Robert M. Rosenbaum

ENZYMIC HISTOCHEMISTRY OF GRANULAR COMPONENTS IN DIGESTIVE GLAND CELLS OF THE ROMAN SNAIL, HELIX POMATIA

1. Cytochemical visualization methods for activity of acid phosphatase, β-glucuronidase, aminopeptidase and non-specific and E-600-resistant esterases were applied to digestive gland tissue from starved and feeding Helix pomatia. Other cytochemical methods used included Bakers acid hematein for phospholipid and the periodic acid Schiff method.2. Calcareous cells stained only for acid phosphatase activity, by both lead-salt and an azo dye method. Calcium granules within these cells did not stain with the azo dye method while false-positive reactions in the granules were always obtained with the lead-salt method. Some increase in enzymic activity was detected in the cytoplasm of feeding animals. Secretory-resorption (SR) cells showed little activity for acid phosphatase.3. The yellow granules in SR cells stained for β-glucuronidase activity in both starved and feeding animals. After feeding, SR cells showed an increase in enzymic activity, both in granules and more diffusely in the cytoplasm. Although some...

Effects of prostaglandins E2 and F2α on lecithin biosynthesis by cultured lung cells

Transformed cells from human lung carcinoma (Line A549), resembling type II pneumocytes, were cultured in monolayer at 37 degrees C and incubated for five hours with 3H-choline and 14C-palmitate in the presence of various concentrations of prostaglandins (PGS) E2 and F2alpha. In the control (no PG) the level of % palmitate incorporation was 13.5 x as high as that of choline, after taking isotope dilution into account. Between the concentrations studied, 0.1 and 10 muM, both prostaglandins stimulated markedly the incorporation of both precursors, though choline up to 3 x better than palmitate. This was indicated by a change in the palmitate/choline incorporation ratio from 13.5 to as low as 4.2. At the lowest PG concentration, 0.1 muM, PGE2 was much more effective than PGF2alpha in stimulating the incorporation of both precursors.

CULTURE CHARACTERISTICS OF CELLS DERIVED FROM TYPE II PNEUMOCYTE ENRICHED FRACTIONS FROM RABBIT AND RAT

SummaryType II cell enriched fractions were isolated from rabbit and rat lungs using density gradient centrifugation. Cultures established from these fractions contained predominantly cells similar in most morphological respects to type II pneumocytes. These were in continuous replicating culture for 1 year and still exhibited contact inhibition. Membrane-bound structures reminiscent of, but no longer strictly identical to, type II cell lamellar cytosomes were seen in cells from these long-term cultures although their numbers were reduced in comparison to lamellar bodies in freshly isolated cells. Mitochondrial numbers and sizes, determined morphometrically, were reduced after culture in comparison to freshly isolated type II cells and those in situ. Phosphatidylcholine was synthesized by these cells and released into the extracellular medium. Application of laser activated electronic sizing data, confirmed by direct micrometry, demonstrated a significant increase in cell size as a function of culture. This sizing data, after prior confirmation by electron microscopy, was used as an aid in identifying type II cells and macrophages in dispersion, especially with those cells derived from rabbit lungs.

Studies with the schistosomacide hycanthone: Inhibition of macromolecular synthesis and its reversal

Abstract The new schistosomacide, hycanthone, was studied with regard to its effects upon macromolecular synthesis in mammalian (HeLa) cells. At concentrations as low as 5 × 10−8M, hycanthone reduces plating efficiency of HeLa cells. At 10−6M, effects on population growth are evident by the second generation. At 5 × 10−6M, RNA synthesis is markedly depressed while DNA and protein synthesis are relatively unaffected. New ribosomal RNA synthesis is rapidly inhibited by hycanthone at 5 × 10−6M, and its processing is markedly delayed. During hycanthone inhibition 28S RNA does not appear in the cytoplasm. Hycanthone inhibition was shown to be readily reversible by washing the cells in fresh medium. The case with which inhibition can be reversed in vitro suggests that it may be necessary to maintain blood levels of this compound if permanent eradication of schistosomal infection is to be attained. Further work must be done to establish this point.

Effects of temperature on the binding of acid and basic dyes, including reference to metachromasy

SummaryWith a number of anionic dyes, a positive temperature effect occurs in the staining of tissues, whereas with a number of cationic dyes, a negative temperature effect occurs. The positive effect involves increased dye binding at 450 as compared to 50; the negative effect involves decreased dye binding at the higher temperature. To obtain these effects, dye concentration must be low and staining must be continued to equilibrium, i. e., for about 24 hours. These facts suggest that the temperature effects may depend in part on the degree of ionization of tissue components and also on competition between tissue components and dye for chromotrope.Deamination of sections depresses acidophilia and enhances basophilia but fails to obliterate the temperature effects.With metachromatic basic dyes, despite reduction in staining of highly acidic compounds at high temperature, the color remains metachromatic. This result differs from that obtained in the test tube and is probably explained by the fact that the chromotropes are relatively fixed in position in tissue sections.ZusammenfassungBei Gewebsfärbungen lassen einige anionische Farbstoffe eine „positive Temperaturwirkung“ erkennen, d. h. eine Zunahme der Farbstoffbindung beim Steigen der Temperatur von 50 auf 450. Bei kationischen Farbstoffen liegt dagegen meist eine „negative Temperaturwirkung“ vor, d. h. eine Verminderung der Farbstoffbindung bei höherer Temperatur. Dieser Effekt kann nur bei niedriger Farbstoffkonzentration und längerer Färbedauer (etwa 24 Std) erzielt werden. Die Temperaturwirkung hängt wohl z. T. vom Grad der Ionisation der Gewebsbestandteile ab wie vom Wettbewerb zwischen Gewebsbestandteil und Farbstoff für Chromotrope.Desaminierung vermindert die Acidophilie und steigert die Basophilie entsprechender Gewebebezirke. Die Temperaturwirkung bleibt erhalten.Trotz Minderung der Anfärbbarkeit von stark sauren Verbindungen bei hoher Temperatur bleibt die Metachromasie mit metachromatisch wirkenden basischen Farbstoffen im histologischen Schnitt erhalten. Im Reagensglasversuch liegen andere Ergebnisse vor. Dieser Unterschied erklärt sich wahrscheinlich aus der ziemlich festen Bindung zwischen Chromotrop und Gewebe.

Gastrular arrest and the control of autolytic activity in the egg of Rana pipiens: the comparative effects of oxygen, supramaximal temperature, and dinitrophenol

Abstract 1. 1. Eggs of R. pipiens exposed to 4–7 atmospheres (abs.) of pure oxygen for from 12 to 24 hours while in pregastrular stages are unable to develop beyond the yolk plug stage. Eggs arrested in this way undergo no visible autolytic changes but remain intact for several weeks provided relatively aseptic conditions exist. Developing eggs treated with sulfhydryl compounds such as reduced glutathione, BAL, and β-mercaptoethylamine causes eggs in gastrular arrest rapidly to undergo autolysis. 2. 2. Employing a casein-urea substrate, the catheptic activity of eggs and embryos of R. pipiens was determined at various stages from four cells to tailbud. The most significant increase in enzyme activity occurred immediately following gastrulation and continuing through the tailbud stage when intensive yolk utilization by the embryo is beginning. 3. 3. The catheptic activity of oxygen-arrested gastrulae was found to be constant and low as long as 100 hours after initiation of the gastrular block. No recovery of enzyme activity could be demonstrated, and these eggs showed no autolytic changes for many days after their return to normal air. The ability of oxygen to inhibit autolysis appears to be a reflection of the sulfhydryl dependency of the cathepsins and is suggestive of a direct effect on endogenous cellular SH by molecular oxygen. 4. 4. Eggs blocked at gastrulation by exposure to supramaximal temperatures or to dinitrophenol underwent rapid autolysis as their catheptic activity markedly increased. 5. 5. No direct relationship between the inhibition of cathepsin by oxygen and the oxygen-induced gastrular arrest could be demonstrated. 6. 6. The probable relationship between proteolytic activity and oxidative metabolism are considered in relation to the comparative effects obtained with gastrular arrests produced by exposure to oxygen, supramaximal temperature, and dinitrophenol. The possibility of oxygen acting on specific subcellular particles and thereby controlling autolysis is presented.

Resistance and Susceptibility to High Oxygen Pressures in the Early Development of the Frog, Rana pipiens

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In vitro activity of metronidazole on Entamoeba histolytica in axenic culture

Abstract Various strains of axenic Entamoeba histolytica were exposed to concentrations of metronidazole ranging from 0·5 μg./ml. to 50 μg./ml. Observations were made every 4 hours with the aid of light and phase microscopy, as well as electron microscopy. Neutral red staining was also employed in an effort to distinguish live from dead amoebae. All strains survived 120 hours in 0·5 and 1·0 μg./ml. without discernible effect. At 2 μg./ml. strains of F-22 and 301 died within 24 hours, but strain HK-9 survived. Amoebae of all strains studied died at 4 μg./ml. within 20 hours. Early non-specific changes were visible by electron microscopy. In vitro amoebacidal levels of metronidazole are noted to be well below the serum concentration attained during therapy for amoebiasis.

Isolation and characterization of plamsa membrane from monolayer cultures of epithelial type II lung cells

Abstract Pneumocyte type II produces a phospholipid, dipalmitoyl lecithin, which is stored in and secreted from the cells inclusion bodies and is indispensable for alveolar stability. Cloned rat lung type II cells were harvested at monolayer confluence and homogenized in swelling buffer. After sequential differential centrifugations, the crude membrane fraction was subjected to discontinuous sucrose density gradient centrifugation at 65,000 × g . Quality of the relevant fractions was monitored by enzyme activities and phase contrast and electron microscopy of two major bands at densities 1.16 and 1.18, respectively. The less dense band contained only small quantities of organelles, little cytochrome c oxidase, and some glucose 6-phosphatase, but had a significant (Na + , K + )-ATPase activity; this and ultrastructural evidence certified the product as a suitable plasma membrane preparation. Upon sodium dodecyl sulfate-poly acrylamide gel electrophoresis, the protein pattern consisted of 11 major protein bands between 13,000 and 68,000 M r ranges, and several minor ones. The lipid pattern was studied by two-dimensional thin layer chromatograpy, followed by various group reactions (e.g., amine, unsaturation, phosphorus, sugars). In the two major phospholipids, phosphatidyl choline and phosphatidyl ethanolamine, palmitic acid was the least abundant of four major fatty acids, accounting for 14.20% in phosphatidyl choline and 5.70% in phosphatidyl ethanolamine, whereas the most abundant were stearic and palmitoleic with about 28% each in phosphatidyl choline, and palmitoleic (29.90%) and oleic (23.05%) in the ethanolamine phosphatide. Apparently, the palmitic acid containing phosphatidyl choline must be in the lamellar inclusion bodies of type II cells and not in their plasma membranes.

Cultured lung cells: effects of isotopic dilution and some undefined stimulation on the incorporation of radiolabeled palmitate and choline into phosphatidyl choline (lecithin).

Abstract Using radiolabels, we studied the effect of certain experimental conditions on the incorporation of choline and palmitate into phosphatidyl choline (lecithin) of cloned type II rat lung cells. When the label was changed from methyl-3H to 1,2-14C the incorporation of choline was reduced to 1/3; in contrast, when the label was moved from 1-14C to 9,10-3H, the incorporation of free palmitate was more than doubled. Removal of choline from the culture medium caused trebling of choline incorporation and appreciable decrease in palmitate uptake, indicating respectively an expected effect of increased choline label concentration in the absence of carrier, and a marked dependence of palmitate on choline incorporation. Removal of fetal calf serum produced more than 2/3 decrease in palmitate incorporation (instead of an expected increase because of palmitate isotope concentration), whereas choline uptake was not affected, meaning respectively that either serum hormones or serum lipids, or both simultaneously, are important for palmitate but not for choline incorporation. This is only the beginning of a host of studies required to clarify the role of fetal calf serum constituents onto lecithin biosynthesis in cultured lung cells and finally gain a full appreciation of the biosynthetic pathways of phosphatidyl choline (“surfactant”) in type II cells of alveolar epithelium.