Inhibition of the formation of lipid-linked intermediates in normal and transformed cells by a purified tunicamycin homologue

Summary The deffects of a purified homologue of tunicamycin (B₂-tunicamycin) on the biosynthesis of lipid-linked intermediates participating in protein glycosylation in normal embryonic fibroblasts, 3T3 and virally transformed (simian virus 40 and polyoma virus) mouse fibroblasts grown in culture were investigated. Long incubations (20 h) with the antibiotic caused a higher degree of inhibition of sugar incorporation into glycoproteins in transformed cells. However, the formation of lipid-linked intermediates was inhibited to a similar level in both cell types. When time dependent inhibition experiments were carried out using transformed cells, an earlier and stronger inhibition of the formation of lipid-oligosaccharides occurred (70% inhibition at 30 min). In 3T3 cells, prolonged incubation (6–8 h) was necessary in order to reach a similar degree of inhibition. Formation of lipid-sugar was also inhibited to a greater extent by B₂-tunicamycin in transformed cells. This inhibition was not clearly time dependent. Analysis of the newly synthesized glycolipids in 3T3 and in transformed cells after B₂-tunicamycin treatment have shown reduction in dolichyl-P-P-sugars as well as in other glycolipids. Dimethylsulfoxide (10%) and linoleic acid (0.5 mg/ml) markedly increased the level of tunicamycin activity in 3T3 cells while phosphatidylcholine (2 mg/ml) partially reversed it. The stronger and faster inhibition of the formation of lipid intermediates of the dolichyl-phosphate cycle caused by B₂-tunicamycin in transformed cells, described here for the first time, may therefore be due to differences in penetration of the antibiotic into these cells.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethylsulfoxide - MF mouse fibroblasts from Balb/c mouse embryos - 3T3 Balb/3T3 mouse fibroblastic line - SV40 Simian virus 40 - PY polyoma virus - TLC thin layer chromatography     

Use of Growth-Attachment Correlation Plots to Analyse Human Melanoma Cell Dependency On 2-Oxocarboxylates and Serum

Modulation of the population-dependent growth of the human melanoma line MM96 by serum and a series of 2-oxocarboxylates was analysed by a method offering several useful features. Initial cell attachment was measured by the use of cells prelabelled with [¹⁴C] thymidine (C) while the size of the replicating cell population on any particular day was monitored by [³H] thymidine (H) incorporation for that particular 24 hr. the ³H incorporation was normalized for the initial cell attachment as the ³H/¹⁴C ratio (H/C), which was found to be little affected by artifacts of precursor equilibration. the method allows large numbers of replicate samples so that quantitation of dose-response relationships is simple and statistically robust, and simultaneous monitoring of cell attachment and growth within the same sample is routine. Not only cell growth, but also attachment to the culture vessel surface was found to be increased by higher seeded population densities, by increasing serum concentration and by pyruvate supplementation. Further investigation of the pyruvate effect showed that all nine of the soluble 2-oxocarboxylates tested were effective for both parameters, with little difference between them which was attributable to molecular structure. Quantitative studies of the potentiation of growth and attachment by increasing cell density or a range of concentrations of 2-oxocarboxylates led to the finding that increments in the growth parameter (H/C) were directly proportional to increments in attachment (C). Such a relationship would be unexpected in ordinary logarithmically-growing cultures. the observation is consistent with the hypothesis that increases in initial cell attachment, whether induced by 2 oxocarboxylates or higher inoculation densities, lead to conditioning of the medium by cell factors which stimulate growth and are produced in amounts proportional to the attached cell numbers. Pyruvate, when present with higher serum concentrations, stimulated growth above the levels accounted for by concomitant increases in cell attachment. Serum also appears to have other potentiating factors in addition to those attributable to its content of 2-oxocarboxylates. Differences in the response to 2-oxocarboxylates of human melanoma cells and human diploid fibroblasts were noted, and suggest that the present observations may be due to unique properties of the melanoma cell.     

Effect of melphalan in vitro on induction of murine suppressor T cells by ConA

Summary The effect of treatment with melphalan in vitro on the activity of spleen cells from BALB/c mice was investigated. Incubation of spleen cells with 1.5–5 g melphalan/1×10⁷ inhibited subsequent mitogenic stimulation by ConA or PHA and the allogeneic response of BALB/c spleen cells against C57B1 target spleen cells. Incubation of spleen cells with ConA led to induction of suppressor T cells which when added to fresh cultures inhibited the allogeneic response. Preincubation of spleen cells with melphalan even at low concentrations (0.15–0.5 g 1×10⁷ cells) which do not directly affect mitogenic stimulation or allogeneic response partially inhibited the generation of suppressor T cells by ConA. Treatment with melphalan had no effect on already induced suppressor T cells as shown by incubation of spleen cells with melphalan (0.15–5 g/1×10⁷ cells) after incubation with ConA. Addition of cells treated with melphalan alone (without ConA) to fresh cultures led to an increase in the allogeneic response.     

Locus determining the human sperm-specific lactate dehydrogenase,LDHC, is syntenic with LDHC

From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA. The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11. The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.     

Isolation, characterization and metal-ion-binding properties of the alpha-subunit from S-100a protein.

The present experiments were undertaken to investigate the role of the phosphoinositides phosphatidylinositol 4-phosphate (PtdIns-4-P) and phosphatidylinositol 4,5-biphosphate (PtdIns-4,5-P2) in the alpha 1-adrenergic stimulation of respiration in isolated hamster brown adipocytes. Exposure of isolated brown adipocytes to the alpha-adrenergic-receptor agonist phenylephrine provoked a breakdown of 30-50% of the PtdIns-4-P and PtdIns-4,5-P2 after prelabelling of the cells with [32P]Pi. Coincident with the breakdown of phosphoinositides was an accumulation of labelled phosphatidic acid, which continued for the duration of the cell incubation. The time course of phosphoinositide breakdown was defined more precisely by pulse-chase experiments. Under these conditions, phenylephrine caused radioactivity in phosphatidylinositol, PtdIns-4-P and PtdIns-4,5-P2 to fall by more than 50% within 30 s and to remain at the depressed value for the duration of the incubation (10 min). This phospholipid response to alpha-adrenergic stimulation was blocked by exposure of the cells to phorbol 12-myristate 13-acetate (PMA); likewise phenylephrine stimulation of respiration was prevented by PMA. beta-Adrenergic stimulation of respiration and inhibition of respiration by 2-chloroadenosine and insulin were, however, unaffected by treatment with PMA. On the assumption that PMA is acting in these cells as an activator of protein kinase C, these results suggest the selective interruption of alpha-adrenergic actions in brown adipocytes by activated protein kinase C. These findings suggest that breakdown of phosphoinositides is an early event in alpha-adrenergic stimulation of brown adipocytes which may be important for the subsequent stimulation of respiration. The results from the pulse-chase studies also suggest, however, that phenylephrine-stimulated breakdown of inositol phospholipids is a short-lived event which does not appear to persist for the entire period of exposure to the alpha 1-adrenergic ligand.     

Lipopolysaccharide core mutants of Salmonella typhimurium containing D-glycero-D-manno-heptose

Mutants resistant to several hydrophobic membrane antagonists were isolated from a "deep rough" (rfaC) mutant of Salmonella typhimurium. The resistance was due to an alteration in the core region lipopolysaccharide composition as evidenced by altered bacteriophage and complement sensitivity and by compositional analysis. The principal change in carbohydrate composition was the predominance of the unusual heptose isomer D-glycero-D-manno-heptose. The unusually wide pleiotropic phenotype of this organism is suggested to be due to a fundamental change in the properties of the bacterial outer membrane.     

Environmental cadmium is not sequestered by metallothionein in rainbow trout

Smooth endoplasmic reticulum vesicles from rat liver display an ATP-supported Ca²⁺ transport which is mediated by a (Ca²⁺ + Mg²⁺)-ATPase. During the catalytic cycle the terminal phosphate from ATP is incorporated to form an acid-precipitable reaction product(118 000-M_r in SDS-gel electrophoresis) with stability characteristics of an acylphosphate. Comparative studies with sarcoplasmic reticulum vesicles from fast-twitch skeletal muscle suggest that the 118 000-M_r phosphopeptide may be identified with the phosphorylated reaction intermediate of a Ca²⁺ transport ATPase in endoplasmic reticulum, similar to that in sarcoplasmic reticulum of muscle.