative effect in treating EISH patients, and could lower the systolic blood pressure, improve quality of life and early renal impairment of the patients, and integrative medical regimen has superiority on account of cooperation, and deserves further study.</p>

Inhibition of collagen-induced arthritis by DNA vaccines encoding TCR Vbeta5.2 and TCR Vbeta8.2 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Arthritogenic T lymphocytes with common T cell receptor (TCR) Vbeta clonotypes, infiltrating in the articulars of rheumatoid arthritis (RA) patients, play a central role in the pathogenesis of RA. TCR Vbeta5.2 and TCR Vbeta8.2 are the main pathogenic T cell clonotypes in the course of collagen-induced arthritis (CIA) progression in Lewis rats. To investigate a TCR-based immunotherapy for RA, we constructed recombinant DNA vaccines encoding TCR Vbeta5.2 and TCR Vbeta8.2, and evaluated the inhibitive effects of the two vaccines on CIA rats.</p><p><b>METHODS</b>Genes encoding TCR Vbeta5.2 and TCR Vbeta8.2 were amplified by RT-PCR from spleen lymphocytes of Lewis rats and cloned into the eukaryotic expression vector pTargeT. The expression of vaccines was confirmed by RT-PCR and immunohistochemistry. The inhibitive effects of the vaccines on articulars of CIA rats were assessed with arthritis index evaluation and histology. Interferon gamma (IFN-gamma) and interleukin (IL)-4 production by spleen lymphocytes were tested with enzyme-linked immunospot assay (ELISPOT) technique, the changes in peripheral CD4(+) and CD8(+) lymphocyte populations were tested by flow cytometry, and the level of anti-CII antibody in serum was assayed by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>Recombinant DNA vaccines pTargeT-TCR Vbeta5.2 and pTargeT-pTCR Vbeta8.2 were successfully constructed. Both vaccines inhibited CIA, which alleviated the arthritis index score (P < 0.05), decreased the level of IFN-gamma (P < 0.05), and reduced the ratio of CD4(+)/CD8(+) lymphocytes (P < 0.05) and the anti-CII antibody in serum (P < 0.05). In addition, the histological change in DNA-vaccinated rats was less serious than CIA rats. Compared to pTCR Vbeta 8.2 and pTCR Vbeta 5.2 groups, the group that was injected with a combination of the two vaccines showed stronger inhibitive effects on CIA than either individual vaccine.</p><p><b>CONCLUSION</b>The recombinant plasmids pTargeT-TCR Vbeta5.2 and pTargeT-TCR Vbeta8.2 have obvious inhibatory effects on CIA rats and better effects could be achieved when the vaccines were used in combination.</p>

Effect of an integrative medical regimen on levels of vascular endothelial function and hypersensitive C-reactive protein in elderly patients with isolated systolic hypertension / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the effect of a integrative medical regimen (IMR), i.e. combined use of Jiangya Capsule (JYC) and Nimodipine (ND), on blood pressure, TCM clinical symptoms, and blood levels of vascular endothelial function and hypersensitive C-reactive protein (hs-CRP) in elderly patients with isolated systolic hypertension (EISH).</p><p><b>METHODS</b>Adopting randomized, double-blinded and controlled principle, a trial was conducted on 135 patients with EISH by randomized them into three groups, they were administered IMR (Group A), JYC plus ND simulator (Group B) and ND plus JYC simulator (Group C) respectively, for 4 weeks. Changes of blood pressure and TCM symptoms, as well as the levels of serum nitric oxide (NO), plasma endothelin-1 (ET-1), 6-keto-prostaglandin 1alpha (6-keto-PGF(1alpha)), thromboxane B2 (TXB2) and hs-CRP were observed before and after treatment.</p><p><b>RESULTS</b>After treatment the systolic blood pressure reduced and clinical symptoms improved, with serum NO and 6-keto-PGF(1alpha) lelels elevated, plasma ET-1, TXB2 and serum hs-CRP decreased in all the three groups (P<0.05 or P<0.01). But the inter-group comparisons showed that the effect in Group A was superior to the other two groups in decreasing systolic pressure, and superior to Group C in improving clinical symptoms, elevating serum NO and decreasing plasma TXB2 (P <0.05).</p><p><b>CONCLUSION</b>The integrative medical regimen of combined use JYC and ND has markedly effect in lowering blood pressure, it could obviously improve the symptoms and vascular endothelial function, and inhibit the level of inflammatory factor in patients with EISH.</p>

Homologous amelogenin gene test of archaeological samples / 法医学杂志

OBJECTIVE@#Based on the sequence differences of Amelogenin homologous gene in the X and Y chromosomes, a pair of specific primers was designed to identify the sex of archaeological samples.@*METHODS@#Ancient DNA fragments were extracted from the bones and teeth of sacrificial slaves with an improved method that combines phenol-chloroform extraction, silicon dioxide adsorption with ultrafiltration concentration. The polyacrylamide gel electrophoresis (PAGE) was used to detect PCR products.@*RESULTS@#Seven in sixteen samples from eight graves showed positive results and the targeted segments were visible: a male with two bands of 106bp (Amel-X) and 112 bp (Amel-Y), while a female with only one band of 106 bp (Amel-X). Ancient DNA analyzing results from tooth samples are more marked than that from bones.@*CONCLUSION@#The improved extraction method is more effective for ancient DNA extraction, which reduced the PCR inhibitors and lowered experimental costs. The sex determination technology based on Amelogenin homologous gene is an important and feasible method in the molecular archaeological research.

Expression of a Novo Human Osteoprotegerin-Mycobacterial Heat Shock 70 Fusion Protein with Baculovirus-Insect Expression System / 中国生物工程杂志

Objective:Osteoprotegerin (OPG) is a key molecule negatively regulating osteoclast differentiation and activation; and the conserved mycobacterial heat shock 70 (HSP70) peptide p111-125 has also been found to inhibit inflammation reactions in chronic arthritis. BaculoDirectTM baculovirus expression system was selected to express recombinant OPG-HSP70 in insect cells.Methods:The human functional fragment (p22-194) of OPG and functional fragment (p111-125) of mycobacterial HSP70 gene were cloned into the transfer vector pENTRTM/SD/D-TOPO. The recombinant plasmid was performed an LR reaction with the BaculoDirectTM Linear DNA to generate recombinant baculovirus DNA. The cultured Sf9 insect cells were directly transferred with the recombinant baculovirus DNA,and the pure recombinant baculovirus was obtained. Then recombinant baculovirus was infected Sf9 insect cells again to express the OPG-HSP70 gene.Results:The target protein was detected at the time of 48h post infection,reached at highest yield at the time of 72h post-infection. A 28kDa protein immunostaining band was detected by Western blotting from lysate of those cells.And the purified protein was obtained by using Ni-NTA system. Functional stuies on the fusion protein showed it significantly reduce osteoclast cell number[(3.10?0.640) cells under each microscope field in treatment group by comparing to (10.70?0.817)cells in the control group] in the osteoclast inhibition test,and reduce the inflammation reaction in a delayed type hypersensitivity (DTH) mice model (P

Expression and Biological Characterization of Human Osteoprotegerin Fused with Mycobacteria Heat Shock Protein 65 / 中国生物工程杂志

A fused functional gene of human OPG and Mhsp65 was amplified by PCR,and cloned into the prokaryotic expression vector pET-28a.The BL21(DE3) strain of E.coli was transformed using the recombinant plasmid pET-28a-OPG-HSP65 and the expected protein was expressed by induction with IPTG.Result of SDS-PAGE indicated that the expected recombinant protein of 23 kDa was expressed with high yield as inclusion body.The fusion protein could be specifically recognized by both the anti-His antibody and anti-human OPG monoclonal antibody in Western blot analysis.The purified and refolded fusion protein could inhibit osteoclast proliferation and bone absorption in vitro.The results of mouse ear swelling assay and expressions of TNF-?,IFN-? and IL-17 mRNAs detected by real-time quantitative PCR demonstrated that the fusion protein had an anti-inflammation activity.The results suggest that the fusion protein of human OPG and Mhsp65 may act as a potential therapeutics for rheumatoid arthritis.

Identification and expression analysis of a full-length cDNA encoding a Kandelia candel tonoplast intrinsic protein / 生物工程学报

Soil salinity is an important issue, as most crop plants are low in salt tolerance. Salt tolerance, a complex, multifactorial, and multigenic process, has been known to be a quantitative trait. The identification of the salt stress responsive genes or salt tolerance genes is essential for the breeding programs. Most recent efforts have been focused on the products of structural genes (transport proteins, ion channels, enzymes of solute synthesis) while little attention were paid to the regulatory aspects of these proteins. Since the first aquaporin gene from plants was cloned and functionally expressed in 1993, there has been a growing interest in the molecular biology of MIPs (membrane intrinsic proteins) and their bearing on the biophysics of water flow across plant membranes. In the last decades, studies on Mangroves, a special kind of wood plants, grow in high-salt and flooding conditions have been concentrated almost exclusively on their physiological and ecological characteristics. Kandelia candel, one of the dominant species of mangroves along the Chinese coast, lacks salt glands or salt hairs used for removal of excess salt in other mangroves. This makes K. candel a perfect model to study the molecular mechanism of salt tolerance in mangrove plants. Using cDNA RDA, a cDNA-specific modification of genomic representational difference analysis, a series of salt responsive genes of Kandelia candel were cloned. Among these gene fragments, a 183 bp fragment (termed as SRGKC1) encoding a tonoplast intrinsic protein (TIP) in Kandelia candel (KCTIP1) was identified. Based on the sequence of SRGKC1, two gene specific primers were designed, and the 3' and 5' end of the KCTIP1 gene were obtained using the SMART RACE cDNA Amplification Kit. RACE products were purified from low-melting agarose, and sequenced directly with GSPs as the sequencing primers. A 500-bp fragment corresponding to the 3'end of this gene was obtained using the GSP1 primer, and a 690 bp fragment corresponding to the 5' end of this gene was obtained using the GSP2 primer. Two primers that flank the putative open reading frame (ORF) were designed to obtain the cDNA containing the complete ORF by RACE PCR reaction. The full-length cDNA of KCTIP1, containing a 756 bp open reading frame (ORF), was approximately 1.1 kb; the start codon was located at the nucleotides of 99-101 and stop codon at the nucleotides of 855-857 followed by a poly (A) tail. The KCTIP1 cDNA sequence in this research was released in GenBank with accession number AF521135. Using ExPASy Proteomics tools provided by EMBL, the isoelectric point and MWt of KCTIP1 are estimated as 5.77 and 26.3 kD respectively. Transmembrane prediction analysis revealed the deduced KCTIP1 protein sequence contains six transmembrane regions at amino acid residues of 20 - 42, 57 - 79, 86 - 108, 113 - 135, 142 - 164 and 217 - 239. Two highly conserved asparagine-proline-alanine (NPA) motifs were located at 85 - 87 and 199 - 201 amino acid residues respectively. KCTIP1 is also predicted to contain the Cys residue (Cys 118) that are shown to confer Hg-sensitivity in Arabidopsis gamma-TIP and delta-TIP. Similarity analysis showed that KCTIP1 shared 77% - 79% amino acid sequence identity with the TIPs from Vitis berlandieri, Brassica oleracea and Arabidopsis thaliana. Expression analyses indicated that KCTIP1 had different expression among species of Mangroves. Expressions of KCTIP1 in Kandelia candel, Rhizophora apoculata and Ceriops tagal were suppressed by salt, and were insensitive to salt stress in unknown species of Mangroves. Previous studied showed that salt conditions might result in large and rapid changes in extracellular water potential and serious disturbance to the cytoplasm. In order to compensate for this imbalance, the relative contribution of water channels to flow across the root could thus vary. K. candel is a species that is native to intertial zone of tropical and subtropical coast and is well-adapted to salt conditions. The coordinated down-regulation of aquaporins in this plant may decrease membrane water permeability and thus increase the cellular water conserva- tion during periods of salt stress. The results reported here are consistent with the postulated roles for tonoplast water channels in regulating the hydraulic permeability of the vacuolar membranes and in adjusting the water homeostasis of the protoplasm under various physiological conditions. The identification of KCTIP1 as one of salt-responsive genes implies that intracellular osmotic equilibration is a part of salt-tolerant mechanisms in Mangroves.

The methods of identifying differences in mRNA expression / 生物工程学报

Identifying changes in gene expression is of prime interest in molecular biology. A variety of methods have been developed for analyzing differentially expressed genes. Among these techniques, differential display reverse transcription PCR (DDRT-PCR), representational difference analysis (RDA) and suppression subtractive hybridization (SSH), which were based on differentially screening and subtractive hybridization technology, are applied most widely. Gene chips (gene microarrays) for analyzing gene expression patterns on larger-scale had also been developed. Here we present a review of these methods.

A STUDY ON FUNCTIONAL CHARACTERS OF BACILLUS THURINGIENSIS SUBSP. ISRAELENSIS TO CHIRONOMUS KIIENSIS TOKUNAGA / 微生物学通报

Aimed at feasibility of microbiological control of Chironomidae larvae in sourcewater, Shenzhen, China, the functional characters of Bacillus thuringiensis subsp. Iaraelensis to Chironomus kiiensis tokunaga were studied for the first time. In this study, bioassays were carried out with third-instar larvae, results showed that the LC 50 s(24h) were 24.2 and 32.6 mg/L for Bacillus thuringiensis subsp. Iaraelensis IPS82 and 187 respectively. Tests in fermentation of IPS82 show good correlations between toxicity, cell density, dissolved oxygen and spore-forming phase. The tests on environmental factors influencing toxicity to Chironomus kiiensis tokunaga showed that sunlight is the most important factor, shortening the half life of Bti from 21 days in dark to 10 days; temperature variations(15～30℃) caused no impact on toxicity, but 35℃ increases 16% of larvae mortality. The toxicity of IPS82 is relatively insensitive to change pH deviated from 7 to 11, due to drop of larvae mortality from 66.7 to 40%, at pH of 3, to 16%; its toxicity is stable in low larva density (