Staining with Tetraphenylporphinesulfonate in Vivo

Tetraphenylporphinesulfonate (TPPS), a synthetic, nonnaturally occurring porphine derivative, was administered parenterally to tumor bearing rats and its in vivo localization was studied with fluorescence microscopy. TPPS was selectively localized in elastica and eosinophilic leukocytes, but not in other tissue sites rich in basic protein. The elastica of the aorta and medium sized arteries, as well as elastic fibers of the subendocardium, paratracheal connective tissue and bronchial walls showed the strongest red fluorescence. The intensity of fluorescence in these sites corresponded with the degree of sulfonation of the TPPS. The tumors showed moderate red fluorescence diffusely in the cytoplasm.     

A combined fluorescent and electron microscopic stain for elastic tissue

Synopsis A histochemical procedure for fluorescent and electron microscopic observations of elastic tissue has been developed. This was accomplished by combining thein vivo andin vitro staining properties of non-metallic tetraphenylporphine sulphonate and its silver derivative. The non-metallic porphyrin gives a bright red fluorescence to elastica after injection, but it does not make the tissue electron dense. However, the metallic derivative imparts a specific electron density to elastica afterin vitro staining, but it does not render elastica either fluorescent or electron dense after injection. Thus, elastic tissue can be viewed with the fluorescence microscope after injection of the nonmetallic porphyrin. Sections from the same block can be stainedin vitro with silver tetraphenylporphine sulphonate and observed in the electron microscope. The nonmetallic porphyrin readily crosses the placental barrier and, therefore, can be used to study the elastica in foetuses by fluorescence microscopy.     

Effect of chloroquine wash-out period of photosensitizers in the skin and selected organs in rats

In the last decade, photodynamic therapy has become an alternative method for the diagnosis and therapy of tumors. In human medicine hematoporphyrin derivatives, sulfonated hydrophilic meso-tetraphenylporphyrin (TPPS4) and an oligomer of hematoporphyrin (Photosan 3), are widely used. Chloroquine is used for the treatment of porphyria cutanea tarda for its power to release porphyrins from the liver tissue. The kinetics of two porphyrin photosensitizers TPPS4 and Photosan 3 in the skin and some organs as well as the effect of chloroquine on the porphyrin excretion and their accumulation in skin and organs of Wistar rats were studied. TPPS4 exhibited maximum fluorescence in skin 48 h after application with decreasing to basal level from the 8th to the 14th day. Maximum fluorescence was reached at 72 hours after Photosan 3 application and it decreased to basal level during 96 hours after application. TPPS4 caused significantly higher fluorescence compared to Photosan 3. Chloroquine after oral administration did not change the fluorescence of skin, but it significantly decreased the TPPS4 concentration in rat organs if chloroquine treatment started 3 days or 2 weeks after TPPS4 application. Chloroquine significantly decreased the serum TPPS4 concentration during the period of 28 days. Fluorescence of skin was significantly higher and lasted longer after application of TPPS4 compared to Photosan 3. Chloroquine after oral administration did not influence the fluorescence of the skin, but it significantly decreased the TPPS4 concentration in rat organs. This effect could be useful in photodynamic therapy for mobilizing exogenous porphyrins from tissues after parenteral photodynamic therapy.     

Simultaneous Demonstration of Connective Tissue elastica and Fibrin by a Combined Verhoeff's Elastic-Martius-Scarlet-Blue Trichrome Stain

A method for the routine combined demonstration of elastica, connective tissue in general, and fibrin is described. Elastica, stained blue-black by Verhoeff's iron hematoxylin, is contrasted with muscle and collagen, stained red and blue or green respectively, by a modification of the Martius-scarlet-blue (MSB) trichrome for fibrin of Lendrum et al. The MSB technique selectively stains fresh, mature and old fibrin orange-yellow, red and blue respectively; the Masson trichrome does not distinguish between erythrocytes and fibrin. Nuclei are stained at the same time as the elastica. The technique takes approximately one and a half hours and is ideal for the study of connective tissue and vascular pathology, especially the necrotizing vasculitides.     

Simultaneous demonstration of connective tissue elastica and fibrin by a combined Verhoeff's elastic-Martius-scarlet-blue trichrome stain

A method for the routine combined demonstration of elastica, connective tissue in general, and fibrin is described. Elastica, stained blue-black by Verhoeff's iron hematoxylin, is contrasted with muscle and collagen, stained red and blue or green respectively, by a modification of the Martius-scarlet-blue (MSB) trichrome for fibrin of Lendrum et al. The MSB technique selectively stains fresh, mature and old fibrin orange-yellow, red and blue respectively; the Masson trichrome does not distinguish between erythrocytes and fibrin. Nuclei are stained at the same time as the elastica. The technique takes approximately one and a half hours and is ideal for the study of connective tissue and vascular pathology, especially the necrotizing vasculitides.     

Nuclear transport of photosensitizers during photosensitization and oxidative stress.

The nuclear transport pathways of the photosensitizers meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4) and meso-tetra(4-N-methylpyridyl)porphyrin (TMPyP) during photosensitization and oxidative stress were characterized in CT-26 murine colon carcinoma cells using fluorescence microscopy and multi-pixel spectral imaging. Prior to irradiation, TPPS4 and TMPyP localized mainly in the lysosomes, while irradiation or H2O2 treatment induced a relocalization into the nucleus and nucleoli. Flow cytometry analysis of isolated nuclei from the treated cells showed an increase in nuclear fluorescence accompanying the relocalization. Isolation and separation of the nuclear proteins according to molecular weight was performed using a sephadex G-100 column. The protein fractions exhibiting high fluorescence were separated by high performance liquid chromatography. Five major classes of proteins with a retention time of 1, 7, 11, 12 and 15 min were obtained. Each photosensitizer was associated with a distinct class of proteins. While TPPS4 fluorescence was detected in the protein fraction with a retention time of 11 min, TMPyP fluorescence was associated with a protein fraction having a retention time of 7 min. We conclude that although oxidative stress triggers entry into the nucleus of both TPPS4 and TMPyP, each sensitizer uses a distinct transport mechanism based on its chemical properties.     

Interaction of monosulfonate tetraphenyl porphyrin, a competitive inhibitor, with acetylcholinesterase

Monosulfonate tetraphenyl porphyrin (TPPS(1)) forms a 1:1 complex with electric eel acetylcholinesterase (AChE) inducing a loss in TPPS(1) absorbance at 402 nm and the appearance of a new absorbance centered at 442 nm. In the presence of AChE, the fluorescence of TPPS(1) at 652 nm is slightly narrowed, with the maximal 652 nm fluorescence shifted from 407 to 412 nm excitation wavelength. The fluorescence peak of TPPS(1) at 712 nm shifts to 716 nm in the presence of AChE. TPPS(1) is a competitive inhibitor of AChE. The addition of acetylcholine iodide (AChI) or the competitive inhibitor tetracaine to the preformed AChE-TPPS(1) complex results in a loss of the 442 nm absorbance band as the porphyrin is displaced from AChE. The absorbance peak does not decrease in the presence of procaine, a non-competitive inhibitor.     

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue

Detection of the initial site of Toxoplasma gondii reactivation in brain tissue is difficult because the number of latent cysts is small and reactivation is a transient event. To detect the early stage of reactivation in mouse brain tissue, we constructed a cyst-forming strain of T. gondii in the tachyzoite stage, specifically expressing red fluorescence. The PLK strain of T. gondii was stably transfected with a red fluorescent protein gene, DsRed Express, under the control of a tachyzoite-specific SAG-1 promoter and the resulting parasite was designated as PLK/RED. Tachyzoites of PLK/RED growing in Vero cells showed red fluorescence. When C57BL/6J mice were i.p. infected with tachyzoites of PLK/RED, red fluorescent tachyzoites were detected in their brains at the fourth day p.i. However, red fluorescent tachyzoites were not detected in BALB/c mice latently infected with PLK/RED, although non-fluorescent cysts were detected in their brains. After treatment of latently infected mice with dexamethasone for 1 month, the mice showed neurological symptoms. In mice with symptoms, red fluorescent tachyzoites were again detected in their brains and in other organs. To detect the initial site of reactivation, BALB/c mice latently infected with the strain were treated with dexamethasone for 3 weeks, and brains were excised before any symptoms appeared. Excised brains were examined for red fluorescence-positive sites. By a histological study of red fluorescent-positive sites, we detected a cyst containing red fluorescent zoites, which still had a PAS stain-positive cyst wall. A few red fluorescent zoites breaking away from the cyst were also observed. The stage-specific expression of fluorescent protein facilitates detection of a rare transient event and makes it possible to detect the initial site of reactivation.     

Functionalization of a protein surface with per-O-methylated β-cyclodextrin

Per-O-methylated β-cyclodextrin (CD) bearing an iodoacetamide group at the 6-position was synthesized to functionalize protein surfaces. Bovine serum albumin (BSA) was quantitatively modified with the CD derivative by the S(N) 2 reaction of iodoacetamide with a cysteine residue (Cys34) on the BSA surface. The resultant CD-functionalized BSA (BSA-CD) spontaneously dimerized upon addition of an anionic tetraarylporphyrin (TPPS) through the supramolecular 1:2 complexation between TPPS and CD on the protein surface. The BSA-CD/TPPS complex further complexed with ferric protoporphyrin IX (hemin) in the hydrophobic pockets of albumin to form a hemin/BSA-CD/TPPS ternary complex in which static fluorescence quenching occurred owing to intramolecular electron transfer from the photoexcited TPPS to hemin.     

Photophysical and electrochemical properties of a dysprosium‐zinc tetra(4‐sulfonatophenyl)porphyrin complex

A dysprosium‐zinc porphyrin, [DyZn(TPPS)H₃O]_n (1) (TPPS = tetra(4‐sulfonatophenyl)porphyrin), was prepared through solvothermal reactions and structurally characterized by single‐crystal X‐ray diffraction analyses. Complex 1 features a three‐dimensional (3‐D) porous open framework that is thermally stable up to 400 °C. Complex 1 displays a void space of 215 ų, occupying 9.2% of the unit cell volume. The fluorescence spectra reveal that it shows an emission band in the red region. The fluorescence lifetime is 39 µsec and the quantum yield is 1.7%. The cyclic voltammetry (CV) measurement revealed one quasi‐reversible wave with E_1/2 = 0.30 V. Copyright © 2015 John Wiley & Sons, Ltd.     

A combination Verhoeff's elastic and Masson's trichrome stain for routine histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeff's technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

A Combination Verhoeffs Elastic and Masson's Trichrome Stain for Routine Histology

A method for the combined staining of elastic, muscle and connective tissue for routine use in histopathology is described. The elastica, stained black by Verhoeffs technique, is contrasted with the muscle and connective tissue stained red and green or blue respectively by a modification of Masson's trichrome. Cell nuclei stain blue-black with Weigert's iron hematoxylin. The procedure takes approximately two hours and is most suitable for the study of vascular pathology in surgical and autopsy sections.     

Spectroscopic study of TPPS4 nanostructures in the presence of bovine serum albumin.

The influence of bovine serum albumin (BSA) on the formation of J-aggregates of meso-tetra(4-sulfonatophenyl)porphine (TPPS4) in aqueous acid solution (pH 1.3) has been investigated by means of absorption and fluorescence spectroscopy. TPPS4 concentration was kept constant at 2 microM while BSA concentration was varied to get TPPS4 : BSA molar ratios from 1 : 0.005 to 1 : 20. In the presence of protein at all used concentrations the intensity of J-aggregates absorption band was higher than that in the pure solution. Spectral changes indicated that the dynamic equilibrium of the aggregated TPPS4 species was highly dependent on the molar ratio between TPPS4 and BSA. Small relative concentrations of BSA (TPPS4 : BSA, 1 : 0.005-1 : 0.1) had a stimulating effect on formation of J-aggregates. Several fractions of J-aggregates located in protein and aqueous moieties were detected in mixed solutions at intermediate BSA concentrations (TPPS4 : BSA, 1 : 0.5-1 : 8), when the absorbance intensity of the J-aggregates was the highest. At the highest used BSA concentrations (TPPS4 : BSA, 1 : 10-1 : 20) the spectral properties of the remaining J-aggregates were similar to those typical for pure porphyrin solution. Additionally, the split of the Soret band into two with peaks at 440 nm and 423 nm was followed by the simultaneous appearance of Q bands and reflected the formation of TPPS4-BSA complexes including both protonated and deprotonated TPPS4 forms.     

Spectrophotometric detection of pentachlorophenol (PCP) in water using immobilized and water-soluble porphyrins

The spectrophotometric properties of porphyrins are altered upon interaction with chlorophenols and other organochlorine pollutants. Meso-tetra(4-sulfonatophenyl)porphyrin (TPPS), zinc meso-tetra(4-sulfonato phenyl)porphyrin (Zn-TPPS), monosulfonate-tetraphenylporphyrin (TPPS1), meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl)porphyrin (C1TPP), meso-tetra(4-carboxyphenyl)porphyrin (C4TPP), and copper meso-tetra(4-carboxyphenyl)porphyrin (Cu-C4TPP) in solution exhibit a broad absorbance in the range 400-450 nm Soret region. The interaction of the above mentioned porphyrins in solution with pentachlorophenol (PCP) induces a red shift in the Soret spectrum with absorbance losses at 413, 418, 403, 405, 407, and 404 nm, respectively, and the appearance of new peaks at 421, 427, 431, 416, 417, and 416 nm, respectively. The intensity of the Soret spectral change is proportional to the pentachlorophenol concentration with a detection limit of 1, 0.5, 1.16, 1, 0.5, and 0.5 ppb, respectively. The interaction of (C4TPP) and (Cu-C4TPP) in solution with PCP shows to concentration dependent for concentrations less than 4 ppb the dependence was log-linear. However, for concentrations greater than 4 ppb the relation was linear. Monosulfonate-tetraphenylporphyrin immobilized as a monolayer on a Kimwipe tissue exhibits an absorbance peak in the Soret region at 422 nm. The interaction of the porphyrin with PCP induces a red shift in the Soret spectrum with absorbance loss at 419 nm and the appearance of new peaks at 446 nm. The intensity of the Soret spectral change is proportional to the log of PCP concentration. The detection limit with immobilized TPPS1 for PCP is 0.5 ppb. These results suggest the potential for development of spectrophotometric chemosensor for PCP residues in water with detection limits less than US EPA maximum contaminate level (MCL) of 1 ppb. The immobilized TPPS1 on the Kimwipe will make it possible to develop a wiping sensors to monitor the PCP or other pesticides residues on the vegetables or wood products.     

Microspectrographic analysis of trypan blue-induced fluorescence in oocytes of the Japanese quail

Summary It was shown that the vital dye trypan blue injected subcutaneously is adsorbed on exogenous yolk and stored in oocytes of Japanese quails. The binding sites of the dye could be visualized by fluorescence microscopy. The spectral distribution of the trypan blue-induced fluorescence emitted by yolk granules was analyzed microspectrographically. The analysis revealed that yolk granules exhibit a deep red fluorescence radiation with a maximum intensity at 670 nm, when blue or green excitation light is used. This fluorescence was exclusively induced by the presence of trypan blue, and not by contaminants of the dye. The fluorescence intensity did not decrease during processing of the tissue throughout the different solvents routinely used in light microscopy, especially after fixation in Heidenhain's fluid, nor did it suffer from pronounced fading during irradiation of the tissue. Model experiments showed that the value of the fluorescence emission maximum was concentration-dependent, and that amounts as little as 5×10^–3 mg trypan blue per ml solution containing an excess of yolk as a substrate for the dye, could clearly be detected and measured.It is suggested that a highly diluted solution of trypan blue can be used without teratogenic effects, as a tracer for exogenous yolk uptake and migration into oocytes, and that fluorescence microscopy is a reliable method for its further localization. A detailed account of the procedure is reported.     

Histochemical Probing of Potato Periderm with Neutral Red: A Sensitive Cytofluorochrome for the Hydrophobic Domain of Suberin

A technique is described which uses the lipid fluorochrome neutral red as a cytochemical probe to detect the hydrophobic domain of the lignosuberin matrix in native and wound periderm of potato tuber. Toluidine blue O is used as a counterstain to quench autofluorescence. The neutral red technique appears to be specific for the hydrophobic/lipid domain of suberin and is significantly more sensitive than Sudan III and IV. The fluorochrome was extensively used on paraffin-embedded tissue with excellent results but also worked on freehand sections of fresh periderm tissue. In tuber tissue undergoing wound-healing, the pattern of suberin fluorescence obtained with the neutral red probe was identical in specificity to the color pattern obtained with Sudan III/IV, but somewhat different than that observed when berberine was used. Results obtained with the neutral red probe and berberine probe visually demonstrated that during ligno-suberin biosynthesis, the depositions of hydrophobic/lipid and phenolic/lignin-like components in potato tuber periderm were separate processes. The deposition of these components does not necessarily require their simultaneous presence because the fluorescence from these probes showed that the components were not consistently present together on the cell walls.     

Induced chirality of binary aggregates of oppositely charged water-soluble porphyrins on DNA matrix

The induced chirality of achiral binary aggregates of meso-tetrakis(4-N-methylpyridyl)porphyrine (TMPyP) and meso-tetrakis(4-sulfonatophenyl)porphyrine (TPPS) on a deoxyribonucleic acid (DNA) matrix was investigated. Although the negatively charged TPPS did not show induced chirality in DNA solution due to the electrostatic repulsion, induced chirality was obtained through the addition of a positively charged TMPyP. It was confirmed that the induced chirality was due to the binary complex formation between TPPS and TMPyP on the DNA matrix. Moreover, the induced chirality depended on the relative molar ratio of TPPS to TMPyP (r) and the binding modes of the complex to DNA. When r<1, induced circular dichroism (CD) spectrum of the ternary complex was similar to that of intercalated TMPyP into DNA. For r=1, the induced CD spectrum showed a reversed biphasic signal due to the complex of TMPyP and TPPS stacking along the DNA surface. At a higher r value (>1), there was an induced CD signal at 482 nm attributed to a lateral shifted arrangement of heteroaggregate of TPPS and TMPyP on DNA matrix where TMPyP acted as a spacer to mediate the growth of heteroaggregates. Increasing the concentration of sodium chloride in the solution would favor the formation of the lateral shifted arrangement of heteroaggregate of TPPS and TMPyP. The resonance light scattering (RLS) spectra confirmed the above results. Analysis of the CD spectral changes in DNA conformation showed that during the binary complex formation of TPPS and TMPyP, the intercalated TMPyP could be 'pulled out' from the base pairs of DNA, which might be useful in gene therapy. A model was proposed to account for these observations.     

Toxic and phototoxic effects of tetraphenylporphinesulphonate and haematoporphyrin derivative in vitro

The toxic and phototoxic effects of tetraphenylporphinesulphonate (TPPS4) and haematoporphyrin derivative (HpD) have been examined in vitro. TPPS4 was found to have less dark toxicity to the cells than HpD as measured by inhibition of cell multiplication and colony formation at comparable extracellular concentrations. TPPS4 was also less effective than was HpD in photoinactivating NHIK 3025 cells by more than a factor 2 which should be expected on the basis of cellular uptake. Spectrofluorometric data suggest that HpD in cells interacts more with lipids than TPPS4. This might explain the large photosensitizing effect of HpD compared to TPPS4 since the lifetime of singlet oxygen is about a factor of 10 longer in a lipid environment than in an aqueous environment. The uptake of TPPS4 and HpD by cancer cells in vitro does not correlate with previous in vivo data, indicating retention of TPPS4 in the tumour stroma. This makes in vitro/in vivo extrapolation difficult with regard to the use of TPPS4 as an agent for photodynamic therapy.     

On the mechanism of Verhoeff's elastica stain: a convenient stain for myelin sheaths.

Verhoeff (1908) recommended an iron-hematein formula containing Lugol's solution for demonstration of elastic tissue; sections are differentiated until desired staining patterns are obtained. Verhoeff's stain colored a variety of tissue structures and showed higher substantivity for myelin sheaths than for elastin. Addition of HCL or omission of Lugol's solution decreased or abolished coloration of pseudo-elastica and thus enhanced selectivity for elastin. Substitution of Fe++ for Fe+++ abolished dye binding by elastin. A review of chemical data indicated interaction of components of Lugol's solution with the dye. Hematein and Fe+++ form a variety of cationic, anionic and non-ionic chelates; the ratio of these compounds changes with time. Dye binding apparently occurs mainly via van der Waals forces and hydrogen bonds. Verhoeff's elastica stain is definitely not specific for elastin and is inferior to orcein and resorcin-fuchsin because of the required differentiation with its inherent bias to produce patterns which conform to expectations. However, Verhoeff's elastica stain is far superior to other metal-hematein technics for myelin sheaths. The combined Verhoeff-picro-Sirius Red F3BA stain can be performed in 30 min and does not require differentiation. It is therefore suggested to reclassify Verhoeff's elastica stain as a method for myelin sheaths.     

An Anionic Porphyrin Binds <Emphasis Type="Italic">β</Emphasis>-Lactoglobulin A at a Superficial Site Rich in Lysine Residues

Binding of small ligands to globular proteins remains a major research topic in biophysics. We have studied the binding of several photoactive dyes to β-lactoglobulin (BLG), as a model to investigate the photoinduced effects of porphyrins on proteins. A combination of optical spectroscopies (fluorescence, circular dichroism) and molecular docking simulations were used to estimate the pH-dependence of the binding parameters and the docking location for meso-tetrakis(sulfonatophenyl)-porphyrin (TPPS). We have observed that the binding of TPPS is not modulated by the pH-mediated conformational transition of the protein (i.e., Tanford transition). Binding of TPPS appears to occur with some degree of negative cooperativity. Moreover, TPPS remains bound even upon partial denaturation of the protein. These results are consistent with a superficial binding site at a location removed from the aperture of the interior β-barrel. Binding occurs through electrostatic interactions between the negative SO₃ ⁻ groups of TPPS and positively charged Lys and Arg residues. This is the first study that explores the interaction of an anionic porphyrin with BLGA in a pH range that spans across the Tanford transition. Establishing the location of the binding site will enable us to explain the photoinduced conformational effects mediated by TPPS on BLG. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.