Effects of HIF prolyl-hydroxylase-2 gene silencing on HCG-induced vascular endothelial growth factor expression in luteal cells.

Vascular endothelial growth factor (VEGF)-dependent angiogenesis plays a crucial role in corpus luteum formation and its functional maintenance in mammalian ovaries. We recently reported that activation of hypoxia-inducible factor (HIF)-1α signaling contributes to the regulation of VEGF expression in luteal cells (LCs) in response to human chorionic gonadotropin (HCG). We examined whether HIF prolyl-hydroxylase (PHD)-2 gene silencing induces VEGF expression in LCs and enhanced its expression induced by HCG in LCs. Using real-time polymerase chain reaction and western blot analysis, we measured the expression of PHD-2 to confirm plasmid PHD-2 shRNA transfection and protein expression and investigated the changes in HIF-1a and VEGF expression after treatment with HCG and PHD-2 shRNA transfection. After PHD-2 shRNA transfection, PHD-2 expression was significantly lower than that in control groups with or without HCG treatment, while a significant increase in VEGF mRNA was observed compared to in controls, indicating that PHD-2 plays an important role in VEGF regulation. Additionally, changes in VEGF mRNA expression were consistent with the expression levels of HIF-1a protein, not HIF- 1a mRNA, which is regulated by HIF prolyl-hydroxylase-mediated degradation. Our results indicate that PHD-2 gene silencing can induce VEGF expression in LCs and HCG-induced VEGF expression can be further enhanced by PHD-2 gene silencing through an HIF-1a-mediated mechanism in LCs. This PHD-2-mediated transcriptional activation may be important for regulating VEGF expression through HIF-1a signaling in LCs during corpus luteum development in mammals.

Diffusion tensor imaging for predicting hand motor outcome in chronic stroke patients.

OBJECTIVE: Previous studies have indicated that diffusion tensor imaging (DTI) values are related to clinical outcome in stroke patients. This prospective study explored whether DTI values were predictive for hand function outcome in chronic stroke patients. METHODS: The DTI parameters (rλ1, rλ23, fractional anisotropy [rFA] and mean diffusivity [rMD]) were investigated in patients with completely paralysed hands (CPH; n=10) or partially paralysed hands (PPH; n=10), by two methods of analysis: segment of the corticospinal tract [sCST] analysis; pure region of interest [ROI] analysis. Spearman's correlation coefficient was used to assess the correlation between the DTI parameters and the following clinical measures: Fugl-Meyer Assessment [FMA]; National Institutes of Health Stroke Scale [NIHSS]. RESULTS: Significant differences were found between CPH and PPH for rFA and rλ23 (sCST analysis) and for rMD and rλ23 (ROI analysis). The rλ23 (sCST analysis) correlated with the NIHSS; the rMD (sCST analysis) correlated with the FMA (hand). CONCLUSION: The three parameters, rFA, rλ23 and rMD may have predictive value for evaluating hand function outcome in chronic stroke patients.

[Synthesis and determination for enantiomeric purity of 6-fluoro-L-DOPA].

AIM: To study the synthesis and determination for enantiomeric purity of 6-fluoro-L-3, 4-dihydroxyphenylalanine (6-fluoro-L-DOPA, 6-FDOPA). METHODS: 2-(2-Fluoro-4, 5-dimethoxybenzyl)-N-(diphenylmethylene) glycine tert-butyl ester (8), a new compound, was synthesized from the starting material nitroveratraldehyde via the nucleophilic displacement, reductive iodination, and chiral catalytic phase-transfer alkylation, and 6-FDOPA was prepared from hydrolysis of 8. The enantiomeric purity of 6-FDOPA was determined by HPLC method using a chiral mobil phase and reversed-phase C18 column. RESULTS: The total time of synthesis was less than 90 min, the overall chemical yield from potassium fluoride was about 33%, and the enantiomeric purity was above 95%. CONCLUSION: Large scale production of 6-FDOPA and automatic synthesis of 6-[18F]fluoro-L-DOPA with excellent chemical and entiomeric purity are available. The practical technique was provided for the radiochemical synthesis and entiomeric purity of 6-[18F]fluoro-L-DOPA.

Lipofuscin-like fluorophores can result from reactions between oxidized ascorbic acid and glutamine. Carbonyl-protein cross-linking may represent a common reaction in oxygen radical and glycosylation-related ageing processes.

Ascorbic acid (AsA), after being oxidized in 0.1 M phosphate (pH 7.0) buffer under the catalytic influence of adventitious iron, reacted with glutamine (Gln) derivatives with the formation of stable fluorophores showing lipofuscin-like blue (350/430 nm) fluorescence. The fluorescence was reversibly quenched by acidity and enhanced by alkaline conditions, and the fluorescence intensity was directly proportional to the Gln and AsA concentrations. Addition of H2O2 considerably increased the velocity of the fluorescence formation. Incubation of AsA/Gln in 0.1 M phosphate buffer at pH 5.0 gave a slower fluorophore formation as compared with incubation at pH 7.0. The iron chelators DTPA and desferrioxamine inhibited the fluorophore development by preventing the iron catalyzed AsA oxidation. This was in contrast to the effects of the chelators ADP and EDTA which did not show such preventive activity. The fluorophores produced by the AsA/Gln reaction are thought to be Schiff bases formed secondary to Maillard reactions involving oxidized AsA. Considering that ascorbic and dehydroascorbic acid are active and common reductones, the oxidation-enhanced carbonyl-protein cross-linking is suggested to be an important chemical reaction which may take place during ageing and be involved in lipofuscinogenesis.

Oxidized ascorbic acid and reaction products between ascorbic and amino acids might constitute part of age pigments.

Fluorescence and non-enzymatic browning were observed in reactions between ascorbic acid (AH2) and amino acids (AA) as well as in reactions involving AH2 autoxidation and/or polymerization in the presence of trace amounts of adventitious iron (less than or equal to 10 microM). These reaction products exhibited fluorescent spectra (400-490 nm) akin to those of extracts from lipofuscin-rich tissues. Lengthy incubation of AH2 at 37 degrees C in phosphate buffer (pH 7.0) caused a concentration- and time-dependent increase in absorbance at 412 nm, paralleling increase in 377/440 nm fluorescence, due to formation of autoxidation/polymerization products. The fluorescences of these substances were increased by acidity and quenched at alkaline conditions but restored by neutralization. The reactions between AH2 (0.1-2.0 mM) and a number of AA (1.0-4.0 mM) were also found to result in products with blue fluorescence. Following TBA test, the AH2 autoxidation/polymerization products and AH2/AA reaction products showed only moderate and slight absorbance at 535 nm, respectively, indicating a little or minute formation of aldehydes with MDA-like reactivity. The findings in this study, nevertheless, suggest possible misinterpretations of results in previous studies dealing with AH2-dependent, oxygen free radical induced, 'lipofuscin-related fluorescence'. Thus, similar to nonenzymatic glycosylation (Maillard) reactions, AH2 autoxidation as well as reactions between AH2 and AA may result in 'lipofuscin-like material', as judged from their fluorescence spectral patterns.

Microfluorometric and fluorometric lipofuscin spectral discrepancies: a concentration-dependent metachromatic effect?

The fluorescent spectral patterns of some lipid peroxidation products and their derivatives have been investigated. A significant concentration-dependent fluorescence shift was found. A variety of suggested age pigment fluorophores, 1,4-dihydropyridines, Schiff base and MDA polymers, all demonstrated a potential for spectral shifts. Along with increased concentration, the fluorescence peaks of these fluorophores shifted from blue (400-490 nm) to golden-yellow or orange-red (500-600 nm). The demonstrated metachromasia is supposed to be an inner-filter effect resulting from molecular polymerization or stacking. Thus, the striking differences between lipofuscin fluorescence spectra obtained by different investigators may be explained as due to large differences in lipofuscin concentration during measurement with different techniques. The pigments are either studied in situ by morphologists and recorded by microscopic fluorometry or by biochemists using spectrofluorometers to measure the extracted and dissolved pigments.

[Comparison of serum trace element spectrum of liver cancer patients and healthy adults].

The contents of 15 trace elements in the sera of 30 liver cancer patients and 30 healthy adults were assayed by ICP-AES method. The data obtained were analysed by routine statistical tests, multi-variate discrimination analysis, multi-variate stepwise regression analysis and non-linear mapping algorithm. The results showed that the contents of copper, vanadium, cadmium, stannum, cobalt, nickel in liver cancer patients were significantly higher than those in healthy adults. The serum trace element spectrum of liver cancer patients was different from that of healthy adults. Hence, the liver cancer patients could be differentiated from healthy adults by serum trace element spectrum.

Measurement of serum transferrin by the gamma-gamma perturbed angular correlation method. Differences between normal and cancer serum.

In this work, the average perturbed angular correlation integral attenuation factor G2 of the transferrin in the serums from the normals and the patients with liver cancer were determined using time integral perturbed angular correlation method (PAC). The average values of G2 were 0.1945 +/- 0.0155 for the cancer serum transferrin and 0.2865 +/- 0.0411 for the normal serum transferrin. The difference between these two groups is significant. Mathematical deduction and theoretical analysis suggested that the equivalent radius of the transferrin molecules in the serum of cancer patients becomes smaller than normal, because of a change in the molecular conformation. Similar results were also observed in experiments with rat serum transferrin.