Biomedical subjects

Wei Cao

Publications and source records attributed to Wei Cao.

At least 55 records · Page 3Linked to original sources

PubMed2005

Improved preservation of human red blood cells by lyophilization.

The lyophilization of human red blood cells has important implications for blood transfusion in clinical medicine. In this study, sugars, human serum albumin, polyvinylpyrrolidone, and dimethyl sulfoxide were used as protective reagents for the lyophilization of red blood cells. Freezing temperature, shelf temperature, and the rehydration conditions were optimized. The results showed that extracellular disaccharides, especially trehalose, did not increase the recovery of hemoglobin. However, when the concentration of human serum albumin was higher than 25%, it had a considerable protective effect on the recovery of lyophilized red blood cells; the cellular hemoglobin recovery was over 70%, which was significantly higher than that in the group without human serum albumin (P<0.01). As the concentration of polyvinylpyrrolidone was increased, the extent of vitrification also increased. But when the concentration of polyvinylpyrrolidone was over 40%, the resulting concentration of free hemoglobin was over 1g/L, which was significantly higher than that with 40% (P<0.01). When lyophilization was carried out after freezing at different temperatures, the recovery of cells and hemoglobin was 70-80% and there were no significant differences among the five groups. When the shelf temperature was higher than -30 degrees C, the samples were partly collapsed, but when the shelf temperature was lower than -30 degrees C, the recovery of cells in the -40 and -45 degrees C groups was significantly higher than in the -30 and -35 degrees C groups (P<0.05). The recovery of cells and hemoglobin after lyophilization and rehydration in solutions containing low concentrations of polymers was over 80%, which is significantly higher than the other groups (P<0.01). In addition, when the temperature was higher than 25 degrees C, the concentration of free hemoglobin was significantly lower than it was at 4 degrees C (P<0.01). In conclusion, our study showed the lyophilization of red blood cells is feasible. Disaccharides have no protective effect on lyophilized cells when they are only extracellular and extensive vitrification may be not beneficial. Although the recovery of cells after lyophilization and rehydration by our method was over 70%, the ultrastructure of the cells may be compromised and some hemolysis does still exist. Further research is required.

Blood Preservation↗

PubMed2005

Protection against hydrogen peroxide-induced cell death in cultured human retinal pigment epithelial cells by 17beta-estradiol: a differential gene expression profile.

It has been demonstrated that estrogen receptors are present in the retinal pigment epithelium (RPE)-choroids complex regardless of sex. This suggests that estrogen could play a functional role in the outer retina, especially the RPE. To gain further insights on the molecular mechanisms differentially activated by 17beta-estradiol (betaE2) in RPE cells, we investigated gene expression changes in response to betaE2 in cultured RPE cells using cDNA microarray technology. A total of 47 genes among 21,329 human genes are significantly altered in response to betaE2 treatment in RPE cells. Among these 47 altered genes, 34 are up-regulated and 13 are down-regulated by betaE2. The products of 34 genes have a known or suspected function. These functions belong to various categories, including caspases; extracellular matrix proteins; metabolism pathway components; GTP/GDP exchangers and G-protein GTPase activity modulators; transcription activators and repressors. Six genes which may contribute to the unique functions of the RPE cells have been validated by both quantitative real-time reverse transcription (RT)-PCR and semi-quantitative RT-PCR. In addition, we also demonstrated that betaE2 quenches H2O2-induced up-regulation of apoptosis-related protein, and protects RPE cell degeneration. These results indicate that estrogen regulates functions of RPE cells and is involved in the maintaining and survival of RPE cells during oxidative stress, and its deficiency during menopause period may be a factor contributing to the development of age-related macular degeneration in elderly women.

Apoptosis↗

PubMed2005

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli.

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.

Amides↗

PubMed2005

Pharmacological characterization of guinea pig chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2).

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor activated by prostaglandin D(2) (PGD(2)), has been identified as a receptor expressed on cell types critical to the pathogenesis of asthma. The cDNA encoding guinea pig CRTH2 was cloned and mRNA expression examined in selected tissues. Transcript profiling of guinea pig CRTH2 indicated relatively high levels of expression in bone marrow, intermediate levels in brain and relatively low levels in lung, spleen, thymus, lymph node, etc. Characterization of the molecular pharmacology of guinea pig CRTH2 revealed that guinea pig CRTH2 exhibited a greater affinity for Delta(12)-PGJ(2), a stable PGD(2) metabolite relative to human CRTH2. The CRTH2 selective agonists 13,14-dihydro-15-keto PGD(2) and Delta(12)-PGJ(2) induced the recruitment of eosinophils following intradermal administration of these ligands in guinea pigs. Chemotaxis of guinea pig eosinophils was elicited by either PGD(2) or Delta(12)-PGJ(2), and was abolished by a CRTH2-specific antagonist. These results indicate that PGD(2) and the stable metabolite, Delta(12)-PGJ(2), play important roles in CRTH2 activation in the guinea pig.

Animals↗

PubMed2005

Shewanella decolorationis sp. nov., a dye-decolorizing bacterium isolated from activated sludge of a waste-water treatment plant.

A highly efficient dye-decolorizing bacterium, strain S12T, was isolated from activated sludge of a textile-printing waste-water treatment plant in Guangzhou, China. The cells were Gram-negative and motile by means of a single polar flagellum. The strain was capable of anaerobic growth either by fermentation of glucose or by anaerobic respiration and utilized a variety of electron acceptors, including nitrate, iron oxide and thiosulfate. The physiological properties, tested by using the Biolog GN2 system, were similar to those of the genus of Shewanella. Analysis of the nearly complete 16S rRNA gene sequence of strain S12T showed the highest similarity (98 and 97 %, respectively) to Shewanella baltica and Shewanella putrefaciens. However, the level of gyrB similarity between strain S12T and S. putrefaciens was 87 %. DNA from strain S12T showed 41.8 and 41.9 % DNA relatedness, respectively, to the DNA of S. baltica DSM 9439T and S. putrefaciens DSM 6067T. The DNA G+C content of strain S12T was 49.3 mol%. The predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The dominant fatty acids were 15 : 0, 16 : 0, iso-15 : 0 and 16 : 1omega7c, similar to the profiles of other Shewanella species. On the basis of its physiological and molecular properties, strain S12T appears to represent a novel species of the genus Shewanella, for which the name Shewanella decolorationis sp. nov. is proposed. The type strain is S12T (=CCTCC M 203093T=IAM 15094T).

Bacterial Typing Techniques↗

PubMed2005

Comparison of expression of monomeric and multimeric adenoregulin genes in Escherichia coli and Pichia pastorias.

Adenoregulin is a 33 amino acid antibiotic peptide who belongs to dermaseptin family which is the first vertebrate family to show lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. Synthetic adenoregulin gene was cloned in 2, 4 and 6 tandem repeats and subcloned in pET32a and pET22b vectors. Recombinant plasmids were transformed into E. coli BL21(DE3), Fusion proteins of Trx-ADR1, Trx-ADR2 and Trx-ADR4 could be expressed after the hosts were induced by IPTG, but the expression level decreased dramatically with the number of tandem repeats increased. ADR1, ADR4 and ADR6 could not be expressed by E. coli without carrier proteins. But for Pichia pastoris GS115, ADR1 and ADR6 in the fermentation broth of the hosts could be detected by ELISA, and the bactericidal activities could also be observed.

Amphibian Proteins↗

PubMed2005

Uptake kinetics of 99mTc-MAG3-antisense oligonucleotide to PCNA and effect on gene expression in vascular smooth muscle cells.

UNLABELLED: To investigate the feasibility of in vivo imaging study of atherosclerotic plaque and restenosis using antisense probe, we evaluated the uptake kinetics of radiolabeled oligonucleotides to the messenger RNA (mRNA) of proliferating cell nucleus antigen (PCNA) in vascular smooth muscle cells (VSMCs) and the effect on gene expression. METHODS: The antisense oligonucleotide to PCNA was radiolabeled with (99m)Tc through bifunctional chelator mercaptoacetyltriglycine (MAG(3)). The labeling efficiency was assessed by Sephadex G25 chromatography. The radiochemical purity, in vitro stability, and ability of the labeled antisense oligonucleotide to hybridize to its complement were analyzed by Sep-Pak C18 column chromatography. The uptake kinetics of (99m)Tc-labeled antisense oligonucleotide and sense oligonucleotide were studied in VSMCs in log and plateau phases. To study whether the antisense probe can hybridize to a respective sequence on the whole PCNA mRNA strand after radiolabeling, we performed reverse-transcriptase polymerase chain reaction to assay the PCNA mRNA level after the VSMCs had been incubated with the (99m)Tc-labeled antisense oligonucleotide and sense oligonucleotide. RESULTS: The labeling efficiency of (99m)Tc-MAG(3)-antisense oligonucleotide was 60.1% (n = 5), the specific activity was 1,960 kBq/microg, and the radiochemical purity was more than 95% after purification. (99m)Tc-MAG(3)-antisense oligonucleotide was stable in vitro and retained the ability to hybridize with its complementary chain. Antisense oligonucleotide showed significantly higher accumulation than sense oligonucleotide in log phase, with peak values of 15.2% +/- 0.58% and 5.6% +/- 0.42%, respectively (P < 0.05). No significant difference was found between uptake of antisense oligonucleotide and uptake of sense oligonucleotide in plateau phase (P > 0.05), but higher accumulation of antisense oligonucleotide was found in log phase than in plateau phase (P < 0.05). The retention rate of antisense oligonucleotide in log phase was much higher than that of sense oligonucleotide (79.6% +/- 0.96% vs. 59.8% +/- 0.75%, P < 0.05) at 240 min. The 2 probes did not significantly differ in plateau phase (P > 0.05). The efflux of antisense oligonucleotide was obviously slower in log phase than in plateau phase (P < 0.05). Compared with (99m)Tc-MAG(3)-sense oligonucleotide, (99m)Tc-MAG(3)-antisense oligonucleotide could inhibit the expression of PCNA mRNA significantly. CONCLUSION: This in vitro study in VSMCs provided evidence that the (99m)Tc-labeled antisense oligonucleotide to PCNA can be used for in vivo imaging of atherosclerotic plaque and restenosis in further study.

Animals↗

PubMed2005

[Optimization of cultural condition of genetic engineering strain for antibiotic peptide adenoregulin and research on its fed-batch cultivation].

33 amino acid antibiotic peptide adenoregulin (ADR), which were firstly isolated from the skin of South America arboreal frog Phyllomedusa bicolor, forms alpha-helix amphipathic structure in apolar medium and has a wide spectrum of antimicrobial activity and high potency of lytic ability. Adr gene was cloned in pET32a and transformed into Escherichia coli BL21(DE3) . The cultural and inductive conditions of E. coli BL21(DE3)/pET32a-adr have been optimized. The effect of three factors which were time point of induction, concentration of IPTG in the culture and time of induction on the expression level of Trx-ADR was investigated. The results indicated that the expression level was affected by the time point of induction most predominantly. 9 veriaties of media in which BL21 (DE3)/pET32a-adr was cultured and induced were tested to achieve high expression level of target protein. It was found that glucose in the medium played an important role in keeping stable and high expression level of Trx-ADR. The optimal inductive condition is as follows: the culture medium is 2 x YT + 0.5% glucose, the time point of induction is OD600 = 0.9, the final concentration of IPTG in the culture is 0.1 mmol/L and the induction time is 4 h. BL21 (DE3)/pET32a-adr was cultivated according to the strategy of constant pH at early stage and exponential feeding at later stage to obtain high cell density. During the entire fed-batch phase, by controlling the feeding of glucose, the specific growth rate of the culture was controlled at about 0.15 h(-1), the accumulation of acetic acid was controlled at low level (<2 g/L), but the plasmid stability could not be maintained well. At the end of the cultivation, 40% of the bacteria in the culture lost their plasmids. As a result, the expression level of the target protein declined dramatically, but 90% of Trx-ADR was in soluble form. The expressed fusion protein showed no antibacterial activity, while the native form of ADR lysed from Trx-ADR showed distinct antibacterial activity.

Amphibian Proteins↗

PubMed2005

[Lyophilization for platelet preservation].

To explore a new lyophilized preservation methods for human platelets, platelets were pre-treated with aldehyde, human albumin or trehalose was added to the system of condensed cooling as protectant to stabilize the structure of platelets. The optimal resuspending buffer was also selected in the study. The morphological changes of platelets were observed by using electron microscopy after lyophilization, and the expression of membrane proteins on platelets was detected also after lyophilization. The results indicated that the recovery rate of platelets treated with aldehyde was generally more than 60%. Aggregative ability was reduced a little than the platelet untreated. 5% of human albumin had an advantage over 40 mmol/L of trehalose in respect of the preservation effect. In the way of keeping aggregative ability, PPP was obviously better than PBS. The results of electron microscopy displayed that organelles including mitochondria and excreted granules could be observed distinctly. Whereas, expression of membrane proteins of platelet treated with aldehyde was evidently dropped as compared with those of the fresh platelet. In conclusion, aldehyde as a novel protective agent, has excellent effects on lyophilization of platelets and is worthy to be further studied.

Albumins↗

PubMed2005

[Analysis of four components in Fufang Danshen Tablets by high performance liquid chromatography with electrochemical and ultraviolet detections].

A rapid and accurate method for the simultaneous separation and determination of protocatechuic acid, protocatechuic aldehyde, caffeic acid and tanshinone II A in Fufang Danshen Tablets by high performance liquid chromatography has been established. The former three constituents were detected by electrochemical array detector (ECD) and the last one by ultraviolet diode array detector (DAD). The separation was performed on a Zorbax SB-C18 column (150 mm x 4.6 mm i.d., 5.0 microm) by gradient elution with methanol and 0.4% aqueous phosphoric acid as mobile phase at a flow rate of 1.0 mL/min. The detections were done at 0.7 V, 270 nm and 30 degrees C. The results showed that the linear ranges were 0.3 - 9.0 mg/L for protocatechuic acid, 1.1 - 54.0 mg/L for protocatechuic aldehyde, 1.1 - 11.1 mg/L for caffeic acid and 5.2 - 52.0 mg/L for tanshinone II A. The average recoveries (n = 6) were 97.8% with relative standard deviation (RSD) of 2.15% for protocatechuic acid, 98.2% with RSD of 2.07% for protocatechuic aldehyde, 97.6% with RSD of 2.18% for caffeic acid and 97.2% with RSD of 2.07% for tanshinone II A. These four components were separated successfully from each other. The sensitivities of detection of protocatechuic acid, protocatechuic aldehyde and caffeic acid with ECD were 11.0 - 25.3 times greater than those with DAD. The study provides a scientific basis for the quality control of Fufang Danshen Tablets.

Abietanes↗

PubMed2004

Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides.

PURPOSE: The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). METHODS: Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5+/-18.5 MBq (99m)Tc-labelled ASON or (99m)Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-myc ASON was evaluated in vivo in atherosclerotic rabbits. RESULTS: Planar imaging revealed accumulation of (99m)Tc-labelled c-myc ASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. CONCLUSION: This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage.

Animals↗

PubMed2004

Temporal kinetics of the light/dark translocation and compartmentation of arrestin and alpha-transducin in mouse photoreceptor cells.

PURPOSE: To determine the temporal kinetics of the simultaneous translocation of arrestin and rod alpha-transducin in mice exposed to different lighting environments and to compare the subcellular compartmentation of cone alpha-transducin with arrestin. METHODS: Double labeling immunofluorescence microscopy and image analysis are used to visualize and quantify the concentrations of rod arrestin and alpha-transducin in the subcellular compartments of the rod outer segments, the rod inner segments and the synaptic terminals. RESULTS: The magnitude of the effects of the translocation are clearly contrasted in images of the retinas of animals that have been maximally light adapted verses retinas that have been maximally dark adapted. The onset of light results in a rapid, simultaneous, translocation of arrestin and alpha-transducin from their respective compartments (alpha-transducin in the rod outer segment and arrestin in the rod inner segment) to the opposite compartment. Almost all of alpha-transducin has translocated in less than two min whereas the translocation of the majority of arrestin requires at least five to six min. Translocation in the opposite direction, from light to dark, occurs more slowly for both proteins with arrestin requiring almost 30 min and alpha-T needing more than 200 min to complete its journey. Under the same lighting conditions, cone arrestin translocation is incomplete. Cone alpha-transducin does not translocate under any the lighting conditions tested. Unlike the frog, continuous exposure of mice to light does not result in arrestin translocating back to the rod inner segment. CONCLUSIONS: These data suggest that there are four mechanisms involved in the translocation of these two proteins. They also support the conclusion that the more important cellular function of the translocation process is to terminate phototransduction in rod and cone photoreceptors, which could provide protection against light damage. The secondary function of translocation is to maximize rod sensitivity to light during dark adaptation. The restricted localization of cone alpha-transducin to the cone outer segment is consistent with the function of cones in bright light, just as the concentration of rod alpha-transducin in dark adapted rod outer segment is consistent with their functioning in dim light.

Animals↗

PubMed2004

Mutational spectra of p53 in geographically localized esophageal squamous cell carcinoma groups in China.

BACKGROUND: Esophageal carcinoma is a particularly interesting tumor because of the dramatic difference in its incidence and geographic distribution among populations of similar ethnic origin. Epidemiologic data have suggested that many environmental exposures may be associated with an increased risk of its formation. METHODS: In this study, 92 samples of esophageal squamous cell carcinoma (ESCC) were collected from patients who resided in 2 geographic areas in China with different incidences of ESCC: Linxian and Zhejiang. Overexpression and mutations of the p53 tumor-suppressor gene were examined by using immunohistochemistry, single-strand conformation polymorphism analysis, and direct sequencing. RESULTS: The rates of point mutation and overexpression of p53 in the ESCC specimens studied were 30.4% (29 of 92 specimens) and 51.1% (47 of 92 specimens), respectively. The overexpression of p53 was associated with tumor metastasis and with 5-year case fatality. Significant differences were found in the rates of overexpression and mutations in patients with clinical T2 tumors between the specimens from Linxian, which is a high-incidence geographic area, and the specimens from Zhejiang, which is a low-incidence area. Furthermore, different mutational spectra were found in the tumor samples from these two geographic areas: In tumor samples from Linxian, the most common substitution mutation was a transversion in exon 5, whereas the most common mutations in tumor samples from Zhejiang were transitions in exon 7. CONCLUSIONS: The data suggest that the mutation and overexpression of p53 may play important roles in the development of ESCC. The changes in p53 may reflect environmental exposure to the different combinations of mutagenic factors and genetic instability demonstrated by the populations in Linxian and Zhejiang. The overexpression of p53 protein may have significance as a prognostic factor for patients with esophageal carcinoma.

Adult↗

PubMed2004

High-efficiency, environment-friendly electroluminescent polymers with stable high work function metal as a cathode: green- and yellow-emitting conjugated polyfluorene polyelectrolytes and their neutral precursors.

A series of aminoalkyl-substituted polyfluorene copolymers with benzothiadiazole (BTDZ) of different content were synthesized by Suzuki coupling reaction, and their quaternized ammonium polyelectrolyte derivatives were obtained through a postpolymerization treatment on the terminal amino groups. Copolymers are soluble in environmentally friendlier solvents, such as alcohols. It was found that the efficient energy transfer occurs by exciton trapping on the narrow band gap BTDZ site under UV illumination. Only 1% of BTDZ content is needed to completely quench a fluorene emission for both the neutral and the quaternized copolymers in the neat film. Absolute PL efficiencies of copolymer films were greatly enhanced as a result of the suppression of excimer formation. Light-emitting devices fabricated from these copolymers show high external quantum efficiencies over 3% and 1% for the neutral precursor and the quaternized copolymers, respectively, with high work function metals such as Al as a cathode. To the best of our knowledge, this is the first report on an electroluminescent polymer which bears the high EL efficiency, the electron-injection ability from high work function metals, and the solubility in environment-friendly solvents at the same time. These features make them a promising candidate for the next generation of light-emitting copolymers in PLED flat panel display application.

Journal Article↗

PubMed2004

Involvement of phosphatidylinositol 3-kinase and insulin-like growth factor-I in YXLST-mediated neuroprotection.

In the present study, we examine the neuroprotective role of the external Qi of YXLST in cultured retinal neurons. Primary retinal neuronal cultures were grown from retinas of 0-2-day-old Sprague-Dawley rats. Cultures were treated directly with external Qi of YXLST 30 min prior to H(2)O(2) exposure in most experiments. Cell viability was measured by 3,(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) assay. Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling TUNEL assay, and by DNA laddering analysis. Northern blot analysis was performed to examine the level of insulin-like growth factor-I (IGF-I) gene expression. Phosphatidylinositol 3-kinase (PI3K) assay was performed to study the PI3K activity. The results showed that treatment of external Qi of YXLST significantly attenuated neuronal death that was induced by 24-h exposure to hydrogen peroxide, and greatly inhibited hydrogen peroxide-induced apoptosis. External Qi of YXLST also upregulated IGF-I gene expression and increased PI3K activity. These observations indicate that external Qi-mediated IGF-I expression and PI3K signaling could be one of the mechanisms in neuroprotection by YXLST.

Animals↗

PubMed2004

Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods.

The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1-8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9-12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOua(R)2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4'maleimidylstilbene-2,2'-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 microM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N -ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein.

Animals↗

PubMed2004

Involvement of insulin/phosphoinositide 3-kinase/Akt signal pathway in 17 beta-estradiol-mediated neuroprotection.

In the present study, we tested the hypothesis that 17beta-estradiol (betaE2) is a neuroprotectant in the retina, using two experimental approaches: 1) hydrogen peroxide (H(2)O(2))-induced retinal neuron degeneration in vitro, and 2) light-induced photoreceptor degeneration in vivo. We demonstrated that both betaE2 and 17alpha-estradiol (alphaE2) significantly protected against H(2)O(2)-induced retinal neuron degeneration; however, progesterone had no effect. betaE2 transiently increased the phosphoinositide 3-kinase (PI3K) activity, when phosphoinositide 4,5-bisphosphate and [(32)gammaATP] were used as substrate. Phospho-Akt levels were also transiently increased by betaE2 treatment. Addition of the estrogen receptor antagonist tamoxifen did not reverse the protective effect of betaE2, whereas the PI3K inhibitor LY294002 inhibited the protective effect of betaE2, suggesting that betaE2 mediates its effect through some PI3K-dependent pathway, independent of the estrogen receptor. Pull-down experiments with glutathione S-transferase fused to the N-Src homology 2 domain of p85, the regulatory subunit of PI3K, indicated that betaE2 and alphaE2, but not progesterone, identified phosphorylated insulin receptor beta-subunit (IRbeta) as a binding partner. Pretreatment with insulin receptor inhibitor, HNMPA, inhibited IRbeta activation of PI3K. Systemic administration of betaE2 significantly protected the structure and function of rat retinas against light-induced photoreceptor cell degeneration and inhibited photoreceptor apoptosis. In addition, systemic administration of betaE2 activated retinal IRbeta, but not the insulin-like growth factor receptor-1, and produced a transient increase in PI3K activity and phosphorylation of Akt in rat retinas. The results show that estrogen has retinal neuroprotective properties in vivo and in vitro and suggest that the insulin receptor/PI3K/Akt signaling pathway is involved in estrogen-mediated retinal neuroprotection.

Animals↗

PubMed2004

Glutathione S-transferase P1 Ile105Val polymorphism, cigarette smoking and prostate cancer.

The enzyme glutathione S-transferase P1 (GSTP1) detoxifies carcinogenic products of tobacco smoke. This exploratory case-control study evaluates the possible effect modification by the GSTP1 Ile105Val polymorphism (replacement of isoleucine by valine at codon 105) on smoking and prostate cancer. Because the Val variant possesses up to a five-fold greater enzymatic activity towards the carcinogenic metabolites of tobacco smoke, the Ile allele is expected to be related to an increase in the risk of prostate cancer among smokers. GSTP1 genotype and epidemiological data were obtained from 122 cases of prostate cancer and 135 healthy males as controls. A logistic regression model was used to estimate odds ratios and 95% confidence intervals. The adjusted OR of homozygous Ile compared to other genotypes for prostate cancer was 1.21 (95% CI: 0.61-2.83). Smoking was not significantly associated with prostate cancer with an adjusted OR of 1.56 (95% CI: 0.78-3.12). However, among individuals with the Ile/Ile genotype, smoking was strongly associated with an increased risk of prostate cancer with an adjusted odds ratio of 4.09 (95% CI: 1.25-13.35). A potential multiplicative interaction was suggested between GSTP1 and smoking on the risk of prostate cancer with the adjusted OR for the interaction of 4.52 (95% CI: 1.07-19.17). To our knowledge, this is the first time that a potential effect modification by the GSTP1 Ile/Ile genotype on smoking and the risk of prostate cancer is suggested.

Adult↗