Fluorescein-5-thiosemicarbazide Research Articles

FTSC-Labeling Coupled with 2DE-LC-MS/MS Analysis of Complex Protein Mixtures for Identification and Relative Quantification of Tissue Carbonylome.

Carbonylation is a nonenzymatic irreversible posttranslational protein modification and the main hallmark of protein oxidative damage. Elevated levels of protein carbonyl groups have been detected in age-related and metabolic diseases such as obesity, diabetes, Alzheimer, Parkinson, and several other oxidative stress-related maladies. Interestingly, many studies have shown that only a subset of proteins is carbonylated under the conditions of oxidative stress, demonstrating that carbonylation is a highly selective process. As a consequence, identifying and quantifying the disease-induced changes on a certain carbonylome are crucial to understanding the etiology and progression of numerous diseases and then designing adequate prevention/palliation strategies. However, the low abundance of carbonylated proteins in vivo, the enormous diversity of reactive species, and their relative lability make the analysis of carbonylated proteins a challenging task for redox proteomic technology. Therefore, we present a proteomic approach based on the labeling of carbonyls formed in vivo on proteins using the fluorescein 5-thiosemicarbazide (FTSC) tag to detect the subset of carbonylated proteins among a complex mixture of proteins regardless of the nature of carbonyl adduct, isolation and relative quantification of carbonylated proteins in 2D gel electrophoresis, and protein identification by LC-MS/MS analysis. This method has been successfully used for the evaluation of in vivo protein carbonylation in very diverse animal tissues (plasma, liver, kidney, skeletal muscle, and adipose tissue) and species (from fish to mammalian) and has also been applied in different research fields (from food technology to nutrition), demonstrating its robustness and reliability.

Modulation of the Liver Protein Carbonylome by the Combined Effect of Marine Omega-3 PUFAs and Grape Polyphenols Supplementation in Rats Fed an Obesogenic High Fat and High Sucrose Diet.

Diet-induced obesity has been linked to metabolic disorders such as cardiovascular diseases and type 2 diabetes. A factor linking diet to metabolic disorders is oxidative stress, which can damage biomolecules, especially proteins. The present study was designed to investigate the effect of marine omega-3 polyunsaturated fatty acids (PUFAs) (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and their combination with grape seed polyphenols (GSE) on carbonyl-modified proteins from plasma and liver in Wistar Kyoto rats fed an obesogenic diet, namely high-fat and high-sucrose (HFHS) diet. A proteomics approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labelling of protein carbonyls, visualization of FTSC-labelled protein on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Results showed the efficiency of the combination of both bioactive compounds in decreasing the total protein carbonylation induced by HFHS diet in both plasma and liver. The analysis of carbonylated protein targets, also referred to as the ‘carbonylome’, revealed an individual response of liver proteins to supplements and a modulatory effect on specific metabolic pathways and processes due to, at least in part, the control exerted by the supplements on the liver protein carbonylome. This investigation highlights the additive effect of dietary fish oils and grape seed polyphenols in modulating in vivo oxidative damage of proteins induced by the consumption of HFHS diets.

Sensitive determination of aldehyde metabolites in exhaled breath condensate using capillary electrophoresis with laser-induced fluorescence detection.

A novel capillary electrophoresis with laser-induced fluorescence detection method has been developed for the analysis of aldehyde metabolism biomarkers for oxidative stress in exhaled breath condensate (EBC), and fluorescein 5-thiosemicarbazide was used as a derivatization reagent. In a simple capillary zone electrophoresis mode, ten low molecular weight aldehydes (LMWAs) could be well separated within 30min. The reaction efficiency was doubled by increasing sample solution pH and magnetic stirring, and the LODs of this method reached 0.16-3.4nM (S/N = 3). Acceptable recoveries (82.1-115%) were obtained for EBC samples, and the RSD data were within 7.9%. This developed method has been applied for the analyses of EBC samples and evaluation of the correlation between smoking and the contents of aldehyde metabolites in EBC. Due to no need of buffer additives and sample preconcentration, this proposed method may provide an appealing alternative for the trace analyses of LMWAs in noninvasive biofluids. Graphical abstract ᅟ.

Protein carbonyl determination by a rhodamine B hydrazide-based fluorometric assay

A new fluorometric assay is presented for the ultrasensitive quantification of total protein carbonyls, and is based on their specific reaction with rhodamine B hydrazide (RBH), and the production of a protein carbonyl-RBH hydrazone the fluorescence of which (at ex/em 560/585 nm) is greatly enhanced by guanidine-HCl. Compared to the fluorescein-5-thiosemicarbazide (FTC)-based fluorometric assay, the RBH assay uses a 24-fold shorter reaction incubation time (1 h) and at least 1000-fold lower protein quantity (2.5 µg), and produces very reliable data that were verified by extensive standardization experiments. The protein carbonyl group detection sensitivity limit of the RBH assay, based on its standard curve, can be as low as 0.4 pmol, and even lower. Counting the very low protein limit of the RBH assay, its cumulative and functional sensitivity is 8500- and 800-fold higher than the corresponding ones for the FTC assay. Neither heme proteins hemoglobin and cytochrome c nor DNA interfere with the RBH assay.

Antiaging and antioxidant effects of topical autophagy activator: A randomized, placebo-controlled, double-blinded study.

Recently, potential roles of autophagy in skin homeostasis received many interests. But, little has been reported for the potential antiaging effects of autophagy activator. With the newly synthesized autophagy activator, heptasodium hexacarboxymethyl dipeptide-12 (Aquatide™) invitro and clinical efficacy of the topical autophagy activator as an antiaging cosmeceutical ingredient was evaluated. Antioxidant effect of Aquatide™ was evaluated by radical scavenging assay. In vitro effect was assessed by measuring the cytotoxicity of hydrogen peroxide in cultured normal human epidermal keratinocytes. Clinical evaluation was performed by a randomized, placebo-controlled, double-blinded study. Antioxidant efficacy was observed by measuring the carbonylated proteins in stratum corneum (SC) by fluorescein-5-thiosemicarbazide (FTZ) staining. Radical scavenging effects of Aquatide were observed with the ABTS assay, and significant reduction in hydrogen peroxide-induced cytotoxicity was observed in Aquatide™-treated cells. Autophagy inhibitor treatment abrogated cytoprotective effects of Aquatide™. In a clinical study, statistically significant increase in skin elasticity was observed after 4 and 8weeks. Quantitative analysis of carbonylated proteins in SC also showed significant reduction in Aquatide™-treated group, which is consistent with the invitro data. These results suggest that autophagy plays important roles in antioxidant system and aging process in skin, and topical autophagy activators can be potential cosmeceutical ingredients for skin antiaging.

Fabrication of fluorescent labeled ginseng polysaccharide nanoparticles for bioimaging and their immunomodulatory activity on macrophage cell lines

Polysaccharides are a major active component of American ginseng root showing various biological activities including anti-carcinogenic, anti-aging, immunostimulatory and antioxidant effects. Although their biological activity has been reported by several groups, no research has explored their cellular uptake and biodistribution, owing to the lack of suitable detection techniques in living cells. This work examines a novel, simple and efficient fluorescent labeling procedure of ginseng polysaccharides (PS), in order to examine their cellular distribution using confocal microscopy. This procedure utilized a one-pot strategy with fluorescein-5-thiosemicarbazide (FTSC) to introduce a thiosemicarbazide group onto the aldehyde group at the reducing saccharide end to form a stable amino derivative through reductive amination. This polysaccharide-FTSC derivative was then characterized by GPC, UV, FTIR, photoluminescence and fluorescence microscopy to confirm attachment and any structural changes. The results demonstrated that the labeled ginseng PS nanostructure showed high fluorescence with minimal changes in PS molecular weight. The labeled PS exhibited almost no cytotoxicity effect against tumor induced macrophage cell lines (RAW 264.7) while retaining high immunostimulating activity similar to the non-labeled ginseng PS. Therefore, the developed approach provides a convenient and highly efficient fluorescent labeling procedure for understanding the mechanism of ginseng PS uptake in macrophage cell lines.

Protein carbonylation during natural leaf senescence in winter wheat, as probed by fluorescein-5-thiosemicarbazide.

Leaf senescence is characterised by a massive degradation of proteins in order to recycle nitrogen to other parts of the plant, such as younger leaves or developing grain/seed. Protein degradation during leaf senescence is a highly regulated process and it is suggested that proteins to be degraded are marked by an oxidative modification (carbonylation) that makes them more susceptible to proteolysis. However, there is as yet no evidence of an increase in protein carbonylation level during natural leaf senescence. The aim of our study was thus to monitor protein carbonylation level during the process of natural senescence in the flag leaf of field-grown winter wheat plants. For this purpose, we adapted a fluorescence-based method using fluorescein-5-thiosemicarbazide (FTC) as a probe for detecting protein carbonyl derivatives. As used for the first time on plant material, this method allowed the detection of both quantitative and qualitative modifications in protein carbonyl levels during the last stages of wheat flag leaf development. The method described herein represents a convenient, sensitive and reproducible alternative to the commonly used 2,4-dinitrophenylhydrazine (DNPH)-based method. In addition, our analysis revealed changes in protein carbonylation level during leaf development that were associated with qualitative changes in protein abundance and carbonylation profiles. In the senescing flag leaf, protein carbonylation increased concomitantly with a stimulation of endoproteolytic activity and a decrease in protein content, which supports the suggested relationship between protein oxidation and proteolysis during natural leaf senescence.

Fluorescein-5-thiosemicarbazide (FTSC) labeling for fluorescent imaging of pectin-derived oligogalacturonic acid transported in living cells by confocal microscopy

Confocal microscopy is a powerful and effective tool for in vivo elucidation of the molecular basis of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrate with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. However, current labeling methods have proved unsuitable for functional pectin-derived oligogalacturonic acid due to the low fluorescence intensity and/or the formation of lactone byproducts. Here, fluorescein-5-thiosemicarbazide (FTSC) was employed for fluorescent labeling of pectin-derived oligogalacturonic acid under mild and neutral reaction conditions to avoid the generation of lactone byproducts. ESI–MS and LC–MS data displayed that pectin-derived pentagalacturonic acid, GalA5, could be quantitatively labeled with FTSC to afford thiosemicarbazones, without any lactone byproducts yielded. Moreover, GalA5-FTSC thiosemicarbazone showed good stability and desirable fluorescence characteristics and exhibited rather low levels of cytotoxicity against sensitive rat thymus cells. Additionally, we have achieved successful fluorescent imaging of GalA5-FTSC thiosemicarbazones transported in living SW-620 and HepG2 cells. The assay illustrates methodological significance in structure–activity elucidation study of functional carbohydrates especially acid oligo-/polysaccharides.

An efficient method for selectively imaging and quantifying in situ the expression of sialylated glycoproteins on living cells

A simple and efficient method for selectively imaging and monitoring in situ the expression of sialylated glycoproteins on living cells has been developed. Treating living cells by mild periodate oxidation to selectively generate aldehydes on sialylated glycoproteins, followed by direct labeling of aldehydes with a commercially available fluorescent tag, fluorescein-5-thiosemicarbazide (FTSC), allows in situ imaging and quantification of sialylated glycoproteins on living cells. Under optimum reaction conditions, the periodate oxidation-based FTSC ligation (PF) strategy could be completed within 40 min. The cells undergoing the PF assay revealed a 91% viability and a fairly high-level of metabolic activity. Compared with current labeling methods, the PF assay proved to be a simpler and faster means of imaging sialylated glycoproteins on living cells. The PF assay has been successfully applied to imaging the location and quantification of the abundance of sialylated glycoproteins on tumor and normal cells. Our results demonstrated the methodological significance in clinical diagnosis and functional elucidation studies.

Reduced protein oxidation in Wistar rats supplemented with marine ω3 PUFAs

The potential effects of various dietary eicosapentaenoic acid (EPA; 20:5) and docosahexaenoic acid (DHA; 22:6) ratios (1:1, 2:1, and 1:2, respectively) on protein redox states from plasma, kidney, skeletal muscle, and liver were investigated in Wistar rats. Dietary fish oil groups were compared with animals fed soybean and linseed oils, vegetable oils enriched in ω6 linoleic acid (LA; 18:2) and ω3 α-linolenic acid (ALA; 18:3), respectively. Fish oil treatments were effective at reducing the level of total fatty acids in plasma and enriching the plasmatic free fatty acid fraction and erythrocyte membranes in EPA and DHA. A proteomic approach consisting of fluorescein 5-thiosemicarbazide (FTSC) labeling of protein carbonyls, FTSC intensity visualization on 1-DE or 2-DE gels, and protein identification by MS/MS was used for the protein oxidation assessment. Albumin was found to be the most carbonylated protein in plasma for all dietary groups, and its oxidation level was significantly modulated by dietary interventions. Supplementation with an equal EPA:DHA ratio (1:1) showed the lowest oxidation score for plasma albumin, followed in increasing order of carbonylation by 1:2 <2:1 ≈ linseed < soybean. Oxidation patterns of myofibrillar skeletal muscle proteins and cytosolic proteins from kidney and liver also indicated a protective effect on proteins for the fish oil treatments, the 1:1 ratio exhibiting the lowest protein oxidation scores. The effect of fish oil treatments at reducing carbonylation on specific proteins from plasma (albumin), skeletal muscle (actin), and liver (albumin, argininosuccinate synthetase, 3-α-hydroxysteroid dehydrogenase) was remarkable. This investigation highlights the efficiency of dietary fish oil at reducing in vivo oxidative damage of proteins compared to oils enriched in the 18-carbon polyunsaturated fatty acids ω3 ALA and ω6 LA, and such antioxidant activity may differ among different fish oil sources because of variations in EPA/DHA content.

Proteomic evaluation of myofibrillar carbonylation in chilled fish mince and its inhibition by catechin

The present study investigates the susceptibility of individual myofibrillar proteins from mackerel (Scomber scombrus) mince to undergo carbonylation reactions during chilled storage, and the antioxidant capacity of (+)-catechin to prevent oxidative processes of proteins. The carbonylation of each particular protein was quantified by combining the labelling of protein carbonyls by fluorescein-5-thiosemicarbazide (FTSC) with 1-D or 2-D gel electrophoresis. Alpha skeletal actin, glycogen phosphorylase, unnamed protein product (UNP) similar to enolase, pyruvate kinase, isoforms of creatine kinase, aldolase A and an isoform of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) showed elevated oxidation in chilled non-supplemented mince. Myosin heavy chain (MHC) was not carbonylated in chilled muscle, but an extensive MHC degradation was observed in those samples. The supplementation of catechin reduced protein oxidation and lipid oxidation in a concentration-dependent manner: control>25>100≈200ppm. Therefore, the highest catechin concentrations (100 and 200ppm) exhibited the strongest antioxidant activity. Catechin (200ppm) reduced significantly carbonylation of protein spots identified as glycogen phosphorylase, pyruvate kinase muscle isozyme, isoforms of creatine kinase. Conversely, catechin was ineffective to inhibit the oxidation of actin and UNP similar to enolase. These results draw attention to the inefficiency of catechin to prevent actin oxidation, in contrast to the extremely high efficiency of catechin in inhibiting oxidation of lipids and other proteins.

Fluorescein-5-thiosemicarbazide as a probe for directly imaging of mucin-type O-linked glycoprotein within living cells

Mucin-type O-linked glycoproteins are known for regulating many aspects of cell activity but remains a challenge to detect under physiological conditions which is due to the diversity of O-glycosylation and the lack of universal method. Here a direct labeling strategy for in situ visualizing of mucin-type O-linked glycoproteins on living cells has been developed. The strategy utilizes the combination of metabolic engineering and chemical probing technologies. Treating cells with an unnatural sugar, 2-keto Ac(4)GalNAc analogue (2-keto isostere of GalNAc) to generate keto groups upon cells, followed by chemoselective ligation of keto groups on cells with a fluorescent tag, fluorescein-5-thiosemicarbazide (FTSC), provides a promising platform to probing mucin-type O-glycosylation on living cells. The FTSC conjugates illustrated very similar fluorescent spectra as FITC, a fluorescent tag widely used in proteomics, indicating good compatibility with commonly used fluorescent equipments. The established method eliminated the need of an additional fluorescent amplification step. Cells after being treated with the method maintained a rather high level of viability of 84.3%. Finally, the assay has been successfully applied to image the expression of mucin-type O-linked glycoproteins within CHO and HeLa cells.

One-pot fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide for imaging polysaccharides transported in living cells

A simple and efficient procedure for the fluorescent labeling of saccharides is a prerequisite step for imaging the transport of polysaccharides in living cells. We report a one-pot strategy for the fluorescent labeling of saccharides with fluorescein-5-thiosemicarbazide (FTSC), which introduces the thiosemicarbazide group of FTSC to the aldehyde group at the reducing end of saccharides to form stable amino derivatives via reductive amination. The Glc-FTSC derivative was characterized by HPLC–MS, HRESIMS and NMR spectroscopy. Saccharides were quantitatively labeled with FTSC at 75 °C for 1 h under optimum reaction conditions. Fluorescence studies illustrated that the conjugation of FTSC to saccharides did not change its florescence properties ( λ ex = 495 nm, λ em = 517 nm), presenting desirable compatibility with commonly used fluorescence equipment. Polysaccharide AAG-FTSC derivatives exhibited rather low levels of cytotoxicity against rat thymus cells, and the fluorescent labeling procedure had slight impact on their anti-tumor activity. Results indicate that the assay neither introduces discernible cytotoxicity against living cells nor obviously alters the functional activities of polysaccharides, and provides a convenient, highly efficient fluorescent labeling approach for imaging the transport of polysaccharides in living cells.

Fish Proteins as Targets of Ferrous-Catalyzed Oxidation: Identification of Protein Carbonyls by Fluorescent Labeling on Two-Dimensional Gels and MALDI-TOF/TOF Mass Spectrometry

Protein oxidation in fish meat is considered to affect negatively the muscle texture. An important source of free radicals taking part in this process is Fenton's reaction dependent on ferrous ions present in the tissue. The aim of this study was to investigate the susceptibility of cod muscle proteins in sarcoplasmic and myofibril fractions to in vitro metal-catalyzed oxidation and to point out protein candidates that might play a major role in the deterioration of fish quality. Extracted control proteins and proteins subjected to free radicals generated by Fe(II)/ascorbate mixture were labeled with fluorescein-5-thiosemicarbazide (FTSC) to tag carbonyl groups and separated by two-dimensional gel electrophoresis. Consecutive visualization of protein carbonyl levels by capturing the FTSC signal and total protein levels by capturing the SyproRuby staining signal allowed us to quantify the relative change in protein carbonyl levels corrected for changes in protein content. Proteins were identified using MALDI-TOF/TOF mass spectrometry and homology-based searches. The results show that freshly extracted cod muscle proteins exhibit a detectable carbonylation background and that the incubation with Fe(II)/ascorbate triggers a further oxidation of both sarcoplasmic and myofibril proteins. Different proteins exhibited various degrees of sensitivity to oxidation processes. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), nucleoside diphosphate kinase B (NDK), triosephosphate isomerase, phosphoglycerate mutase, lactate dehydrogenase, creatine kinase, and enolase were the sarcoplasmic proteins most vulnerable to ferrous-catalyzed oxidation. Moreover, NDK, phosphoglycerate mutase, and GAPDH were identified in several spots differing by their pI, and those forms showed different susceptibilities to metal-catalyzed oxidation, indicating that post-translational modifications may change the resistance of proteins to oxidative damage. The Fe(II)/ascorbate treatment significantly increased carbonylation of important structural proteins in fish muscle, mainly actin and myosin, and degradation products of those proteins were observed, some of them exhibiting increased carbonylation levels.

A novel clean-up method for urine analysis of low-molecular mass aldehydes by capillary electrophoresis with laser-induced fluorescence detection

A rapid clean-up method using ultra-filtration was developed to remove sample matrix in the determination of low-molecular mass aldehydes in human urine. The ensuing filtrate was derivatized with fluorescein 5-thiosemicarbazide and the labelled aldehydes determined by capillary zone electrophoresis with laser-induced fluorescence detection. Practical aspects related to the effect of the urine sample matrix on the label chemistry and the electrophoretic separation showed that the urine samples must be diluted 20-fold after their ultra-filtration. By using synthetic urine, linear ranges were established in the range of 15–5000 μg/l with limits of detection between 4.5 and 9 μg/l. The intra- and inter-assay precision (relative standard deviation, %) of the aldehydes ranged from 4.1% to 8.4% and 6.1%–9.6%, respectively, and the average specific uncertainty was 149 ± 12 ng. The average recoveries performed on two levels by enriching synthetic urine samples ranged between 94% and 100%. Finally, the proposed method was applied to check low-molecular mass aldehydes in the human urine of a female volunteer to obtain information about the risk in her exposure to these chemicals in the workplace.

Analysis of low‐molecular mass aldehydes in drinking waters through capillary electrophoresis with laser‐induced fluorescence detection

The potential of CZE with LIF detection in the separation and determination of low-molecular mass aldehydes involving precolumn derivatization with fluorescein 5-thiosemicarbazide was investigated. Different variables that affect derivatization (pH, fluorescein 5-thiosemicarbazide concentration, time and temperature) and separation (pH and concentration of the BGE, kind and concentration of surfactants at levels higher and lower than CMC, and applied voltage) were studied. The separation was conducted within 16 min by using borate buffer (60 mM; pH 10) with 10 microM polyethylene glycol tert-octylphenyl ether as modifier. Good linearity relationships (correlation coefficients ranged from 0.9978 to 0.9994 for aldehydes) were obtained between the peak areas and concentration of the analytes (0.5-100 microg/L). The LODs for aldehydes were achieved at submicrogram-per-liter level (0.15-0.35 microg/L), which indicated that the proposed method surpassed other electrophoretic alternatives in terms of LOD, in many cases even at ca. 1000-fold. The inter-day precision (RSD, %) of the aldehydes ranged from 5.2 to 8.3%. Finally, the method was successfully applied to bottled drinking-water samples, and the aldehydes were readily detected at 0.6-4.4 microg/L levels with average recoveries ranging from 99.1 to 103.5%.

Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5

Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9-13-nucleotide window. This sequence is remarkably abundant in the 3'-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation.

A simple and non‐invasive visualization for assessment of carbonylated protein in the stratum corneum

Stratum corneum (SC) is the interface of body and environment and is continuously exposed to oxidative stress, resulting in oxidative modification of proteins. Consequent carbonylated proteins (CPs) have so far been labeled with 2,4-dinitrophenyl (DNP) hydrazine and subsequently detected with anti-DNP antibody. We developed a simpler, non-invasive method to assess CP level in the SC and applied it to following research. SC was collected by adhesive tape stripping and its carbonyl groups were labeled with fluorescein-5-thiosemicarbazide (FTZ). The staining image was observed by fluorescence microscopy and the average fluorescence intensity of the SC extracted from the image was calculated as stratum corneum carbonylated protein (SCCP) level. By reaction with FTZ, carbonyl groups in the SC could be detected easily. Relatively strong fluorescence was observed in exfoliating scales. Lipid removal from the SC in vitro or in vivo did not show any change in fluorescence intensity, suggesting that carbonyl groups were mainly derived from proteins, not from lipids. SCCP level was higher in the upper layer than the lower layer, and higher in the cheek (sun-exposed) than the inside of upper arm (unexposed), positively correlated with age especially in male cheek, positively correlated with transepidermal water loss, negatively correlated with water content, and showed a subtle correlation with sebum level. On the other hand, SC collected by cyanoacrylate resin and labeled with FTZ revealed strong fluorescence around the pores in the cheek and on the grooves in the upper arm, suggesting the role of sebum in the generation of SCCP. SCCP was assessed in a simple and non-invasive method, and suggested to be a novel indicator that reflects some aspect of skin condition.

Detection of protein carbonyls in aging liver tissue: A fluorescence-based proteomic approach

Protein carbonyls are commonly used as a marker of protein oxidation in cells and tissues. Currently, 2,4-dinitrophenyl hydrazine (DNPH) is widely used (spectrophotometrically or immunologically) to quantify the global carbonyl levels in proteins and identify the specific proteins that are carbonylated. We have adapted a fluorescence-based approach using fluorescein-5-thiosemicarbazide (FTC), to quantify the global protein carbonyls as well as the carbonyl levels on individual proteins in the proteome. Protein carbonyls generated in vitro were quantified by labeling the oxidized proteins with FTC followed by separating the FTC-labeled protein from free probe by gel electrophoresis. The reaction of FTC with protein carbonyls was found to be specific for carbonyl groups. We measured protein carbonyl levels in the livers of young and old mice, and found a significant increase (two-fold) in the global protein carbonyl levels with age. Using 2-D gel electrophoresis, we used this assay to directly measure the changes in protein carbonyl levels in specific proteins. We identified 12 proteins showing a greater than two-fold increase in carbonyl content (pmoles of carbonyls/μg of protein) with age. Most of the 12 proteins contained transition metal binding sites, with Cu/Zn superoxide dismutase containing the highest molar ratio of carbonyls in old mice. Thus, the fluorescence-based assay gives investigators the ability to identify potential target proteins that become oxidized under different pathological and physiological conditions.

Exploration of RNA 3' terminal locations in rat ribosome.

The locations of the 3' ends of RNAs in rat ribosome were studied by a fluorescence-labeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3' ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe - fluorescein 5-thiosemicarbazide (FTSC), indicating that the 3' termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3' terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3' end of 5 S RNA was located on the interface of two subunits. However, no fluorescence-labeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3' end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3' ends of ribosomal RNAs including oxidation with NaIO4, reduction with KBH4 and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3' ends of RNAs were not involved in the translation activity of ribosome.