Phenotypic exclusion in mouse melanoma-rat hepatoma hybrid cells: pigment and albumin production are not reexpressed simultaneously.

Hybridization of cells of defined and different histotypes has been carried out to investigate whether the expression (or reexpression) of parental functions is mutually exclusive, as is expected if the generally assumed rule of discreteness of differentiation applies to hybrid cells. A cross of pigmented mouse melanoma cells and albumin-producing rat hepatoma cells gave rise to hybrids containing essentially one set of chromosomes from each parent and producing neither melanin nor albumin. Cells of one hybrid clone are shown to retain the potential to reexpress both parental differentiations. Successive subclonings of this hybrid have shown that cells which reexpress one function may retain the potential to reexpress the other, and that freshly isolated, morphologically homogeneous subclones may produce pigment or albumin, but not both; there successive and exclusive shifts of phenotype are documented, and in these cases, chromosome loss is very slight. The use of immunoadsorbed antisera has revealed that most (if not all) of the albumin produced by the hybrid cells is of the mouse type. We conclude that both parental determinations are retained by the hybrid cells, and that the parental differentiations are reexpressed only in a mutually exclusive fashion.     

The use of a phospholipase A-less Escherichia coli mutant to establish the action of granulocyte phospholipase A on bacterial phospholipids during killing by a highly purified granulocyte fraction

Phospholipase A₂ present in a highly purified, potently bactericidal, fraction from rabbit granulocytes produces net bacterial phospholipid degradation during killing of a phospholipase A-less strain of Escherichia coli. In the wild-type parent strain phospholipid breakdown is caused not only by the action of phospholipase A₂ but also by phospholipase A₁, indicating activation of the most prominent phospholipase of E. coli. This activation occurs as soon as the bacteria are exposed to the granulocyte fraction. Phospholipid breakdown by both phospholipases A is dose dependent but reaches a plateau after 30–60 min and at higher concentrations of the fraction.Phospholipid degradation is accompanied in both strains by an increase in permeability to actinomycin D that is also dose dependent. Even though net hydrolysis of phospholipids is greater in the parent strain than in the mutant, the increase in permeability is the same in the two strains.The addition of 0.04 M Mg²⁺, after the effects on phospholipids and permeability have become manifest, initiates in both strains the restoration of insensitivity to actinomycin D, the net resynthesis of phospholipids, and the disappearance of monoacylphosphatides and the partial disappearance of free fatty acids that had accumulated. Loss of ability to multiply is not reversed by Mg²⁺ in either strain. Less than 5 μg of granulocyte fraction causes loss of viability of from 90 to 99% of $\text{1 × 10}{}^8$ microorganisms of both strains. However, at lower concentrations the parent strain is considerably more sensitive to the bactericidal effect of the granulocyte fraction than the mutant strain.     

A heme-nonapeptide tracer for electron microscopy

Summary A heme-nonapeptide (H-9-P)¹, applicable to electron microscopic cytochemistry via peroxidase-like activity, was prepared by passing horse heart cytochrome c through a column with Sepharose and covalently attached trypsin. After purification by column chromatography (Sephadex G50 Superfine, Biogel P-2) a maximal yield of 50% and purity of >99% was achieved. A concise schedule allows for inexpensive preparation of H-9-P with standard laboratory equipment. H-9-P has the following properties: Its structure is (14) Cys-Ala-Gln-Cys-His-Thr-Val-Glu-Lys (22) with heme attached to Cys (14) and (17). MW=1630, pI=4.95, _E(max) ^{pH 7} = 397.5 nm, _{22 °C, pH 7} ^{397.5 nm} = 1.11 × 10⁵ [Liter/Mole x cm]. With the use of a diaminobenzidine-H₂O₂-medium — as applied for cytochemistry — we determined spectrophotometrically a pH_opt=12.5 and an apparent K₅ = 3.14 × 10^{– 3} [M]. Glutardialdehyde leads to considerable de-activation and, according to SDS-polyacrylamide-gel-electrophoresis, to diffuse crosslinking accompanied by a shift of the active pH-region towards neutral pH values. An attempt was made to optimize the cytochemical assay. The peroxidase-like activity of H-9-P is well comparable to that of other heme-tracers; only horseradish peroxidase has a higher turnover number. When injected to mice or added to cell suspensions, even high concentrations of H-9-P did not entail any signs of toxicity.Abbreviations AAA amino acid analysis - AHC ammoniumhydrogencarbonate - BSA bovine serum albumin - Cyt c cytochrome c - DAB 5,3-diaminobenzidine - GA glutardialdehyde - H-8-P heme-octapeptide - H-9-P heme-nonapeptide - H-11-P heme-undecapeptide - HR-POX horseradish peroxidase - MW molecular weight - PAGE polyacrylamid-gel-electrophoresis - pI isoelectric point - SDS sodiumdodecylsulphate - SG-TLC silicagel-thin-layer-chromatography This work was supported by the Österreichische Forschungsfonds     

Differentiation is not restored in hybrids between independent variants of a rat hepatoma

Crosses have been undertaken between cells of three independent clones of dedifferentiated rat hepatoma variants to investigate whether "complementation" leading to restoration of the original differentiation would occur. Hybrids were examined between ten days and two months after fusion for the presence of intracellular albumin and for their ability to proliferate in glucose-free medium where survival requires activity of the liver-specific gluconeogenic enzymes. In none of the three possible crosses involving the three variants was evidence of reexpression of hepatic functions obtained.     

Difference in chromatin sensitivity to heparin of chronic atrophic gastritis, of carcinoma-free and carcinoma-bearing stomach in comparison with normal gastric mucosa and gastric carcinoma as revealed by flow cytometry

DNA distribution patterns from gastric mucosal cells corresponding to four groups defined by histological examination were measured by flow cytometry before and after treatment with heparin, a polyanion. Group I comprised normal gastric mucosal cells; group II, chronic atrophic gastric mucosal cells originating from a carcinoma free stomach; group III, chronic atrophic gastric mucosal cells originating from a carcinoma bearing stomach; and group IV, malignant gastric mucosal cells. The heparin concentrations used were 1.25, 1.5, and 5 U/ml cell suspension. Heparin caused increases in fluorescence intensity and in coefficients of variation, which are interpreted as a reflection of alterations in chromatin structure. For the four groups investigated, the heparin-initiated changes were dependent, in varying degree, on concentration and time. Group I showed a much more extensive sensitivity to heparin than group IV. Group II and III reacted similarly to group I or group IV, depending on the source, i.e., either a carcinoma-free stomach or a carcinoma-bearing stomach. Further extension of this method might yield information concerning the real premalignant potential of a specific case of chronic atrophic gastritis.     

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.     

Sialic acid metabolism in sialuria fibroblasts

Sialuria is a rare inborn error of metabolism caused by excessive synthesis of sialic acid (N-acetylneuraminic acid, NeuAc). Fibroblasts cultured from the three known cases of sialuria contained 70-200-fold increases in soluble sialic acid, but normal concentrations of bound sialic acid. The sialic acid appeared in the cytosolic fraction of the cells on differential centrifugation, and was susceptible to borohydride reduction, suggesting that accumulated sialic acid was in the form of NeuAc and not CMP-NeuAc. In biochemical studies, CMP-NeuAc (50 microM) inhibited the UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase of normal fibroblasts by 84-100%, but inhibited the epimerase from sialuria cells by only 19-31%. Feeding sialuria cells up to 5 mM D-glucosamine for 72 h increased free sialic acid content 20-60%, but normal cells were unaffected by this treatment. Cytidine feeding (5 mM, 72 h) reduced the NeuAc content of sialuria cells, initially 112, 104, and 266 nmol/mg protein, by 63-71 nmol/mg protein; CMP-NeuAc concentrations, initially 4, 2, and 5 nmol/mg protein, increased by 14-33 nmol/mg protein. Consequently, the total cellular content of soluble sialic acid (NeuAc + CMP-NeuAc) was lowered 14-46% by cytidine feeding. The inheritance pattern of sialuria has not been determined. However, cells from both parents of one sialuria patient contained normal concentrations of free sialic acid, and the parental epimerase activity also responded normally to CMP-NeuAc. We conclude that the basic biochemical defect in all known cases of sialuria is a failure of CMP-NeuAc to feedback-inhibit UDP-GlcNAc 2-epimerase and cytidine feeding can lower the intracellular soluble sialic acid concentration of sialuria cells.