Dye Front Research Articles

USING N-OVEL COATED SAND AS REACTIVE BED IN PERMEABLE BARRIER FOR ELIMINATION OF METHYL ORANGE DYE FROM GROUNDWATER*

This study investigates the efficacy of sand coated with calcium / aluminum (Ca/Al)-layered double hydroxide (LDH) in the elimination of methyl orange dye from simulated groundwater using method of permeable reactive barrier. For green (sustainable) manufacturing of this sand, aluminum prepared from dissolution of alum (cheap coagulant) can be interacted chemically with calcium extracted from cement kiln dust byproduct to create nanoparticles of Ca and Al that precipitate on the filter sand. Sorption measurements for interaction of methyl orange dye and coated sand was well formulated by Langmuir model with highest value of correlation (determination coefficient= 0.999) and lowest errors (sum of squared errors= 0.0122). Also, the maximum capacity of adsorption was valued of 0.9453 mg/g and the sorption curve can classify as “favorable” type. According to desorption measurements, little percentage of dye (0.91%) can desorb from exhausted coated sand through washing with water due to strength of bonding between sorbent and dye molecules. Also, the exhausted sorbent can regenerate with efficiency not less than 85% after eight of regeneration cycles. Finally, measurements of breakthrough curves in the continuous mode operation using column setup proved that the propagation of dye front (and consequently the longevity of barrier) would increase significantly with thicker barrier and lowest values of influent concentration and flow rate. COMSOL (computer solution) package can use effectively in the simulation of these curves.

Characterization of Effective Diffusion within Viscoelastic Fluids with Elastic Instabilities

We considered effective diffusion, characterized by magnitudes of effective diffusion coefficients, in order to quantify mass transport due to the onset and development of elastic instabilities. Effective diffusion coefficient magnitudes were determined using different analytic approaches, as they were applied to tracked visualizations of fluorescein dye front variations, as circumferential advection was imposed upon a flow environment produced using a rotating Couette flow arrangement. Effective diffusion coefficient results were provided for a range of flow shear rates, which were produced using different Couette flow rotation speeds and two different flow environment fluid depths. To visualize the flow behavior within the rotating Couette flow environment, minute amounts of fluorescein dye were injected into the center of the flow container using a syringe pump. This dye was then redistributed within the flow by radial diffusion only when no disk rotation was used, and by radial diffusion and by circumferential advection when disk rotation was present. Associated effective diffusion coefficient values, for the latter arrangement, were compared to coefficients values with no disk rotation, which were due to molecular diffusion alone, in order to quantify enhancements due to elastic instabilities. Experiments were conducted using viscoelastic fluids, which were based on a 65% sucrose solution, with different polymer concentrations ranging from 0 ppm to 300 ppm. Associated Reynolds numbers based on the fluid depth and radially averaged maximum flow velocity ranged from 0.00 to 0.5. The resulting effective diffusion coefficient values for different flow shear rates and polymer concentrations quantified the onset of elastic instabilities, as well as significant and dramatic changes to local mass transport magnitudes, which are associated with the further development of elastic instabilities.

PULSATILE FILLING OF DILATED CHOROIDAL VESSELS IN MACULAR WATERSHED ZONES.

To describe pulsatile filling of dilated choroidal veins in the watershed zones and propose an alteration in choroidal perfusion pressure. Retrospective review of original and digital subtraction indocyanine green angiography. We observed pulsating blood flow within choroidal vein segments in the posterior pole in 14 eyes (diagnosis of polypoidal choroidal vasculopathy, central serous chorioretinopathy, or neovascular age-related macular degeneration). Pulsating dye front was observed in single or multiple large choroidal vein(s) in a location that is ordinarily a watershed zone between the segmental areas of venous drainage, and vessels proximal and distal were often dilated. The pulsatile venous segments filled more slowly than the neighboring veins. In digital subtraction indocyanine green angiography, the dye front advanced in an incremental fashion or oscillated in a back-and-forth manner during several cardiac cycles during the filling of these larger choroidal veins. With indocyanine green angiography, we observed dilated choroidal veins that violated the macula watershed zone, localized bulbous dilations, and arteriole-over-vein crossings with apparent compression. These novel observations suggest the pressure gradient for flow in the affected veins varied from low gradients when the filling was slow to high gradients when the filling was faster. The vessels violated the physiological watershed zone and seem to function as anastomoses between the ordinarily segmented venous drainage of the choroid. The dilated segments may result in pooling of venous blood as part of venous outflow abnormalities that may be operative in these diseases.

Correlation of choriocapillaris hemodynamic data from dynamic indocyanine green and optical coherence tomography angiography

To investigate the correlation between posterior pole choroidal blood flow evaluated with digital subtraction indocyanine green angiography and enface optical coherence tomography angiography (OCTA). Imaging in animal study. The anatomy of 2 cynomogulus monkeys was studied. Each monkey was given a 0.75 mg/kg injection of indocyanine green in the saphenous vein. The dynamic angiographic filling sequence was recorded at 15 frames per second using the Heidelberg Spectralis. After image registration, sequential frame subtraction was used to image the dye front moving through the choroid. The OCTA was obtained by frame averaging nine separate choriocapillaris slab flow images obtained from the Zeiss Plex Elite 9000. Posterior pole choriocapillaris filling pattern in relation to the choriocapillaris anatomy as imaged by OCTA. In the posterior pole, the choriocapillaris fills in the pattern of discrete units with variable sizes and shapes. The cycle of dye filling begins in the peripapillary area and progresses toward the periphery in a wavelike manner. This filling pattern repeats in a cyclical manner, consistent with the cardiac cycle. OCTA shows a uniform mesh of vessels. While OCTA shows a uniform meshwork appearance of the choriocapillaris, the dynamic dye angiography suggests an irregular configuration of functional units partitioned by pressure gradients as opposed to structural boundaries. Disturbance of local perfusion pressure within choroidal vasculature may result in abnormal flow patterns, which could be evaluated in the clinic using commercially available equipment.

Correction factor for measuring mean overland flow velocities on stony surfaces under rainfall using dye tracer

Dye tracing is a common and simple method for measuring surface velocity of overland flow. However, a correction factor is needed to convert the velocity of the dye front to mean velocity. Selecting the appropriate correction factor for particular conditions is complex and its behavior has not been well studied in sheet flow under rainfall on actively eroding surfaces. A series of simulated rainfalls were conducted on a 2 m by 6 m plot with gravelly sandy loam soil. The plot was progressively eroded to examine a wide range of hydraulic conditions. A total of 178 velocities (maximum and mean) were measured using electrolyte and dye tracers over three travel distances (1.65, 3.5, and 5.8 m), and three slope gradients (5%, 12% and 20%). Discharge, sediment yield, random roughness, rock cover, and a range of hydraulic variables were also determined. Mean flow velocity was derived from the centroid of the electrolyte breakthrough curve. Our findings suggest that the velocity correction factor is a dynamic, site specific property. A linear model was proposed to estimate the correction factor based on readily available predictor variables, those being travel distance, unit discharge, and maximum velocity. The results will benefit further investigations of overland flow hydraulics on semiarid rangelands by providing more comprehensive correction factors to determine mean velocity.

Influence of Hydrogen Ion Concentrations on the Protein Mobility in Native-PAGE Buffers

The study demonstrates the effect of hydrogen ion concentrations (pH) of Tank Buffer (TB) and resolving gel buffer (RGB) in a native-PAGE system on protein samples. A pH range of 7.8 to 9.3 for RGB and 8.3 to 9.3 for TB were used. Proteins in the samples under native-PAGE at varying pH of RGB such as 7.8, 8.3 and 8.8 with pH of TB, 8.3 and 8.8 were resolved well. The total running time for the samples to reach the end of the dye front ranged between 2.30 h to 4.30 h at the above pH combinations. It was observed as 3.30 h as total running time under normal pH conditions (RGB, pH 8.8; TB, pH 8.3). At the same time, buffers at higher pH or the combination of extreme pH (7.8 vs. 9.3) in the buffers were not favored good protein mobility/resolution, and bands were diffused. Longer running time was observed in various combinations of pH of RGB and pH of TB with 18.00 h being a longest with a pH of 8.3 and 9.3 for RGB and TB, respectively. This indicated the importance of pH of electrophoresis buffers for ionization of various proteins for better separation.

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Anti-stress phytohormones impact on proteome profile of green gram (Vigna radiata) under salt toxicity

Diffusiophoretic enhancement of mass transfer by nanofluids

Observations of an enhanced mass transfer and an irregular mixing when a small drop of dye is gently placed on a nanofluid dispersion have led to several propositions for the underlying cause, but none of them have been conclusively established. Here, we reproduce the enhancement phenomenon within a glass capillary containing fluorescein di-sodium dye solution on one side and alumina nanoparticle suspension on the other, avoiding the inertial interference present earlier. The enhancement is explained by the convective motion (and not a diffusion) of the dye solution counter to the direction of the diffusiophoretic motion of the nanoparticles towards a higher concentration of the dye. The initial velocity of the dye front in the capillary and the dye-drop experiment agree quantitatively with independent theoretical estimates from the diffusiophoretic velocity of alumina nanoparticles. With a suitably chosen nanofluid, it should now be possible to effect a desired level of enhancement (or suppression) of mass transfer of any solute. In micro-environments requiring fast mixing, such as in nanofabrication, lab-on-chip, drug-delivery, and chemotaxis, the effects can be substantial.

Development of an Experimental and Digital Cardiovascular Arterial Model for Transient Hemodynamic and Postural Change Studies: "A Preliminary Framework Analysis".

The ultimate goal of the present work is to aid in the development of tools to assist in the treatment of cardiovascular disease. Gaining an understanding of hemodynamic parameters for medical implants allow clinicians to have some patient-specific proposals for intervention planning. In the present work an experimental and digital computational fluid dynamics (CFD) arterial model consisting of a number of major arteries (aorta, carotid bifurcation, cranial, femoral, jejunal, and subclavian arteries) were fabricated to study: (1) the effects of local hemodynamics (flow parameters) on global hemodynamics (2) the effects of transition from bedrest to upright position (postural change) on hemodynamics, and (3) diffusion of dye (medical drug diffusion simulation) in the arterial system via experimental and numerical techniques. The experimental and digital arterial models used in the present study are the first 3-D systems reported in literature to incorporate the major arterial vessels that deliver blood from the heart to the cranial and femoral arteries. These models are also the first reported in literature to be used for flow parameter assessment via medical drug delivery and orthostatic postural change studies. The present work addresses the design of the experimental and digital arterial model in addition to the design of measuring tools used to measure hemodynamic parameters. The experimental and digital arterial model analyzed in the present study was developed from patient specific computed tomography angiography (CTA) scans and simplified geometric data. Segments such as the aorta (ascending and descending) and carotid bifurcation arteries of the experimental and digital arterial model was created from online available patient-specific CTA scan data provided by Charite' Clinical and Research Hospital. The cranial and coronary arteries were simplified arterial geometries developed from dimensional specification data used in previous work. For the patient specific geometries, a MATLAB code was written to upload the CTA scans of each artery, calculate the centroids, and produce surface splines at each discrete cross section along the lumen centerline to create the patient specific arterial geometries. The MATLAB code worked in conjunction with computer aided software (CAD) Solidworks to produce solid models of the patient specific geometries and united them with the simplified geometries to produce the full arterial model (CAD model). The CAD model was also used as a blueprint to fabricate the experimental model which was used for flow visualization via particle imaging velocimetry (PIV) and postural change studies. A custom pulse duplicator (pulsatile pump) was also designed and developed for the present work. The pulse duplicator is capable of producing patient-specific volumetric waveforms for inlet flow to the experimental arterial model. A simple fluid structure interaction (FSI) study was also conducted via optical techniques to establish the magnitude of vessel diameter change due to the pulsatile flow. A medical drug delivery (dye dispersion and tracing) case was simulated via a dye being dispersed into the pulsatile flow stream to measure the transit time of the dye front. Pressure waveforms for diseased cases (hypertension & stenotic cases) were also obtained from the experimental arterial model during postural changes from bedrest (0°) to upright position (90°). The postural changes were simulated via attaching the experimental model to a tile table the can transition from 0° to 90°. The PIV results obtained from the experimental model provided parametric data such as velocity and wall shear stress data. The medical drug delivery simulations (experimental and numerical) studies produce time dependent data which is useful for predicting flow trajectory and transit time of medical drug dispersion. In the case of postural change studies, pressure waveforms were obtained from the common carotid artery and the femoral sections to yield pressure difference data useful for orthostatic hypotension analysis. Flow parametric data such as vorticity (flow reversal), wall shear stress, normal stress, and medical drug transit data was also obtained from the digital arterial model CFD simulations. Although the present work is preliminary work, the experimental and digital models proves to be useful in providing flow parametric data of interest such as: (1) normal stress which is useful for predicting the magnitude of forces which could promote arterial rupture or dislodging of medical implants, (2) wall shear stress which is useful for analyzing the magnitude of drug transport at the arterial wall, (3) vorticity which is useful for predicting the magnitude of flow reversal, and (4) arterial compliance in the case of the experimental model which could be useful in the efforts of developing FSI numerical simulations that incorporates compliance which realistically models the flow in the arterial system.

“Dye front reciprocation” in combined central retinal vein occlusion with cilioretinal artery infarction

“Dye front reciprocation” in combined central retinal vein occlusion with cilioretinal artery infarction

Dispersion in two-dimensional turbulent buoyant plumes

Using high-resolution imaging and dye studies, we investigate experimentally the mixing of a tracer by the eddies within a two-dimensional turbulent buoyant plume. Instantaneously, the plume consists of a series of eddies, and at each point along the centreline of the plume, the along-plume speed of the leading edge of the eddies $w_{e}\approx 1.3f^{1/3}$, where $f$ is the buoyancy flux, while the product of the length scale, $A$, and frequency, ${\it\omega}$, of the eddies ${\it\omega}A\approx 0.15f^{1/3}$. The circulation and flow associated with the eddies lead to longitudinal mixing relative to the mean flow. To illustrate this mixing, we analyse the evolution of the horizontally averaged dye front produced by adding a constant flux of dye to a steady plume for times $t>0$. We show that the centre of mass of the horizontally averaged dye front has an along-plume speed ${\approx}1.04f^{1/3}$. This is consistent with the predictions of a time-averaged model for the evolution of the horizontally averaged mass, momentum and buoyancy flux in the plume. The new data also show that the longitudinal spreading of the horizontally averaged dye front can be described in terms of a dispersivity ${\approx}\,0.02f^{1/3}z$, where $z$ is the vertical distance below the source. This model of longitudinal mixing enables calculation of the residence time distribution of material in the plume, which may be key to modelling the products of a reaction in which the reaction time is comparable to the travel time in the plume.

Observing Solute Transport in the Capillary Fringe Using Image Analysis and Electrical Resistivity Tomography in Laboratory Experiments

Five laboratory experiments were conducted to study solute transport in the capillary fringe in a sand‐filled glass tank containing an artificial groundwater zone, an unsaturated zone, and a capillary fringe in between. Dye‐stained water, applied at the soil surface, moved downward through the unsaturated zone and then horizontally in the capillary fringe. The horizontal velocity of the dye plume front was calculated using optical image analysis and electrical resistivity tomography (ERT) measurements. Both methods gave similar velocities if an appropriate value of the threshold ratio describing the dye front was used. The hydraulic model HYDRUS‐2D was used to simulate dye movement in the glass tank. After calibrating the dispersivity value in the hydraulic model, the horizontal velocity was found to be in the range −10 to 17% compared with the values measured using image analysis and −12 to 24% compared with the values measured by ERT; the differences could probably be attributed to uncertainties in the hydraulic parameters and soil heterogeneities. Both experimental and numerical results showed that the horizontal velocity of the capillary fringe is more or less identical to the one in the saturated zone. Thus, from a water transport perspective, the results suggest that the capillary fringe should be treated as a part of the saturated zone in hydrologic modeling.

Measurement of flow velocity and inference of liquid viscosity in a microfluidic channel by fluorescence photobleaching.

We present a simple, noninvasive method for simultaneous measurement of flow velocity and inference of liquid viscosity in a microfluidic channel. We track the dynamics of a sharp front of photobleached fluorescent dye using a confocal microscope and measure the intensity at a single point downstream of the initial front position. We fit an exact solution of the advection diffusion equation to the fluorescence intensity recovery curve to determine the average flow velocity and the diffusion coefficient of the tracer dye. The dye diffusivity is correlated to solute concentration to infer rheological properties of the liquid. This technique provides a simple method for simultaneous elucidation of flow velocity and liquid viscosity in microchannels.

Troubleshooting forum. SDS-page. What is the best way to separate proteins of similar molecular weight? (Thread 33111).

BioTechniquesVol. 56, No. 3 Troubleshooting ForumOpen AccessSDS-PAGEKristie NyboKristie NyboSearch for more papers by this authorPublished Online:3 Apr 2018https://doi.org/10.2144/000114144AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail This month's question from the Molecular Biolog y Forums (online at molecularbiology.forums.biotechniques.com) comes from the “Protein Methods” section. Entries have been edited for concision and clarity. Mentions of specific products and manufacturers have been retained from the original posts, but do not represent endorsements by, or the opinions of, BioTechniques.Molecular Biology Techniques Q&AWhat is the best way to separate proteins of similar molecular weight? (Thread 33111)Q I need to separate 2 proteins with molecular weights of 86 kDa and 80 kDa; however, I haven't obtained a good separation yet, even on a 6% polyacrylamide gel. To make matters worse, the two proteins are isoforms, and there are no commercially available antibodies against either one.I'm considering using a 4% gel or maybe a 2-D gel, although both isoforms seem to have the same isoelectric point (pI). Can anyone recommend a better technique for separating these proteins?A You're going the wrong way with the gel concentration; you want to be more restrictive. Try a 10% polyacrylamide gel with a narrow range gradient, such as 10-12%. You might also add 4 M urea to the gel.Q I did try 10% polyacrylamide on several occasions but still didn't get good separation. I will try again, this time adding urea to the sample as you suggest.Will the urea compress the protein bands so that they are distinguishable from bands with similar molecular weights?A Remember, your samples are already denatured (SDS and reducing agent). I don't think the urea will further denature the sample, but urea does enhance charge separations and sharpens banding.You should add urea to the gel, not just to the the sample. If you want to add urea to the sample, add it after the heating and cooling steps.Q I tried a 4 M urea SDS-PAGE but saw fuzzy bands after probing. Could that be because I did not add urea to my samples? What amount of urea should I add?A It is best if the urea concentration in the sample and gel is the same, but you can use a higher concentration in the sample if necessary.Were your bands separated enough to distinguish between the isoforms?Fuzzy bands can be the result of glycosylation, diffusion (from standing around too long before fixing or transfer, or soaking too long in the transfer buffer), or insufficient stacking, among other reasons.Q I ran another 6 M urea, 10% polyacrylamide gel with 6 M urea-containing samples and saw rather compact bands; however, I think the fuzzy bands could be due to differences in protein expression levels. I'm using cells from lymphoid organs and cell lines with varying expression levels of my protein isoforms of interest. It looks like I will need to titrate the amount of protein loaded since the separation is still not clear.Is it a good idea to let the dye front run off and only retain the protein of interest?A That depends on what you want to show. If you are only interested in those two bands, then run the dye out of your gel. If you want reproducible results, run the gels for a specific number of volt-hours.Q Since the separation is still not clear, does it make sense to use a higher percentage of urea?A 8 M urea may improve the separation and make the bands sharper, but you may want to try a 12% gel, or even better, a 10-12% gradient gel.Q Is a 30 min incubation of the samples in 8 M urea sufficient?A Incubation with urea is not necessary. Also, be sure never to heat the urea; it will decompose and carbamylate your protein.Q Even with these modifications, I see only minimal separation. One problem might be that I am loading too much protein on the gel. I will try reducing the input to reduce the thickness of the band.A We saw improved resolution of our proteins using two electrode buffers instead of Laemmli buffer, along with running and stacking gel buffers.A A longer gel with proper stacking or a gradient should work without too much band broadening, especially if you add some urea to the gel.FiguresReferencesRelatedDetails Vol. 56, No. 3 Follow us on social media for the latest updates Metrics History Published online 3 April 2018 Published in print March 2014 Information© 2014 Author(s)PDF download

Biochemical and Functional Characterization of the Klotho-VS Polymorphism Implicated in Aging and Disease Risk

Klotho (KL) is an age-regulating protein named after the Greek goddess who spins the thread of life. Mice deficient in KL are normal throughout development, but rapidly degenerate and display a variety of aging-associated abnormalities that eventually lead to decreased life expectancy. While multiple genetic association studies have identified KL polymorphisms linked with changes in disease risk, there is a paucity of concrete mechanistic data to explain how these amino acid substitutions alter KL protein function. The KLVS polymorphism is suggested to lead to changes in protein trafficking although the mechanism is unclear. Our studies have sought to further investigate the functional differences in the KLVS variant that result in increased risk of many age-related diseases. Our findings suggest that the F352V and C370S substitutions lead to alterations in processing as seen by differences in shedding and half-life. Their co-expression in KLVS results in a phenotype resembling wild-type, but despite this intragenic complementation there are still changes in homodimerization and interactions with FGFR1c. Taken together, these studies suggest that KLVS leads to altered homodimerization that indirectly leads to changes in processing and FGFR1c interactions. These findings help elucidate the functional differences that result from the VS polymorphism, which will help clarify how alterations in KL function can lead to human disease and affect cognition and lifespan.

Proteomics of Genetically Engineered Mouse Mammary Tumors Identifies Fatty Acid Metabolism Members as Potential Predictive Markers for Cisplatin Resistance

In contrast to various signatures that predict the prognosis of breast cancer patients, markers that predict chemotherapy response are still elusive. To detect such predictive biomarkers, we investigated early changes in protein expression using two mouse models for distinct breast cancer subtypes who have a differential knock-out status for the breast cancer 1, early onset (Brca1) gene. The proteome of cisplatin-sensitive BRCA1-deficient mammary tumors was compared with that of cisplatin-resistant mammary tumors resembling pleomorphic invasive lobular carcinoma. The analyses were performed 24 h after administration of the maximum tolerable dose of cisplatin. At this time point, drug-sensitive BRCA1-deficient tumors showed DNA damage, but cells were largely viable. By applying paired statistics and quantitative filtering, we identified highly discriminatory markers for the sensitive and resistant model. Proteins up-regulated in the sensitive model are involved in centrosome organization, chromosome condensation, homology-directed DNA repair, and nucleotide metabolism. Major discriminatory markers that were up-regulated in the resistant model were predominantly involved in fatty acid metabolism, such as fatty-acid synthase. Specific inhibition of fatty-acid synthase sensitized resistant cells to cisplatin. Our data suggest that exploring the functional link between the DNA damage response and cancer metabolism shortly after the initial treatment may be a useful strategy to predict the efficacy of cisplatin.

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) (1,2). CSF is a protein rich fluid that is essential for normal brain development and function(3-6). In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes (7,8), while claudin 5a is necessary for tight junction formation (9). Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation. To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab(10), to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2). This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.

Degradation of an Old Human Protein

Long-lived proteins exist in a number of tissues in the human body; however, little is known about the reactions involved in their degradation over time. Lens proteins, which do not turn over, provide a useful system to examine such processes. Using a combination of Western blotting and proteomic methodology, age-related changes to a major protein, γS-crystallin, were studied. By teenage years, insoluble intact γS-crystallin was detected, indicative of protein denaturation. This was not the only change, however, because blots revealed evidence of significant cross-linking as well as cleavage of γS-crystallin in all adult lenses. Cleavage at a serine residue near the C terminus was a major reaction that caused the release of a 12-residue peptide, SPAVQSFRRIVE, which bound tightly to lens cell membranes. Several other crystallin-derived peptides with double basic residues also lodged in the cell membrane fraction. Model studies showed that once cleaved from γS-crystallin, SPAVQSFRRIVE adopts a markedly different shape from that in the intact protein. Further, the acquired helical conformation may explain why the peptide seems to affect water permeability. This observation may help explain the changes to cell membranes known to be associated with aging in human lenses. Age-related cleavage of long-lived proteins may therefore yield peptides with untoward biological activity.

Proteomic Analysis of Human Saliva From Lung Cancer Patients Using Two-Dimensional Difference Gel Electrophoresis and Mass Spectrometry

Lung cancer is often asymptomatic or causes only nonspecific symptoms in its early stages. Early detection represents one of the most promising approaches to reduce the growing lung cancer burden. Human saliva is an attractive diagnostic fluid because its collection is less invasive than that of tissue or blood. Profiling of proteins in saliva over the course of disease progression could reveal potential biomarkers indicative of oral or systematic diseases, which may be used extensively in future medical diagnostics. There were 72 subjects enrolled in this study for saliva sample collection according to the approved protocol. Two-dimensional difference gel electrophoresis combined with MS was the platform for salivary proteome separation, quantification, and identification from two pooled samples. Candidate proteomic biomarkers were verified and prevalidated by using immunoassay methods. There were 16 candidate protein biomarkers discovered by two-dimensional difference gel electrophoresis and MS. Three proteins were further verified in the discovery sample set, prevalidation sample set, and lung cancer cell lines. The discriminatory power of these candidate biomarkers in lung cancer patients and healthy control subjects can reach 88.5% sensitivity and 92.3% specificity with AUC = 0.90. This preliminary data report demonstrates that proteomic biomarkers are present in human saliva when people develop lung cancer. The discriminatory power of these candidate biomarkers indicate that a simple saliva test might be established for lung cancer clinical screening and detection.

Electrophoretic isolation of saponin fractions from Saponinum album and their evaluation in synergistically enhancing the receptor-specific cytotoxicity of targeted toxins

Saponinum album (SA) is a commercial mixture of saponins isolated from Gypsophila species. In the previously published work, we reported that SA dramatically improves the inhibition of tumor growth by targeted toxins in mice in a synergistic way. Here we report a simplified electrophoretic method for the isolation of a highly effective fraction of SA with a relative electrophoretic mobility to the dye front (R(f) ) of 0.63 from the mixture. In total, four different fractions were separated at a preparative scale, and evaluated by ESI-MS, HPLC and TLC analysis. Electrophoretic mobility and electrochemical properties of the different fractions of saponins from SA were set into relation to their ability to enhance the cytotoxicity of epidermal growth factor (EGF)-based targeted toxins. We here treated HER-14 cells, which are NIH-3T3 Swiss mouse embryo cells transfected with the human EGF receptor. Untransfected NIH-3T3 cells served as control. The major bulk of SA (72.3%) (R(f) =0.78) migrated the farthest and was found to be significantly ineffective (p<0.05) in enhancing the cytotoxicity of the targeted toxin, while the second fraction (R(f) =0.63) showed an enhancement of 9800-fold. The third (R(f) =0.56) had an enhancement factor of 3200, the fourth (R(f) =0.08) was again significantly ineffective (p<0.05) in exhibiting any enhancement of cytotoxicity.