TF Content Research Articles

Antioxidant potency of white ( Brassica oleracea L. var. capitata) and Chinese ( Brassica rapa L. var. pekinensis (Lour.)) cabbage: The influence of development stage, cultivar choice and seed selection

The accumulation of total phenols (TP, Folin-Ciocalteu method) and total flavonoids (TF, colorimetric assay with AlCl 3) and the evolution of antioxidant capacity (FRAP assay, DPPH and ABTS radical scavenging assays) have been monitored in juices of Croatian white cabbage ( Brassica oleracea var. capitata) cultivars Varaždinski and Ogulinski, as well as Chinese cabbage ( Brassica rapa var. pekinensis), at various developmental stages. Measurements were performed every four weeks, starting from planting to full maturity, throughout the course of eight months. In the first 8–12 weeks, the TP and TF contents as well as antioxidant capacity increased significantly in all analyzed juice samples and in most even doubled. This rapid increase was followed by a gradual decrease in values derived from all assays, over the course of 12–30 weeks, to the final values which were in all cases lower than the values measured at week 4. The results also point to significant variability in TP and TF contents and antioxidant capacity at the fully mature stage between white and Chinese cabbage juices and between juices extracted from cultivars Ogulinski and Varaždinski.

Platelet Tissue Factor Activity In Patients with Hypercholesterolemia: Modulation of Procoagulant Activity by Statins

AbstractAbstract 156Human platelets store TF that expresses procoagulant activity (PCA) associated with platelet activation (Panes O. et al, Blood 2007;109:5242-50). We have also observed that platelet TF-induced PCA is specifically and rapidly induced by activation of GPIb complex by VWF-Ristocetin (Panes O. et al. Blood 2008;112:A113). Hypercholesterolemia has been associated with increased platelet function and hypercoagulability, but the mechanisms are still unknown. The cholesterol content in cell membranes is directly related with the plasma concentration of the sterol. Inhibition of HMG-CoA reductase by statins reduces cardiovascular risk by decreases in plasma cholesterol and by other independent (“pleiotropic”) effects.We studied the relationship between total and LDL plasma cholesterol with platelet TF content and TF-dependent PCA of washed platelets activated for 5 min with VWF-Ristocetin (VWF-R). We also examined these variables after atorvastatin or rosuvastatin intake (80 and 20 mg/day × 1 month, respectively) in 25 subjects with hypercholesterolemia. TF was measured by ELISA and PCA by FXa generation (chromogenic assay) in washed platelet suspensions before and after activation. We found no significant differences between both statins regarding the decrease in plasma LDL-cholesterol levels and platelet TF-dependent PCA; both statins had any significant effects on the levels of blood inflammatory markers (usCRP, fibrinogen and VWF). Thus, we analyzed the pooled data of patients receiving either one or the other statin. Total and LDL-Chol dropped from 271±41 and 185±40 to 167±30 and 87±23 mg dL-1, respectively. This was associated with decrease in basal PCA (non-stimulated platelets) from 34±27 to 22±14 nmol of FXa/2*107platelets (p=0.02). After VWF-R stimulation, a mean 3.48-fold increase in platelet PCA was observed in hypercholesterolemic patients before taking statins. Surprisingly, a mean 20-fold increase in FXa generation in activated platelets was observed after 1 month on statin therapy, and this difference was statistically significant (p=0.018). These changes in PCA were not associated with significant changes in TF content of platelet membranes (621±481 to 555±300 pg/mg prot). We also found that plasma LDL-Chol was negatively correlated with platelet PCA induced by VWF-R activation (r = −0.239, p = 0.026) and with the ratio of PCA between activated and non-activated platelets (r = −0.27, p = 0.011). The striking post-statin increase of platelet-PCA without change in the total platelet TF protein suggests that PCA depends more on the fraction of platelet TF available for activation than on the total mass of platelet TF. The decrease in basal PCA of non-stimulated platelets after statin therapy suggests that lower concentration of cholesterol in platelet membranes makes the platelets more stable during the isolation procedure. The higher increase after stimulation would reflect a better hemostatic response to a specific stimulus. Thus, changes in plasma cholesterol, possibly through decrease in platelet membrane cholesterol, modulates the PCA of platelets, either by making platelets more stable under resting conditions and more responsive after specific activation. Disclosures:No relevant conflicts of interest to declare.

Identification of radiolysis products of solid thiamphenicol

Spectroscopic and chromatographic methods (HPLC, HPLC–MS, NMR) were used to observe, separate and identify products of radiolysis of thiamphenicol (TF), irradiated in the solid state at room temperature and atmospheric pressure with an electron beam from a linear accelerator to doses between 25 and 800 kGy. Nine products of radiolysis of thiamphenicol were identified, among them were TF amine, dichloroacetic acid, 4-methylsulfonylbenzoic acid, demono- and dedichloroderivative of TF, 2,2-dichloro- N-{3-hydroxy-1-[4-(methylsulfonyl)phenyl]-1-oxopropan-2-yl}acetamide and 3-({1,3-dihydroxy-1-[4-(methylsulfonyl)phenyl]propan-2-yl}amino)-3-oxopropanoic acid. The process of radiodegradation of TF was proposed as consisting of several parallel primary reactions (dehalogenation, oxidation of the OH group at C 1, hydrolysis of the amide bond, a rapture of the C 2–C 3 bond of propan-1-ol) and secondary reactions (carboxylation and oxidation). The use of high doses, well above the sterilization dose of 25 kGy, allowed observation of changes of TF content as a function of radiation dose, calculation of radiolytic yield (G −TF) and kinetic parameters of the degradation reaction. It was found that the standard sterilizing dose lowers the content of TF by only 0.1% and the radiolytic efficacy of the process of radiodegradation is 0.76 molecules/100 eV. Further increase in the dose lowers the content of TF to 92.1% for 800 kGy dose and leads to an increase in the value of G −TF. It was also found that the summative process of radiodegradation of TF exposed to a beam of electrons of 10 kGy/s follows the first order reaction kinetics with a degradation constant of k = 0.001 s −1. On the basis of the experiments conducted it can be stated that the radiolysis of TF in the presence of an E-beam, in substantia, follows multidirectional course in the same way as radiolysis of chloramphenicol. TF exposed to the standard sterilizing dose of 25 kGy degrades only by 0.1%, the amount acceptable by the ICH, and forms only one product of radiolysis (TF amine) and therefore we conclude that it can be sterilized by ionizing radiation under the conditions described above.

Mechanism of active targeting in solid tumors with transferrin-containing gold nanoparticles

PEGylated gold nanoparticles are decorated with various amounts of human transferrin (Tf) to give a series of Tf-targeted particles with near-constant size and electrokinetic potential. The effects of Tf content on nanoparticle tumor targeting were investigated in mice bearing s.c. Neuro2A tumors. Quantitative biodistributions of the nanoparticles 24 h after i.v. tail-vein injections show that the nanoparticle accumulations in the tumors and other organs are independent of Tf. However, the nanoparticle localizations within a particular organ are influenced by the Tf content. In tumor tissue, the content of targeting ligands significantly influences the number of nanoparticles localized within the cancer cells. In liver tissue, high Tf content leads to small amounts of the nanoparticles residing in hepatocytes, whereas most nanoparticles remain in nonparenchymal cells. These results suggest that targeted nanoparticles can provide greater intracellular delivery of therapeutic agents to the cancer cells within solid tumors than their nontargeted analogs.

Melatonin exerts a more potent effect than S-adenosyl- l-methionine against iron metabolism disturbances, oxidative stress and tissue injury induced by obstructive jaundice in rats

Melatonin and S-adenosyl- l-methionine (SAMe) prevent oxidative stress and tissue dysfunction in obstructive jaundice (OJ). Lipid peroxidation is exacerbated in the presence of trace amounts of iron (Fe). The study investigated the regulation by melatonin and SAMe the induction of oxidative stress, iron metabolism disturbances and tissue injury in an experimental model of OJ. Different parameters of lipid peroxidation, antioxidant status, tissue injury and Fe metabolism were determined in liver and blood. OJ induced Fe accumulation in liver, and increased transferrin (Tf) saturation and loosely bound Fe content in blood. Melatonin, and SAMe at lesser extent, enhanced protein Tf content in liver and blood, that reduced loosely bound Fe content in blood. Melatonin and SAMe did not affect ferritin (FT) and Tf mRNA expression, but reduced Tf receptor (TfR) mRNA expression in liver. In conclusion, the effect of melatonin and SAMe on Fe metabolism may be included in the beneficial properties of these agents on lipid peroxidation and tissue injury induced by OJ.

Polyol pathway mediates iron-induced oxidative injury in ischemic–reperfused rat heart

Recent studies have shown that the polyol pathway is involved in ischemia–reperfusion (I/R)-induced myocardial infarction, but the mechanism is unclear. We previously found that lack of aldose reductase (AR), the first enzyme of the polyol pathway, attenuated the increase in transferrin (Tf) level in I/R brain, suggesting that AR contributes to iron-catalyzed free radical-induced damage. We therefore investigated if this mechanism occurs in I/R hearts. We found that inhibition of AR or sorbitol dehydrogenase (SDH), the second enzyme of the polyol pathway, both attenuated the I/R-mediated increases in HIF-1α, Tf, TfR, and intracellular iron content and reduced the I/R-induced infarct area of the heart. Further, administration of niacin, which replenishes NAD +, the cofactor for SDH, also normalized TfR and HIF-1α levels in I/R hearts. These results suggest that during I/R polyol pathway activity increases the cytosolic NADH/NAD + ratio. This activates HIF-1α that induces the expression of TfR, which in turn increases Tf uptake and iron accumulation and exacerbates oxidative damage that increases the lipid peroxidation. This was confirmed by the fact that administration of the iron chelator deferoxamine attenuated the I/R-induced myocardial infarction.

Variability of total flavonoids in Crataegus — Factor evaluation for the monitored production of industrial starting material

European Hawthorn species ( Crataegus spp.) are traditionally used for their demonstrated cardioprotective benefits. Most products are based on the compendial leaf and flower drug (LFD), which is standardised on the total flavonoid content (TF). In order to reduce variability associated with the wild plant origin and to ease product standardisation, we set out to develop a sustainable source of high quality raw material. Firstly, the LFD of wild trees in Germany was screened in terms of TF content (spectrophotometric analysis, calculated as hyperoside) according to the current pharmacopoeia requirements. Secondly, eleven high value provenances were selected, propagated, cultivated and the grafted clones were reanalysed. Thirdly, major environmental and sourcing influences were assessed: the year of harvest, the growing location, exposure to sunlight, the harvest time and the portion of leaf, flower and wood within the LFD. We found the TF in LFD of 150 wild grown Crataegus ranged between 0.28% and 1.92% (average 1.15%). Pure single styled Crataegus monogyna and hybrid trees represented the major portion (57%) of all screened trees. The hybrids with mainly two-styled flowers (pure Crataegus laevigata and hybrids) showed slightly higher TF values. The selected clones proved to maintain a high TF profile in cultivation, although superiority was attenuated when not only O-glycosides, but also C-glycosides were covered by the assay. Environmental influence surpassed in part the genetic variation of the selection in this study. In conclusion, cultivated high performance trees under central European conditions produced LFD exceeding the Pharmacopoeia standards. Careful monitoring of production – particularly in terms of the harvest time and actual LFD location and composition – has decisive impact to guarantee consistent high TF values.

Effect of ground variability on acoustic-to-seismic transfer function and false alarms in landmine detection

The spatial variability of ground properties leads to fluctuations in the acoustic-to-seismic transfer function (A/S TF), the ratio of the normal particle velocity on the ground to sound pressure. In some cases, these fluctuations may lead to false landmine detection alarms. This work shows that small variations in the ground properties may cause strong variations in the A/S TF. Experimental measurements of the A/S TF performed at a US Army eastern temperate site are presented and a correlation between high magnitudes of the A/S TF (false alarms) and moisture content on the surface is shown. A simple model of the ground explaining this correlation is suggested. This model was used to describe spatial distribution of high magnitudes in the TF and natural spatial variability of the TF. Results of calculations were compared with experimental data. Two frequency modulation scales in the A/S TF are observed at positions on the ground where land mines are not located. It was hypothesized that these are due to influence of wheeled vehicular traffic on the acoustic parameters in the ground layers. A comparison between numerical modeling of the A/S TF and the experimental data was performed. Direct measurements of acoustic parameters confirmed conclusions obtained from analysis of the A/S TF.

Polyphenol constituents and antioxidant activity of grape pomace extracts from five Sicilian red grape cultivars

The methanolic extracts (MeOH) obtained from de-stemmed grape pomace samples of five Sicilian red grape cultivars (Nero d′Avola-NA, Nerello Mascalese-NM, Nerello Cappuccio-NC, Frappato-FR and Cabernet Sauvignon-CS) were evaluated for their DPPH and ABTS − radical scavenging capacity, and submitted to HPLC–UV–DAD and HPLC–MS–ESI analysis to determine the main polyphenolic constituents, namely anthocyanins and flavonols. All the MeOH extracts showed significant antioxidant activity, with some differences between the two methods employed. The NM sample was the most active in both tests. A large variability in the total anthocyanin (TA) and flavonol (TF) contents of the MeOH extracts, as well as in the quantitative distribution of the single anthocyanins and flavonols was observed. Statistically insignificant correlations between the TA + TF content and antioxidant activity, as measured by DPPH and ABTS − model systems ( r 2 = 0.0607, P > 0.05; r 2 = 0.3471, P > 0.05), were established, but the most active sample, NM, showed the highest content of anthocyanins including a free catechol moiety in their structure.

Storage of olives ( Olea europaea) under CO 2 atmosphere: Effect on anthocyanins, phenolics, sensory attributes and in vitro antioxidant properties

Green, unripe olives were subjected to post-harvest treatment under a CO 2 atmosphere for a period of 12 days. The total polyphenol (TP), total flavonoid (TFd) and total anthocyanin (TA) contents, along with the antioxidant and sensory characteristics, were continuously monitored during the treatment on a 24 h-interval basis, in order to identify possible changes in the quality of olives related mainly to changes in the polyphenolic contents. The storage of olives under CO 2 atmosphere resulted in pronounced increases in TP and TF contents, mainly within the first 3-5 days, but TA exhibited a different pattern of evolution. Furthermore, storage under CO 2 contributed to flavour appearance with the development of fruity/floral notes, and reduced bitterness. The in vitro antioxidant properties of the CO 2-treated sample showed notable increases compared with the sample stored under regular atmospheric conditions. It was concluded that storage of olives under a CO 2 atmosphere resulted in the appearance of desired sensory attributes, by decreasing bitterness and developing aroma and colour, and the functional (antioxidant) properties were improved. This approach may be used as an alternative, chemical-free means of table olive debittering.

Quantification of beta-trace protein and detection of transferrin isoforms in mixtures of cerebrospinal fluid and blood serum as models of rhinorrhea and otorrhea diagnosis.

Different mixtures from a serum pool and a cerebrospinal fluid (CSF) pool were used as models to study CSF contamination in secretions by determining two CSF specific proteins: beta-trace protein (beta-TP) and the asialo-transferrin (a-Tf) band which was detected by isoelectric focusing (IEF) with Tf specific immunofixation. Beta-TP and total Tf were measured immunonephelometrically. Secretion/serum ratios of beta-TP content > 2.0 indicated CSF contaminations with > or = 5% (v/v) CSF; this was confirmed by detecting the a-Tf band by IEF. Reliable a-Tf bands were only revealed with secretion/serum rations of Tf contents < 0.1, indicating an interference of major sialo-Tf fractions with the a-TF band detection in the sample. For CSF detection in rhinorrhea and otorrhea, complementary use of beta-TP assay and a-Tf assay is recommended. Preanalytically, dilution or concentration of the sample as well as denaturation of Tf and beta-TP should be prevented by optimizing sample collection.

Transferrin in After-Cataract and as a Survival Factor for Lens Epithelium

The Fe-transport protein, transferrin (Tf), is synthesized and secreted by whole lenses and cultured lens epithelial cells. Because of Tf's central role in cell growth and proliferation, its participation in lens cell proliferation following cataract extraction was explored using a rabbit model of after-cataract. Varying amounts of the central anterior lens capsule were removed (0, 35, or 80%) following extraction of the lens. The Tf content of and secretion by after-cataract lens capsular sacs containing regenerated lens tissue was determined ex vivo at 0, 3, 5, 7 and 9 weeks post-surgery. In all cases Tf content of and secretion by the lens sacs was higher than that of their contralateral controls (whole lenses). Tf secretion was up to 5-fold higher and metabolic labeling studies indicated secretion of newly synthesized Tf. The sacs contained up to 10 times the concentration of Tf as the control lenses. Human lens after-cataract capsular bags also secreted Tf.The function of Tf as a survival factor was tested on cultured lens epithelial cells. Cells cultured in serum-free medium had a survival rate of only 20–34% if the medium was changed each day. If the medium was never changed during this period, the survival rate was 43–52%, suggesting secretion of essential growth factors by these cells. Addition of 200 μg ml-1Tf to the medium during each daily change increased survival to levels attained when the medium was not changed. Addition of Tf antibodies to the culture medium during each daily change decreased cell survival to 14%. Apparently Tf acts as a survival factor for lens epithelia and its synthesis is up-regulated in after-cataract lens sacs. These factors suggest that Tf may play an important role in the pathogenesis of lens epithelial cell proliferation and after-cataract formation following cataract surgery.

Androgens in seminal plasma: markers of the surface epithelium of the male reproductive tract

Androgens in seminal plasma have been investigated in relation to other markers of the male reproductive system. Testosterone and dihydrotestosterone (DHT) were measured by radioimmunoassay after paper chromatography in 104 semen samples. Concentrations of testosterone and DHT were significantly lower in samples from patients with abnormal sperm characteristics than in men with normozoospermia. Both testosterone and DHT were correlated significantly with sperm concentration (r = 0.40, p < 0.001; r = 0.41, p < 0.001, respectively), motile sperm concentration (r = 0.35, p < 0.01; r = 0.35, p < 0.01) and ATP concentration (r = 0.59, p < 0.001; r = 0.45, p < 0.001). In addition, the total amounts of testosterone and DHT were correlated with total activity of alpha-glucosidase (r = 0.49, p < 0.001; r = 0.58, p < 0.01), and gamma-glutamyltransferase (r = 0.49, p < 0.001; r = 0.48, p < 0.001) in seminal plasma. Transferrin (Tf) concentration in seminal plasma was significantly lower in samples without spermatozoa, and total Tf content was lower in oligozoospermic samples (p < 0.05). Testosterone and DHT were correlated positively with Tf levels in seminal plasma (r = 0.48, p < 0.0001; r = 0.78, p < 0.0001 respectively). Testosterone, DHT and Tf in seminal plasma were higher in the first than in the second fraction of split ejaculates, and DHT, but not testosterone, increased significantly in each of six patients treated with tamoxifen. Testosterone, DHT, and Tf in seminal plasma were not correlated with serum concentrations of LH, FSH, testosterone or prolactin, but the DHT : T ratio in seminal plasma was correlated with serum LH (r = 0.36, p < 0.05). It is concluded that the concentration of androgens in seminal plasma is related predominantly to the activity of the seminiferous epithelium and, to a lesser extent, to the function of the accessory sex glands.

Evidence for nonvectorial, retrograde transferrin trafficking in the early endosomes of HEp2 cells.

We have previously characterized the trafficking of transferrin (Tf) through HEp2 human carcinoma cells (Ghosh, R. N., D. L. Gelman, and F. R. Maxfield, 1994. J. Cell Sci. 107:2177-2189). Early endosomes in these cells are comprised of both sorting endosomes and recycling compartments, which are distinct separate compartments. Endocytosed Tf initially appears in punctate sorting endosomes that also contain recently endocytosed LDL. After short loading pulses, Tf rapidly sorts from LDL with first-order kinetics (t1/2 approximately 2.5 min), and it enters the recycling compartment before leaving the cell (t1/2 approximately 7 min). Here, we report a second, slower rate for Tf to leave sorting endosomes after HEp2 cells were labeled to steady state with fluorescein Tf instead of the brief pulse used previously. We determined this rate using digital image analysis to measure the Tf content of sorting endosomes that also contained LDL. With an 11-min chase, the Tf in sorting endosomes was 24% of steady-state value. This was in excess of the amount expected (5% of steady state) from the rate of Tf exit after short filling pulses. The excess could not be accounted for by reinternalization of recycled cell surface Tf, implying that either some Tf was retained in sorting endosomes, or that Tf was delivered back to the sorting endosomes from the recycling compartment. The former is unlikely since nearly all sorting endosomes contain detectable Tf after an 11-min chase, even though more than one third of the sorting endosomes were formed during the chase time. Furthermore, while observing living cells by confocal microscopy, we saw vesicle movements that appeared to be fluorescent Tf returning from recycling compartments to sorting endosomes. The slow rate of exit after steady-state labeling was similar to the Tf exit rate from the cell, suggesting an equilibration of Tf throughout the early endosomal system by this retrograde pathway. This retrograde traffic may be important for delivering molecules from the recycling compartment, which is a long-lived organelle, to sorting endosomes, which are transient.