Use Of Rhodamine Research Articles

Role of proteoglycans and cytoskeleton in the effects of TGF-β1 on renal proximal tubule cells

Transforming growth factor-beta (TGF-beta) is a critical cell regulatory protein which influences cell growth, cell differentiation and cell chemotaxis. TGF-beta 1 has been previously shown to promote a migratory and adherent transformation of monolayers of renal proximal tubule cells in primary culture to form solid clusters of cells. To better understand the cellular basis of this TGF-beta 1 effect, these studies evaluated the influence of TGF-beta 1 on the synthesis of proteoglycans and on cytoskeleton rearrangement in rabbit renal proximal tubule cells in primary culture, and their role in this transformation effect of TGF-beta 1. Biosynthetic labeling of proteoglycans with 35S sulfate and enzyme digestion studies demonstrated that TGF-beta 1 promoted the synthesis of heparan sulfate proteoglycans in these cells. The importance of proteoglycan synthesis induced by TGF-beta 1 in this migration and aggregation process was demonstrated with the use of two chemically-dissimilar proteoglycan synthesis inhibitors: xyloside and galactosamine. Both compounds inhibited TGF-beta 1 stimulation of proteoglycan synthesis and diminished TGF-beta 1 promoted transformation of proximal tubule cells as assessed by quantitative morphometry. Further experiments evaluated the influence of TGF-beta 1 on actin microfilaments with the use of rhodamine conjugated phalloidin staining and immunofluorescent microscopy, and demonstrated that TGF-beta 1 provoked a dramatic rearrangement of actin microfilaments into stress fibers. The use of actin microfilament disrupting agents, cytochalasin B and D, attenuated the stress fiber formation promoted by TGF-beta 1 and inhibited the TGF-beta 1-induced morphologic transformation of these cells. Further studies evaluated these effects on the rate of DNA synthesis in these cells, as assessed with 3H-thymidine incorporation. Proteoglycan synthesis inhibitors significantly diminished the maximal proliferative response of these epithelial cells to epidermal growth factor (EGF). In contrast, actin microfilament disaggregation with cytochalasin B or D did not change the rate of DNA synthesis in response to EGF but did attenuate the antiproliferative effect of TGF-beta 1 on EGF-induced DNA synthesis cells. These studies demonstrate that the TGF-beta 1 promoted synthesis cells. These studies demonstrate that the TGF-beta 1 promoted an increase in the production of proteoglycans and a higher ordered structure of the cytoskeleton. Both effects were instrumental in the adhesive migratory response of proximal tubule cells to TGF-beta 1 as well as the DNA synthesis rate response to both EGF and TGF-beta 1.

Use of rhodamine 123 to identify viable cells isolated from embryonic chick calvaria

A technique is described for the determination of viable bone cell numbers isolated from embryonic calvaria. The protocol utilized postisolation staining of the initial cell pellet with 10 µg/ml rhodamine 123. Using phase contrast and epifluorescence microscopy, this vital dye allowed for direct confirmation of viable osteogenic cells. Viability assessments comparing trypan blue exclusion with rhodamine 123 uptake varied with cell source. Estimates of cells cultured for 4 days were identical using the two methods, whereas trypan blue estimates of primary isolate cells were twofold greater than estimates using rhodamine 123. To ascertain the effects of rhodamine pretreatment on growth and differentiation of the osteogenic cells in culture, treated and untreated control cultures were compared. Over 21 days in culture, no differences were observed between the two groups. These data suggest that rhodamine 123 is an efficient tool for measuring viability of osteogenic cells which does alter phenotypic expression in vitro.

Use of rhodamine 123 to assess P-glycoprotein activity in cultured rat hepatocytes

Use of rhodamine 123 to assess P-glycoprotein activity in cultured rat hepatocytes

Use of rhodamine 123 to label and lesion interstitial cells of Cajal in canine colonic circular muscle

The role of interstitial cells of Cajal (ICC) is difficult to determine because these cells are not easily identified by light microscopy, and there are no compounds available to specifically lesion ICC. Ultrastructural studies have shown an abundance of mitochondria in ICC. Therefore, we have used rhodamine 123, a fluorescent dye that is specifically accumulated by mitochondria, to identify ICC in canine proximal colon. This technique provided good discrimination between ICC and smooth muscle cells, but enteric neurons were labeled with rhodamine 123. This compound has cytotoxic properties in some cells. Therefore, we treated intact muscle strips with rhodamine 123 while recording intracellular electrical activity from circular muscle cells. Uptake of rhodamine 123 by ICC was associated with an alteration in electrical rhythmicity. These data suggest that rhodamine 123 may be a useful tool for visualizing and perhaps chemically lesioning ICC.

Use of rhodamine 123 in the treatment of the pollard iii rat prostate adenocarcinoma

Significant destruction of the Pollard III rat prostate adenocarcinoma was achieved by treatment with rhodamine 123 at a dosage of 15 mg/kg every other day for 33 to 38 days. Injection of tumor remnants into untreated animals produced no tumor regrowth. Our data have demonstrated rhodamine 123 to be a significant drug for the management of the very aggressive Pollard III tumor.

Use of Rhodamine 123 to investigate alterations in mitochondrial activity in isolated mouse liver mitochondria

The fluorescent dye Rhodamine-123, which selectively stains mitochondria depending on the mitochondrial membrane potential, was used with flow cytometry to evaluate alterations in activity of mitochondria isolated from mouse liver. Under in vitro conditions, with succinate and ADP present in the buffer, mitochondrial activity was affected by a variety of metabolic inhibitors that modify membrane potential. These results demonstrate clearly that flow cytometric techniques using Rhodamine-123 can be employed to study activity in isolated mitochondria.

Mitochondrial analysis in living cells: the use of rhodamine 123 and flow cytometry.

Flow cytometry combines the advantages of microscopy and biochemical analysis in a single highly sensitive technique for a rapid examination of numerous individual living cells. It has become a potent and essential tool in the studies of the physiology of the whole cell and its organelles. Rhodamine 123 is a vital fluorescent dye used in flow cytometry. As it is specifically concentrated in mitochondria because of the transmembrane potential that these organelles maintain in living cells, rhodamine 123 is thus a useful probe for monitoring the abundance and activity of mitochondria. A critical survey of the routine use of rhodamine 123 together with flow cytometry in mitochondrial research is presented.

Cytoplasmic lipid droplets as the possible eventual cellular fate of active forms of cyclosporin

The activity of cyclosporins can be defined with regard to their ability to inhibit the proliferation of susceptible lymphoblastoid cell clones. All active cyclosporins cause the emergence of highly refringent globular bodies, independently of cell susceptibility to cyclosporin. Cyclosporins devoid of proliferation-inhibition capability do not cause such alterations in cell morphology. The use of dansylated active cyclosporins suggests that within a few hours of treatment in vitro, most if not all cyclosporin is contained within those globular bodies. By using cytochemical methods allowing differential staining, we show here that these cyclosporin-containing structures are neither normal mitochondria (shown by use of rhodamine 123) nor normal lysosomes (shown by use of acridine orange under stringent staining conditions) but most probably lipid droplets (shown by use of perylene and various dansylated hydrophobic probes).

KELAINAN PATOLOGI PADA MENCIT DAN TIKUS DISEBABKAN ZAT WARNA RHODAMINE B DAN METANIL YELLOW

PATHOLOGICAL LESIONS IN MICE AND RATS CAUSED BY DYESTUFFS RHODAMINE B AND METANIL YELLOW. Rhodamine B and Metanil Yellow are 2 non-edible dyestuffs which are widely used for coloring snacks and drinks in Jakarta. These substances are reportedly toxic for human beings, however no data on acute or chronic intoxications are available so far. Groups of mice and rats were fed with either Rhodamine B or Metanil Yellow. The doses of these dyestuffs varied from 0.5 mg to 1350 mg per kilogram body weight. These animals were dcvided into 3 different experimental groups, respectively acute, subacute and chronic toxicity tests. Clinical signs included discoloration of the skin and its intensity depended upon the concentration of the dyestuffs used. The body weight gain of the test animals were consistently lower than those of the controls. Some animals became agressive and cannibalism occurred. Pathological lesions consisted of unthriftiness, focal liver inflammation, hydronephrosis, hepatoma and lymphoma. Considering the results of the experiments, it is justified to warn that the wide use of Rhodamine B and Metanil Yellow for food coloring might be hazardous for human health.

Localization of mitochondria in living cells with rhodamine 123.

The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment.

Rhodamine immunohistofluorescence applied to plant tissue.

Tissue slices from the roots and seeds of sanifoin (Onobrychis viciifolia, Scop.) exhibit bright autofluorescence when illuminated with blue (495 nm) light. This autofluorescence is indistinguishable from the fluorescence emission of fluorescein, the commonly used fluorochrome in immunohistochemical staining procedures. Rhodamine isothiocyanate, when coupled to immunoglobulin, and excited with green light at 546 nm, exhibits a reddish-orange fluorescence with an emission maximum at 590 nm. Plant tissue has little or no autofluorescence when illuminated at this wavelength and viewed with a 580 nm barrier filter. Therefore, use of rhodamine for immunohistochemical localization in plant tissue avoids interpretative complications due to inherent autofluorescence.

Phagocytosis of Candida albicans by eosinophilic leukocytes

The percentage of eosinophils phagocytosing Candida albicans spores approximates that of neutrophils in in vitro experiments. The phagocytic activity of the eocinophils is shown to parallel anti-Candida IgG serum antibody titers and to involve the complement system. By means of an immunofluorescent technique, with the use of rhodamine B conjugated to bovine serum albumin to block nonspecific staining, the presence of antibody on the phagocytosed spores is demonstrated.

A procedure for tracing of kraft mill effluent from an ocean outfall by constituent fluorescence

WATER pollution control agencies are concerned with tracing pollutants discharged from ocean outfalls for the following reasons: (1) the information obtained on the concentration distribution in the environment can be used to improve the fundamental understanding of dispersion and will lead to improved methods for designing ocean outfalls; and (2) as a means of surveillance activities to assure that operating systems are meeting water quality requirements. Several methods are available for performing this task which have utility for both objectives. HhRREMO~S (1966) used radioactive bromine for tracing drift plumes which developed below the surface. Rhodamine dye has been used by a number of investigators (BAUMGARTNER et al., 1969; STEWART et al., 1969). Coliform bacteria have also been used (FoxWORTHY and KNEELING, 1969), either alone or with an additional tracer such as rhodamine, to aid in correcting the coliform counts which are influenced not only by dilution, but also by variable regrowth and die-offpatterns. One objection to the use of rhodamine is on the basis of its cost, because the useful technical attributes are well recognized (ANON, 1968). Assume that in a typical plume study, a minimum rhodamine WT concentration desired at the initial point in the study is 10 parts per billion by weight (ppb). If dilution on the order of 100 times will be achieved in the vertical rise of the waste from the diffuser to the surface, the amount of stock rhodamine solution which must be added to the pipeline is 13 ml of 20 per cent rhodamine per minute per million gallons per day (mgd) of waste flow. Hence, for a study of a 10 mgd discharge over a 10-h period, 20 gal. of 20 per cent rhodamine would be required; at a cost of $10 per gallon in 50 gal. drums, this would amount to an expenditure of $200. The limitations for the use of radionuclides, in addition to the cost, are the special equipment required for counting, the problem of dosing with high energy stock solutions, the licensing requirement of state and federal regulations, and the local public concern. The most desirable situation is to find some constituent of the waste stream itself which, because it is either conservative or the rate of decay is well established, can be used as a tracer.

Application of Fluorescent Tracing Techniques to Hydrologic Studies

This article discusses the use of Rhodamine B as a suitable tracer in hydrologic studies.

The use of rhodamine .B in analytical chemistry: The determination of small quantities of gold

Using the technique already developed for determination of antimony with rhodamine B a method has been derived for determining small quantities of gold in the range 0–30 μg. Owing to the stability of the higher valency of gold no oxidation stage is required; extraction of the complex is only satisfactory with isopropyl ether in contrast with the case of antimony which gives good results with benzene as well as the isopropyl ether.

The use of rhodamine b in analytical chemistry: The determination of small quantities of antimony

A method for determining antimony is given. The antimony is separated from other ions by collection on a manganese dioxide precipitate which is redissolved. After oxidation to the pentavalent state the antimony is determined with rhodamine B. In the absence of thallium and large amounts of platinum or vanadium the method appears to be specific.