Evidence of morphological and physiological transformation of mammalian cells by strong magnetic fields.

Cultures of L-929 and WI-38 cells, frozen to 4.2 degrees K and exposed for 4 to 8 hours to 5000-oersted magnetic fields, were markedly inhibited in their growth as compared to controls. In cultures grown on cover slips, approximately 7 days after exposure, morphologically distinct cells emerged and were propagated from generation to generation; 3 weeks later, in flask cultures, contact inhibition was abolished. It is concluded that under certain experimental conditions, strong magnetic fields induce morphological and physiological transformations of target cells.

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. The natural killer (NK) cell fraction of the PHA-stimulated and unstimulated AIDS cultures comprised approximately 20% as compared to 10% in controls. However, no changes in the percentages of T-lymphocytes were detected in the AIDS cell cultures. Throughout the culture period, viability of the unstimulated AIDS lymphocytes exceeded 90%, whereas in stimulated cultures it fluctuated within the 65-90% range. It is concluded that the liability of AIDS lymphocytes to mitogens is probably a direct consequence of the human immunodeficiency virus (HIV) infection.

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.

Quantitative determination by bioassay of photoactive 8-methoxypsoralen in serum.

Photoactive 8-methoxypsoralen (8-MOP) levels in human and animal sera were determined by bioassay with Staphylcoccus aureus serving as the test organism. 8-MOP was extracted from sera with as 9:1 (V/V) ethyl acetate-n-hexane mixture and reconstituted in an aqueous medium following evaporation of the extractant. Bacterial suspensions containing extracted 8-MOP were irradiated for predetermined intervals with longwave ultraviolet light (UVA) at an intensity of 2.1 mw/cm2. Cultures containing known amounts of 8-MOP were used as standards and cytotoxicity (i.e., drug levels) determined by colony counts. The detection limit for 8-MOP was 5 ng/ml with an accuracy of +/- 10% above 10 ng/ml. Concomitant determinations of 8-MOP levels by high pressure liquid chromatography (HPLC) were in excellent agreement with the bioassay results.

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.

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.

Permanent fluorescent staining of nucleic acids in isolated cells.

Staining of fixed cells, blood smears and chromosomes with 0.1% w/v of 3, 5, 7, 2', 4'-pentahydroxyflavanol (morin dehydrate) in 70% ethanol after brief mordanting with 5% w/v aluminum ammonium sulfate results in permanent fluorescence of cellular nucleic acids. Incubation in ribonuclease solution before mordanting, or 5-min hydrolysis with INHCL at 60 degrees C selectively abolishes RNA fluorescence, while the incubation in deoxyribonuclease solution abolishes DNA fluorescence. The morin-mordant complex bound to nucleic acids is stable to photodegradation and up to five years of storage.

Selective histochemical localization of polysaccharides in tissue sections oxidized by acetic anhydride in dimethyl sulfoxide.

Oxidation of tissue sections by 25-30% (v/v) acetic anhydride (AA) in dimethyl sulfoxide (DMSO) resulted in facile induction of tissue carbonyls readily localized with Schiff's reagent and o-dianisidine but not with the 3-hydroxy-2-naphthoic acid hydrazide-tetraazotized diorthoanisidine method. Carbonyls generated by AA-DMSO oxidation were confined predomintly to substrates containing pyranosides. Oxidized furanosides, as represented by deoxyribonucleic acid and ribonucleic acid, gave only a residual color reaction. The AA-DMSO method possesses an advantage in that the oxidation of tissue polysaccharides does not proceed beyond the formation of carbonyly and is particularly suited for use after formol fixation.

The in situ determination of melting-solidification points of lipid inclusions in fixed cultured cells.

Melting-solidification points of microscopic lipid inclusions in fetal human kidney cultures were determined visually by gradual heating of formol-fixed cell monolayers while immersed in dilute aqueous solutions of Nile blue A. Intracellular lipid inclusions stained red when their melting point was reached and reverted to blue color at the solidification temperature. The melting-solidification points of intracellular lipid inclusions in kidney cells were determined to fall within 19-25, 40-50, and 60-65 degrees C range.

Dimethyl sulfoxide-lead tetraacetate method for histochemical oxidation of polysaccharides.

Oxidation of fixed tissues and unfixed peripheral blood smears by 1% (w/v) lead tetraacetate in dimethyl sulfoxide for 10 to 60 min resulted in facile induction of tissue carbonyls readily demonstrable with Schiff's reagent and by sodium bisulfite addition reaction, followed by toluidine blue staining at controlled pH. Tissue carbonyls represented a broad range of oxidation-labile substrates and included neutral polysaccharides (glycogen). The oxidation procedures were not destructive to tissues and were characterized by technical simplicity and consistent reproducibility, thus affording a substantial improvement over the hitherto used methods of histochemical oxidation by lead tetraacetate.

Oxidation of tissue polysaccharides by periodic acid in dimethyl sulfoxide and its anhydrous and aqueous mixtures.

Periodic acid (1% w/v) solvated by anhydrous dimethyl sulfoxide (DMSO) readily induced a strong Schiff reaction in a variety of structures containing polysaccharides, but not glycogen. With the increasing amounts of water added to DMSO, glycogen was also oxidized, while the selective localization of other polysaccharides remained unimpaired. Periodate, solvated in the anhydrous acetic acid-DMSO mixture, rapidly induced concomitant oxidation of nucin and glycogen-containing structures. Sodium bisulfite addition derivatives of carbonyls, induced by periodate oxidation in DMSO, were stained meta- and orthochromatically with toluidine blue at controlled pH. Certain metachromatic tissue components were strongly birefringent in polarized light in contrast to the identical structures oxidized by aqueous periodate. Marked differences in staining reactions elicited in identical structures by periodate in DMSO as compared with aqueous periodate suggest that DMSO-periodate method considerably enhances the range of histochemical oxidations by periodate.

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.

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.

Kinetics of [14C-5] 8-methoxypsoralen distribution in rabbits.

Distribution, kinetics and localization of 8-methoxypsoralen (8-MOP) was determined in rabbits over 24 h following i.v. administration of [14C-5] labeled and carrier 8-MOP at respective concentration of 50 muCi and 5 mg/kg. Peak levels were reached by kidneys at 1 h (18,500 ng/g) at 2 h by liver (2,470 ng/g) and at 8 h by bile (64,000 ng/g). Muscles, lymphatic tissues and brain showed low drug uptake (800 ng/g). Endocrine organs also had low drug concentration. In gastrointestinal tract the maximum level of 8-MOP was 1,500 mg/g at 1 h in jejunum. Plasma 8-MOP concentration was 3,700 ng/ml at 5 min post injection with 100 ng/ml still detectable at 24 h. Urine label concentration peaked at 1 h (4 x 10(5) cpm/ml) and was 2 x 10(2) cpm/ml at 24 h. The intact skin concentration was at the maximum during the first 30 min (1,958 ng/g) declining progressively thereafter to 155 ng/g at 24 h. The UVA irradiated skin (320-380 nm at the rate of 14.2 mW/cm2 x s-1 for 1 h) had a higher 8-MOP concentration (2,834 ng/g at 1 h and 280 ng/g at 24 h).

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.

The interaction of bisulfite and its free radical analog-nitroxyldisulfonate with periodic acid-oxidized lymphocytes and their effect on blastogenesis.

Nitroxyldisulfonate [Fremy's salt; (KSO3)2NO.] and bisulfite (NaHSO3) have abolished periodic acid (H5IO6)-induced blastogenesis of human peripheral blood lymphocytes (HPBL), but only inhibited the blastogenic response of H5IO6-oxidized rat and mouse lymphocytes, as determined by the rates of nucleic acids synthesis, BrdUrd incorporation and by cell numbers in S + G2 + M phases of the cell cycle. The viability of the intact human, rat and mouse lymphocytes remained essentially unimpaired by 30 min pulses of 1 mM Fremy's salt or bisulfite. The marked inhibition of periodic acid-induced blastogenesis, exerted by Fremy's salt and by bisulfite, was attributed to the effect of the corresponding carbonyl addition derivatives formed in situ of the oxidized cell membranes. Consequently, it is concluded that Fremy's salt like bisulfite possibly forms addition derivatives with membrane carbonyls of viable target cells.

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.

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.

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. Of the five patients in Group 2 with high serum calcitonin, one had medullary cancer of the thyroid, one had multiple endocrine neoplasia, one had acromegaly, and two remained undiagnosed. Increased serum calcitonin levels were also found in seven of 12 normotensive relatives of a patient with pheochromocytoma. It is therefore concluded that high serum calcitonin levels in patients with thyroid disorders strongly suggest the presence of C-cell neoplasia or medullary cancer of the thyroid.

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.