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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Establishment of primary cell cultures from human and canine parathyroid gland explants.

Primary parathyroid cell cultures were established from 22 canine and 20 human parathyroid glands explanted into Connaught Medical Research Laboratory-1415 (CMRL-1415) medium containing fetal calf serum (FCS). Initiation of cellular outgrowth and the rate of cellular propagation through the first subcultures were evaluated in various media. These included Roswell Park Memorial Institute-1640 (RPMI-1640), minimal essential medium (MEM), medium-199 (M-199) and Coon's modified Ham's F-12 (F12) and CMRL-1415 media.

Aneuploidy of glandular epithelial cells in histologically normal prostate glands.

The percentage of aneuploidy in normal prostate glands from subjects 13-38 years old and 45-66 years old ranged from 0-78% and 0-63%, respectively. In contrast to adults, aneuploidy was absent in fetal and postnatal prostates. It is concluded that aneuploidy is a fundamental attribute of histologically normal adult prostate glands.

Effect of mitogens on the cell cycle progression and the quantification of T-lymphocyte surface markers in acquired immune deficiency syndrome.

The cell cycle progression and viability of stimulated and intact lymphocytes from 20 subjects with acquired immune deficiency syndrome (AIDS) was determined by flow cytometry. As compared to controls, 62% less AIDS lymphocytes, cultured for 72 hr in the presence of lectins (Con-A, PHA, PWM), had entered the proliferative phases of the cell cycle, while the respective value for periodic-acid (H5IO6)-stimulated cells was 34%. The helper-suppressor ratios and natural kill cell percentages of the unstimulated and PHA-activated AIDS lymphocytes increased approximately 3-fold after 72 hr in culture.

A modified periodic acid-thiocarbohydrazide-silver proteinate staining sequence for enhanced contrast and resolution of glycogen depositions by transmission electron microscopy.

Oxidation of araldite-embedded liver sections by 1% w/v aqueous H5IO6 for 15 min and a 5-min reaction of carbonyls with 1% w/v thiocarbohydrazide in 10% v/v acetic acid was employed for subsequent staining of glycogen with silver-proteinate (S-P). The network of branching intracellular glycogen aggregates was revealed by 15-min staining with S-P, whereas 24 hr incubation in S-P was necessary to enhance the contrast of glycogen inclusions. We conclude that the proposed modification of glycogen staining readily affords the means for its localization at a desired level of contrast and resolution.

Serum calcitonin in thyroid disorders and in pheochromocytoma kindred.

Serum calcitonin was determined by RIA in 59 healthy subjects (Group 1), 49 randomly selected patients with treated or untreated thyroid disorders (Group 2), and in 12 kindred of a pheochromocytoma index case (Group 3). Although most subjects in Group 2 had normal calcitonin levels, there were significant (p less than 0.001) differences between all three groups.