(Na (+) + K (+)-stimulated ATPase of human kidney, normal and adenocarcinoma. Phosphorylation and inhibition by antitumor proteins

(Na⁺+K⁺)-ATPase was purified from human kidney of normal and tumor tissue with specific activities of 100.0 and 16.6 μmol Pi/mg/h, respectively. The antitumor proteins, macromomycin, largomycin, and NSC 327459 (50 μg/ml each) caused 70 to 90% inhibition of (Na⁺+K⁺)-ATPase from tumor tissue, whereas auromomycin had no effect. (Na⁺+K⁺)-ATPase from both sources could be phosphorylated by rabbit muscle protein kinase; there was 3 to 6-fold stimulation of phosphorylation by cyclic AMP. Phosphorylation resulted in 70 to 80% decrease in (Na⁺+K⁺)-ATPase activity, and caused the normal enzyme to become sensitive to inhibition by macromomycin.     

Local measurement of lateral motion in erythrocyte membranes by photobleaching technique

The lateral diffusion coefficients ($\text{D}$) of the molecular fluorescence probe 3,3′-dioctadecylindocarbocyanine iodide (DII) in the membrane of discoid erythrocyte ghosts has been measured with the photobleaching technique between 7°C and 40°C. A fluorescence microscope which allows bleaching experiments within small local fields (approx. 1 μm²) at high magnification (X1600) has been used for these measurements. The diffusion coefficient increases from $\text{D = 9 · 10}{}^{\mathrm{?10}}\text{cm}{}^2\text{/s}$ to $\text{D = 7.5 · 10}{}^{\mathrm{?9}}\text{cm}{}^2\text{/s}$ from 7 to 40°C. An increase in membrane fluidity between 12°C and 17°C indicates a conformational change of the lipid bilayer moiety in this temperature region. The diffusion coefficient measured in the regions between the spicules of echinocytes is appreciably smaller than in the untransformed discoid ghosts. In the myelin tubes originating from cells, the lateral diffusion is somewhat larger (about a factor of 2) than in the non-transformed ghosts. With the fluorescence probe technique the rate of growth of myelin tubes of 0.3 μm diameter has been estimated.     

Propidium: induction of petites and recovery from ethidium mutagenesis in Saccharomyces cerevisiae.

It was shown that petite induction in growing cells of $\text{Saccharomyces}$$\text{cerevisiae}$ by ethidium was strongly stimulated by the presence of propidium, a phenanthridinium dye of similar structure to ethidium. Propidium itself also induced petites in growing but not in resting cells. Furthermore, propidium could prevent petite induction in resting cells and caused recovery from ethidium induction with prolonged incubation. A possible mode of action of propidium in the ethidium-induced petite mutagenesis is discussed.     

Induction of a chloride conductance in gastric vesicles by limited trypsin or chymotrypsin digestion or ageing

Transport activity of the hog gastric (H⁺ + K⁺)-ATPase system was measured either as the formation of a proton gradient using the dye probe acridine orange or as the formation of a proton diffusion potential using the cyanine dye 3,3′-diethyloxdicarbocyanine iodide in the presence of the protonophore tetrachlorosalicylanilide. The development of these gradients has been compared in K⁺ media in the presence of either Cl^? or SO₄^2? as the anionic species. This comparison of proton diffusion potential formation to proton gradient formation has been used to demonstrate that a Cl^? conductance in this vesicular system results from limited enzymic digestion with either trypsin or α-chymotrypsin and from the ageing process itself. The possible significance of this finding is discussed.     

Lymphangitic sporotrichosis: An uncommon bilateral localization

Sporotrichosis is a mycotic disease caused by cutaneous inoculation of the dimorphic fungus Sporothrix schenckii. The primary lesion can spread and often develop a unilateral lymphocutaneous lesions or, rarely, disseminated disease. We report a lymphangitic sporotrichosis case with ulcerated erythematous nodules distributed bilaterally on the posterior and medial aspect of the both legs, probably due to multiple inoculations. The treatment with oral potassium iodide was satisfactory. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mitochondrial dysfunction in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize: analysis at the population and individual cell level

Summary The urf13TW gene, which is derived from the mitochondrial T-urf13 gene responsible for Texas cytoplasmic male sterility in maize, was expressed in Saccharomyces cerevisiae by targeting its translation product into mitochondria. Analysis by oxygraphy at the population level revealed that in the presence of methomyl the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory substrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrate oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in significant inhibition of oxygen uptake. This inhibition is eliminated and oxygen uptake is stimulated by subsequent addition of NAD⁺. Using 3,3-dihexyloxacarbocyanine iodide [DiOC₆(3)] as probe, interactive laser scanning and flow cytometry, which permit analysis at the individual cell level, demonstrated that specific staining of the mitochondrial compartment is obtained and that DiOC₆(3) fluorescence serves as a measure of the membrane potential. Finally, it was shown that, as in T cytoplasm maize mitochondria, HmT toxin and methomyl dissipate the membrane potential of yeast mitochondria that carry the foreign protein. Furthermore, the results suggest that the HmT toxin and methomyl response is related to the plasmid copy number per cell and that the deleterious effect induced by HmT toxin is stronger than that of methomyl.     

Characterization of Pneumocystis carinii Preparations Developed for Lipid Analysis

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10⁸-10⁹ organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.     

Flow cytometric analysis of intracellular pH in cultured opossum kidney (OK) cells

Summary Suspensions of OK cells (a continuous renal epithelial cell line originating from the opossum kidney) were examined by flow cytometry. Three parameters were evaluated simultaneously; cell integrity as assayed by propidium iodide fluorescence, cell size as measured by time-of-flight, and intracellular pH as measured by fluorescence of 2,7-bis-(2-carboxyethyl)-5,6 carboxyfluorescein (BCECF). The suspension was shown to be composed of both intact singlets and doublets of cells, and no difference was noted in the behavior of these two populations with respect to the resting intracellular pH, or of the response of intracellular BCECF to changes in pH. Evidence suggests that using NH₄ prepulses to create an acid load broadens the intracellular pH distribution. The population of OK cells demonstrates a recovery from this acid load which is very homogeneous with respect to its sensitivity to Na⁺ removal or EIPA (ethylisopropyl-amiloride), suggesting that virtually all cells utilize Na⁺/H⁺ exchange for this recovery. The data also suggest heterogeneity in the cellular pH recovery from an acid load with respect to the observed rates of Na⁺/H⁺ exchange. Despite this heterogeneity, the Na⁺/H⁺ exchanger is observed to focus the resting intracellular pH of the population to approximately pH 7.4–7.5. The response of the population to PTH suggests that the majority of cells respond to the hormone, and that the total Na⁺/H⁺ exchange in individual cells is only partially inhibited even in the presence of saturating PTH concentrations.     

Celecoxib induces p53-PUMA pathway for apoptosis in human colorectal cancer cells

Celecoxib, a clinical non-steroidal anti-inflammatory drug, displays anticarcinogenic and chemopreventive activities in human colorectal cancers, although the mechanisms of apoptosis by celecoxib are poorly understood. The existence of functional p53 but not securin in colorectal cancer cells was higher on the induction of cytotoxicity than the p53-mutational colorectal cancer cells following celecoxib treatment. The p53-wild type HCT116 cells were more susceptible to increase ∼25% cell death than the p53-null HCT116 cells after treatment with 100 μM celecoxib for 24 h. Transfection with a small interfering RNA of p53 reduced the celecoxib-induced cytotoxicity in the RKO (p53-wild type) colorectal cancer cells. Celecoxib (80-100 μM for 24 h) significantly increased total p53 proteins and the phosphorylated p53 proteins at serine-15, -20, -46, and -392 in RKO cells. However, the phospho-p53 (serine-15, -20, and -392) proteins were presented on the nuclei of cells but the phospho-p53 (serine-46) protein was located on the cytoplasma of apoptotic cells following treatment with celecoxib. Interestingly, the p53 up-regulated modulator of apoptosis (PUMA) protein, which located on the mitochondria, was induced by celecoxib in the p53-functional colorectal cancer cells but not in the p53-mutational cells. Together, this study provides the first time that celecoxib induces the various phosphorylated sites of p53 and activates p53-PUMA pathway, which potentiates the apoptosis induction in human colorectal cancer cells.