Effects of dimethylsulfoxide on the ultrastructure of fixed cells.

It has been reported that the use of dimethylsulfoxide (DMSO) as a solvent for fixatives enhances preservation of cellular ultrastructure. By contrast, we have shown that DMSO alters the ultrastructural integrity of glutaraldehyde fixed cells. The cell membrane, nuclear envelope, endoplasmic reticulum, ribosomes, microtubules and intracytoplasmic organelles are most susceptible to the action of DMSO. We hypothesize that DMSO exerts intracellular alterations via its interaction with remnant interfacial water in fixed cells. DMSO-induced alterations of these and related cellular components may result in the formation of artefactual structures and networks. Thus, it appears that DMSO containing glutaraldehyde neither accelerates fixation nor enhances stabilization of cellular ultrastructure. For these reasons, addition of DMSO to fixatives is not recommended.

Microscopic and histochemical manifestations of hyaline cartilage dynamics.

Structure and function of hyaline cartilages has been the focus of many correlative studies for over a hundred years. Much of what is known regarding dynamics and function of cartilage constituents has been derived or inferred from biochemical and electron microscopic investigations. Here we show that in conjunction with ultrastructural, and high-magnification transmission light and polarization microscopy, the well-developed histochemical methods are indispensable for the analysis of cartilage dynamics. Microscopically demonstrable aspects of cartilage dynamics include, but are not limited to, formation of the intracellular liquid crystals, phase transitions of the extracellular matrix and tubular connections between chondrocytes. The role of the interchondrocytic liquid crystals is considered in terms of the tensegrity hypothesis and non-apoptotic cell death. Phase transitions of the extracellular matrix are discussed in terms of self-alignment of chondrons, matrix guidance pathways and cartilage growth in the absence of mitosis. The possible role of nonenzymatic glycation reactions in cartilage dynamics is also reviewed.

Transmission light microscopy of structurally colored semithin cartilage sections.

Conversion of osmified tracheal cartilage constituents into an array of laminar interference gratings has been attained by three tandem reactions. Oxidation of semithin, LR white-embedded cartilage sections by acetic anhydride in dimethyl sulfoxide is the first step in the conversion process. Subsequent addition reactions of oxidized cartilage pyranoses and furanoses with thiocarbohydrazide constitutes the second step. Reduction of silver proteinate by thiocarbohydrazones and the concomitant coating of cartilage constituents with silver gratings completes the conversion of cartilage sections into a system of layered interference filters. In transmitted light, all components of converted cartilage display vivid structural colors, which allow detailed microscopic analysis of structurally colored cellular and extracellular cartilage constituents.

Effects of psoralen on the structural integrity of cultured osteoblasts. Phase contrast, immunofluorescent, and electronmicroscopic evaluation.

The 72-h dark interaction of cultured osteoblasts with 0.5-1.0 microgram/ml 8-methoxypsoralen (8-MOP) resulted in the accumulation of cytoplasmic lipid droplets (steatosis) in target cells. These methanol-extractable lipid droplets and 8-MOP, however, had no microscopically detectable effect on the organization of alpha-actin and beta-tubulin-containing filaments. On the ultrastructural level, psoralen effects ranged from negligible to unambiguous structural alterations of target cells. The latter consisted of blebbing, segmental deletions of the nuclear envelope, and structural unraveling of nucleoli. Moreover, the dilatation of the rough endoplasmic reticulum, the decrease in the number of ribosomes, and the extensive vacuolation of cytoplasm constituted yet another hallmark of the 8-MOP effect in the absence of light. Whereas psoralen has induced a number of structural alterations in some, but not in all osteoblasts, more than 95% of the target cells have, nonetheless, remained viable. Taken together, our results suggest that the dark reaction of psoralen with osteoblasts results mainly in transient structural alterations of affected cells.

Interaction of cultured rat osteoblasts with 8-methoxypsoralen during irradiation with long-wave ultraviolet light.

Rat osteoblasts in monolayer cell cultures have been irradiated with long-wave ultraviolet light (UVA) in the presence and without 8-methoxypsoralen (8-MOP). In the absence of 8-MOP, the exposures to UVA (3 x 10(-3)W.cm-2) for up to 30 min have not affected cellular viability, the rate of 14C-acetate incorporation, and alkaline phosphatase (AP) activity. However, it depressed 3H-TdR incorporation rates by osteoblasts. In the presence of 15 to 100ng of 8-MOP/ml, even 5-min irradiation of osteoblasts was sufficient to reduce DNA synthesis. Much higher (0.5 to 1.0 micrograms/ml) 8-MOP concentrations were required to depress lipid synthesis, AP activity, and the viability of irradiated cells. These results suggest that in osteoblasts the machinery of DNA synthesis is especially labile to photosensitization with 8-MOP and UVA, whereas UVA light by itself exerts a less potent inhibitory effect.

Morphologic and metabolic changes in rat osteoblast cultures during the dark reaction with 8-methoxypsoralen.

The dark reaction of 8-methoxypsoralen (8-MOP) with cultured rat osteoblasts did not cause significant changes in cellular replication rates or in the synthesis of RNA and proteins. Microscopic examination, however, revealed that the dark reaction resulted in massive accumulation of perinuclear lipids and in the statistically significant enhancement of alkaline phosphatase activity. A sharp, and statistically significant, upsurge of lipid synthesis in osteoblasts preceded microscopically detectable accumulation of lipids and occurred only during the initial, but not during the subsequent stages of the dark reaction. These results suggest that in the course of the dark reaction the plasma membrane of osteoblasts is a target of psoralen.

Lead tetraacetate-thiocarbohydrazide-silver proteinate method for light microscopy of polysaccharides.

Lead tetraacetate-thiocarbohydrazide-silver proteinate reaction sequence for light microscopy of polysaccharides was evaluated on Carnoy's fixed rat liver sections. The results of this evaluation suggest that, on the light microscopic level, the lead tetraacetate-thiocarbohydrazide-silver proteinate method may serve as a practical and histochemically specific alternative to the lead tetraacetate-Schiff reaction for the localization of tissue carbohydrates.

Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen.

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.

Calcium is required for the mitogenic activation of lymphocytes by periodic acid oxidation.

This investigation of Ca2+ requirements for the mitogenic activation of lymphocytes by periodic acid has shown that oxidation by periodate causes an immediate and transient increase of Ca2+ influx and efflux in oxidized cells. Oxidized lymphocytes maintained in the medium containing 0.2 mM Ca2+ failed to proliferate or to produce IL-2, whereas a 1.4 mM Ca2+ concentration was shown to be sufficient to sustain cellular proliferation and IL-2 secretion. These results indicate that mitogenic activation of lymphocytes by periodic acid oxidation is Ca(2+)-dependent.

Cytometric and electron microscopic studies of the direct interaction of divalent nickel with intact and chemically modified HuT-78 lymphoblasts.

Cytometric and ultrastructural studies on 24 hr cultures of intact, 1.0 mM H5IO6, and 0.1 mM SeO2-oxidized HuT-78 lymphoblasts were performed after their direct, 30 min interaction with 1.0 mM NiCl2. Except for moderately depressed cell viability, divalent nickel did not alter the progression of intact and oxidized target cells through the phases of the cell cycle. Although the plasma membrane remained structurally intact, marked distortion of mitochondria structure and increased osmiophilia were an invariable attribute of all nickel-pulsed cells. Moreover, numerous electron-opaque, intracellular depositions were detected in SeO2-oxidized, nickel-pulsed cells. It is concluded that the initial state of plasma membrane, and the interaction of nickel with other trace elements, have jointly determined the response of HuT-78 cells to brief and direct, divalent nickel pulses.

Rapid microscopic detection of malaria parasites permanently fluorochrome stained in blood smears with aluminum and morin.

Intra- and extracellular Plasmodium parasites in fixed blood smears are easily identifiable by fluorescence microscopy after brief mordanting with aluminum ammonium sulfate and staining with morin (3,5,7,1',4'-pentahydroxyflavanol). The intensely fluorescent preparations of stained parasites are strongly resistant to photodegradation and remained essentially unimpaired for two years.

Direct oxidation of lymphocytes by chromic acid does not induce blastogenesis.

Blastogenic and cytotoxic effects of hexavalent chromium were evaluated by direct, 2 and 20 min oxidation of lymphocytes by 10.0 to 0.0005 mM CrO3 at 0 degrees C. Oxidized cells exhibited concentration-dependent cytotoxicity and the inhibition of tritiated thymidine incorporation rates. When lymphocytes were oxidized first by 1.0 mM periodic acid (H5IO6) and thereafter by 1.0 mM CrO3, the viability and [3H]-TdR incorporation rates of sequentially oxidized cells were identical to the corresponding indicators of lymphocytes oxidized only by CrO3. The reversal of the oxidation sequence restored [3H]-TdR incorporation to control levels and increased cell survival. It is therefore concluded that direct interaction of hexavalent CrO3 with plasma membrane of lymphocytes results in concentration-dependent cytotoxicity and the inhibition of [3H]-TdR incorporation, but it does not induce blastogenesis.

Ultrastructural modification of the plasma membrane in HUT 102 lymphoblasts by long-wave ultraviolet light, psoralen, and PUVA.

Ultrastructural alterations of the plasma membrane in HUT 102 lymphoblasts were assessed after a 2-h interaction with a suprapharmacologic (15 micrograms/ml) concentration of 8-MOP, 2-h irradiation with UVA (2.1 mW/cm2), and the exposure of the HUT 102 cells to PUVA under the same conditions. The dark reaction of HUT cells with 8-MOP resulted in the disappearance of microvilli, the emergence of plasma-membrane-associated spherical bodies, formation of lamellar fungiform membrane evaginations, and, in approximately 1% of the cells, formation of uropods and cell capping. Except for uropod formation and cell capping, UVA has induced the same plasma-membrane alterations, and was more deleterious to structural cytoplasmic integrity than 8-MOP. Morphologic changes of the plasma membrane in PUVA-exposed cells tended to replicate structural alterations elicited independently during the dark reaction by suprapharmacologic 8-MOP concentrations. Partial retention of microvilli by cells after PUVA was the sole exception. In light of all available evidence we conclude that psoralen during the dark reactions interacts with plasma membrane lipids by as yet undisclosed mechanisms and that in addition to lipids, membrane proteins are also the primary target of the initial interaction of HUT 102 cells with psoralen during PUVA treatment.

Production of interleukin-1 beta by periodic acid-oxidized human peripheral blood mononuclear cells.

Interleukin-1 (IL-1) production by periodic acid (H5IO6)-oxidized human peripheral blood mononuclear (PBMN) cells was assessed by the thymocyte co-mitogenesis assay. Maximum IL-1 levels (approximately 1.2 U/ml) in the conditioned media of PBMN cells were registered within the first 24 hrs post-oxidation, whereas no IL-1 was detected in the media from 24 hrs control cultures. Thymocyte proliferation, driven by periodic acid-induced IL-1, was abolished by an antibody to IL-1 alpha and IL-1 beta. Quantitative analysis of IL-1-containing medium by radioimmunoassay (RIA) indicated that IL-1 beta comprised about 80% of total IL-1. Partial characterization of H5IO6-induced IL-1 beta indicated that it was identical to IL-1 produced by lipopolysaccharide-stimulated macrophages. It is concluded that oxidation of human PBMN cells by H5IO6 triggers synthesis and release of IL-1, most of which was in its IL-1 beta form.

Cytometric analysis of the proliferative capacity of HUT 102 lymphoblasts exposed to long-wave UV light and psoralen.

Cultures of HUT 102 lymphoblasts, comprised of near-diploid and hyperdiploid cells (11 and 22 pg of DNA/nucleus), were irradiated for 2 h by long wave ultraviolet light (UV-A) (2.1 mW/cm2), reacted for the same time in the dark with suprapharmacologic concentrations of 8-methoxypsoralen (8-MOP) (15 micrograms/ml) and exposed to 8-MOP and UV-A (PUVA) under identical conditions. Flow cytometric determinations of cellular DNA content and IdUrd incorporation indicated that UV-A irradiated cells incorporated IdUrd at the control levels and that the number of cells in S and G2 + M phases of the cell cycle likewise were identical to controls. The dark reaction of HUT 102 cells with 8-MOP has, however, resulted in a transient increase of IdUrd incorporation, but it has not affected cellular proliferation rates. On the other hand, no IdUrd incorporation was detected after PUVA. The analysis of the DNA content of intact and PUVA-exposed cultures showed that the number of near-diploid S-phase cells was greater 24 h after PUVA, whereas the number of hyperdiploid cells in all phases of the cell cycle had decreased. Under stated conditions the viability of HUT 102 cells and their cell cycle disturbances by PUVA are determined in part by the amount of DNA in target lymphoblasts.

Enhanced protein phosphorylation, IL-2 receptor expression, and IL-2 production precede cellular proliferation in lymphocytes oxidized by periodic acid.

Increased rates of protein phosphorylation, IL-2 receptor (IL-2R) expression, IL-2 production and DNA synthesis were quantified in rat lymphocytes oxidized by periodic acid (H5IO6). Enhanced phosphorylation of 97, 59 and 37 to 29 kDa proteins in lymphocytes was detected at 18 and 36 hrs post-oxidation, whereas IL-2R expression and IL-2 elaboration were at their maximum by 24 hrs. The number of oxidized cells entering the S-phase of the cell cycle and their thymidine incorporation rates reached their maximum at 72 hrs. These results indicate that H5IO6-induced blastogenesis elicits a progression of responses temporally similar to those of lymphocytes stimulated by lectins.

Ultrastructural cytochemistry of hepatic lysosomes and their protein components is selectively revealed by the ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate reaction.

Proteins in lysosomal membranes, lysosomes and within the transtubular network are readily accessible for electron microscopic analysis by a new three-step method. Oxidative deamination of tissue-bound amino acids by ninhydrin in aqueous dimethyl sulfoxide and the concomitant formation of corresponding carbonyl groups comprise the first step. The addition reaction of thiocarbohydrazide to tissue-bound carbonyl groups comprises the second step, while the reduction of silver proteinate by tissue-bound thiocarbohydrazones is the final step of this sequential method. Glutaraldehyde-fixed and osmified ultrathin sections of rat liver embedded in LR White were oxidatively deaminated for 24 h by 1% w/v ninhydrin in aqueous 75% v/v dimethyl sulfoxide (DMSO). They were then incubated for 40 min in aqueous 1% w/v thiocarbohydrazide (TCH) and stained for 30 min at 50 degrees C with silver proteinate (SP). The ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate (N-DMSO-TCH-SP) reaction proved to be chemically specific and highly selective for ultrastructural resolution of the internal structure of lysosomes and their protein components. We conclude that the N-DMSO-TCH-SP reaction is the method of choice for cytochemical elucidation of the protein ultrastructure of lysosomes and their enzymatic aggregates.

Localization of carbohydrates in thick and ultrathin LR white-embedded tissue sections oxidized by acetic anhydride in dimethyl sulfoxide.

The applicability of acetic anhydride (AA) in dimethyl sulfoxide (DMSO) for the oxidation of polysaccharide and their subsequent visualization with thiocarbohydrazide (TCH) and silver proteinate (SP) was evaluated on LR White-embedded thick and ultrathin liver sections. The results of these studies indicated that AA-DMSO-TCH-SP reaction is chemically specific on LR White-embedded tissues and that it offers distinct advantages for the localization of minute glycogen aggregates.

Periodic acid-induced blastogenesis of lymphocytes is independent of phosphoinositide turnover.

The blastogenic transformation of lymphocytes by periodic acid was investigated to determine if blastogenesis induced by this mitogen was preceded by phosphoinositide turnover as previously shown for the lectins. Although periodate oxidation stimulated nucleic acid synthesis and interleukin-2 production, no changes in phosphoinositide turnover could be detected when compared to control lymphocyte cultures. These data indicate that increased phosphoinositide turnover is not an absolute prerequisite for lymphocyte proliferation.