The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.     

The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

Comparative effects of adenine analogs upon metabolic cooperation between Chinese hamster cells with different levels of adenine phosphoribosyltransferase activity

Colony formation by variant Chinese hamster cells highly resistant to adenine analogs and deficient in adenine phosphoribosyltransferase (APRT) activity was measured after co-cultivation with APRT⁺, CHO-K1 cells in medium containing one of three different adenine analogs. Depending upon the density of APRT⁺ cells and the specific adenine analog, large differences in the recovery of APRT^? colonies were observed. The particular adenine analog and APRT⁺ cell density were more significant factors in the recovery of APRT^? colonies than the concentration of the analog or the level of APRT activity. The number of wild-type cells (CHO-K1) required to inhibit formation of APRT^? colonies by 50% (mean lethal density; MLD₅₀) with 65 μg/ml 8-aza-adenine (AzA) as the selective drug was 8.0 × 10⁵ cells/100 mm dish (1.5 × 10⁴/cm²). With 100 μg/ml 2,6-diaminopurine (DAP) the MLD₅₀ for CHO-K1 was 4.0 × 10⁵ cells/100 mm dish (7.3 × 10³/cm²). The MLD₅₀ for CHO-K1 when the DAP concentration was decreased to 50 μg/ml was only slightly higher, 5 × 10⁵ cells/100 mm dish (9.1 × 10³/cm²). The most toxic effect was observed with 2-fluoroadenine (FA). The MLD₅₀ for CHO-K1 in 2 μg/ml FA was 4.5 × 10⁴ cells/100 mm dish (8.2 × 10²/cm²), a cell density which permits minimal direct contact between APRT⁺ and APRT^? cells. The toxic effects of FA on individually resistant, APRT^? cells were found to be mediated by metabolites released into the medium by dying APRT⁺ cells. This metabolite toxicity to APRT^? cells was also demonstrated in mixtures with cells having only 8% of wild-type APRT activity. The MLD₅₀ for these APRT⁺ (8%) cells in 2 μg/ml FA was 7.5 × 10⁴ cells/100 dish (1.4 × 10³/cm²), a small difference from the MLD₅₀ for cells with wild-type levels of APRT activity. The differences in the recovery of APRT^? colonies from mixtures with APRT⁺ cells in these three adenine analogs are critical to the design of procedures for the selection of APRT^? cells from populations of APRT⁺ cells and emphasize the importance of establishing the parameters of metabolic cooperation, not only in terms of cell density but also with regard to the particular selective agent, in any experiment designed to determine precise mutation rates or to test putative mutagens upon mammalian cells in culture.     

Inhibition of rat mixed lymphocyte cultures by suppressor macrophages.

Normal rat spleens contain suppressor cells which can inhibit proliferative and cytotoxic responses of lymphocytes to alloantigens in vitro. The suppressor cells are adherent, phagocytic, resistant to treatment with ATS and C, radioresistant, resistant to treatment with mitomycin C, apparently absent from the thymus, and found in very high concentrations in peritoneal exudates. These characteristics indicate that the suppressor cell is a macrophages and not a T cell. When suppressor cells were removed from spleen cell suspensions, strong in vitro proliferative and cytotoxic responses to alloantigens could consistently be observed.     

Neurological and electroencephalographic changes in newborns treated with prostaglandins E2 and E2

Six newborns with obstructive right heart lesions were examined neurologically and electroencephalographically during treatment with prostaglandin (PG) E₁ or E₂ given to maintain patency of the ductus arteriosus and to increase pulmonary blood flow. PG was administered intravenously or intraarterially in the aortic isthmus proximal to the ductus arteriosus. Besides a rise in arterial oxygen saturation, all patients had some sign of central nervous system involvement. The electroencephalogram showed minor changes suggestive of sedation. In addition, three patients in whom PG given intravenously presented various combinations of neurological abnormalities (“myoclonic jerks”, apnoeic spells, hiccup) of subcortical origin. Side-effects subsided after stopping the treatment anf posed no problem in the management of the patients. These findings confirm the usefulness and safety of the PG therapy and indicate that the intraaortic route of administration is preferable.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Opposing effects of human peripheral blood lymphocytes on the growth of cultured leukemia cell lines

Natural killing of two human leukemia cell lines (K562 and Molt 4) in a soft-agar, clonagenic assay was shown to be the result of two opposite yet concurrent processes: target cell colony stimulation and inhibition. The stimulatory effect was demonstrable when the effector lymphocytes and target cells were separated in contiguous agar layers, suggesting mediation by a soluble factor. Similarly, stimulation occurred when the effector lymphocytes and target cells were combined at low effector-target cell ratios that do not favor direct cell contact. Target colony inhibition was found to be dominant when large E:T ratios were employed. Both target-effector binding and natural killing were significantly reduced in medium devoid of divalent cations.