Human Cell Metabolism Research Articles

The effect of actinomycin D on RNA metabolism in human cells

Terms to be familiar with before you start to solve the test: pre-mRNA (hnRNA); 45 S pre-rRNA; 28 S and 18 S rRNA; pulse-chase labeling; [(3) H]uridine; RNA isolation; separation methods; centrifugation methods; chromatography methods; tRNA; 5 S rRNA; RNA synthesis; RNA processing; RNA transport; polyadenylation. (1) The abbreviations used are: hnRNA, heterogeneous nuclear RNA; MCQ, multiple-choice questions.

Cellular target proteins of quercetin and flavonoid metabolism in human cells

Cellular target proteins of quercetin and flavonoid metabolism in human cells

Effect of the delta6-desaturase inhibitor SC-26196 on PUFA metabolism in human cells.

The objective of this study was to determine the effect of 2,2-diphenyl-5-(4-[[(1 E)-pyridin-3-yl-methylidene]amino]piperazin-1-yl)pentanenitrile (SC-26196), a delta6-desaturase inhibitor, on PUFA metabolism in human cells. SC-26196 inhibited the desaturation of 2 microM [1-14C] 18:2n-6 by 87-95% in cultured human skin fibroblasts, coronary artery smooth muscle cells, and astrocytes. By contrast, SC-26196 did not affect the conversion of [1-14C]20:3n-6 to 20:4 in the fibroblasts, demonstrating that it is selective for delta6-desaturase. The IC50 values for inhibition of the desaturation of 2 microM [1-14C] 18:3n-3 and [3-14C]24:5n-3 in the fibroblasts, 0.2-0.4 microM, were similar to those for the inhibition of [1-14C 18:2n-6 desaturation, and the rates of recovery of [1-14C]18:2n-6 and [3-14C]24:5n-3 desaturation after removal of SC-26196 from the culture medium also were similar. SC-26196 reduced the conversion of [3-14C]22:5n-3 and [3-14C]24:5n-3 to DHA by 75 and 84%, respectively, but it had no effect on the retroconversion of [3-14C]24:6n-3 to DHA. These results demonstrate that SC-26196 effectively inhibits the desaturation of 18- and 24-carbon PUFA and, therefore, decreases the synthesis of arachidonic acid, EPA, and DHA in human cells. Furthermore, they provide additional evidence that the conversion of 22:5n-3 to DHA involves delta6-desaturation.

Secretion and Intracellular Generation of Truncated Aβ in β-Site Amyloid-β Precursor Protein-cleaving Enzyme Expressing Human Neurons

Insoluble pools of the amyloid-beta peptide (Abeta) in brains of Alzheimer's disease patients exhibit considerable N- and C-terminal heterogeneity. Mounting evidence suggests that both C-terminal extensions and N-terminal truncations help precipitate amyloid plaque formation. Although mechanisms underlying the increased generation of C-terminally extended peptides have been extensively studied, relatively little is known about the cellular mechanisms underlying production of N-terminally truncated Abeta. Thus, we used human NT2N neurons to investigate the production of Abeta11-40/42 from amyloid-beta precursor protein (APP) by beta-site APP-cleaving enzyme (BACE). When comparing undifferentiated human embryonal carcinoma NT2- cells and differentiated NT2N neurons, the secretion of sAPP and Abeta correlated with BACE expression. To study the effects of BACE expression on endogenous APP metabolism in human cells, we overexpressed BACE in undifferentiated NT2- cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated Abeta as a result of normal processing of endogenous APP, BACE overexpression increased the secretion of Abeta1-40/42 and Abeta11-40/42 in both NT2- cells and NT2N neurons. Furthermore, BACE overexpression resulted in increased intracellular Abeta1-40/42 and Abeta11-40/42. Therefore, we conclude that Abeta11-40/42 is generated prior to deposition in senile plaques and that N-terminally truncated Abeta peptides may contribute to the downstream effects of amyloid accumulation in Alzheimer's disease.

Effects of modulators of protein phosphorylation on heme metabolism in human hepatic cells: induction of delta-aminolevulinic synthase mRNA and protein by okadaic acid.

Effects of modulators of protein phosphorylation on delta-aminolevulinic acid (ALA) synthase and heme oxygenase-1 mRNA were analyzed in the human hepatic cell lines Huh-7 and HepG2 using a quantitative RNase protection assay. Okadaic acid was found to induce ALA synthase mRNA in a concentration-dependent fashion in both Huh-7 and HepG2 cells. The EC(50) for induction of ALA synthase mRNA in Huh-7 cells was 13.5 nM, with maximum increases occurring at okadaic acid concentrations of 25-50 nM. The EC(50) for induction of ALA synthase mRNA in HepG2 cells was 35.5 nM, with maximum increases occurring at okadaic acid concentrations of 50 nM. Concentration-dependent induction of ALA synthase mRNA paralleled the increase in ALA synthase protein. Maximum induction of ALA synthase was observed between 5 and 10 h post-treatment in both cell lines. Induction of ALA synthase mRNA in Huh-7 cells, but not HepG2 cells, was associated with an increase in ALA synthase mRNA stability. Okadaic acid also induced heme oxygenase-1 mRNA in both cell lines, but the magnitude of induction was only twofold, and was rapid and transient. Okadaic acid and phorbol 12-myristate 13-acetate significantly decreased heme-mediated induction of heme oxygenase-1 mRNA in both Huh-7 and HepG2 cells. Wortmannin diminished the heme-mediated induction of heme oxygenase-1 mRNA in HepG2 cells, but not Huh-7 cells. These results report a novel property of okadaic acid to affect heme metabolism in human cell lines.

Arylsulfatase D gene in Xp22.3 encodes two protein isoforms.

The human genome contains six arylsulfatase genes (ARSA-ARSF), of which four are clustered in a distal region of the short arm of the X chromosome (Xp22.3). They were probably generated by a series of evolutionary duplication events; their exon-intron boundaries are identical. Nevertheless, different transcript lengths and the absence of cross-hybridizations point to a specific function of each gene in human cell metabolism, and multiple transcripts suggest the coding of protein isoforms. We identified a novel protein isoform of the ARSD gene by isolation of a series of cDNA clones from a human testis cDNA library. The clones were only partially identical to another series of ARSD clones isolated earlier (now designated ARSDalpha clones). Their specific C-terminal region (1160 nt) encodes a novel ARSD peptide of 48 amino acids and was identified as part of intron 6 of the ARSD gene in Xp22.3. We therefore designate them ARSDbeta clones. Expression analyses of ARSDalpha and ARSDbeta by semiquantitative RT-PCR revealed the presence of both in multiple human tissues, although in different quantities. A physiologic substrate for arylsulfatase D proteins is not known. We therefore estimated their sulfatase activities in vitro with the aid of the 4-methylumbelliferyl sulfate (4-MUS) assay. Surprisingly, neither ARSD protein isoform demonstrated any sulfatase activity alone or in combination, although their catalytic peptide domain is strongly conserved in comparison with that of the other X-chromosomal arylsulfatase enzymes (ARSC, ARSE, ARSF), all of which are functionally active in the 4-MUS assay.

Mitochondrial role in life and death of the cell.

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.

Regulation of Energy Metabolism in Human Cells in Aging and Diabetes: FoF1, mtDNA, UCP, and ROS

Recent advances in bioenergetics consist of discoveries related to rotational coupling in ATP synthase (FoF1), uncoupling proteins (UCP), reactive oxygen species (ROS) and mitochondrial DNA (mtDNA). As shown in cloned sheep, mammalian genomes are composed of both nuclear DNA (nDNA) and maternal mtDNA. Oxidative phosphorylation (oxphos) varies greatly depending on cellular activities, and is regulated by both gene expression and the electrochemical potential difference of H+ (Δ μH+). The expression of both mtDNA (by mtTFA) and nDNA for oxphos and UCP (by NRFs, etc.) is coordinated by a factor called PGC-1. The Δ μH+ rotates an axis in FoF1 that is regulated by inhibitors and ATP-sensitive K+-channels. We cultured human ρo cells (cells without mtDNA) in synthetic media and elucidated relationships among mtDNA, nDNA, Δ μH+, UCPs, ROS, and apoptosis. These cells lack oxphos-dependent ROS formation and survive under conditions of high O2. Cells cultured in the absence of ROS scavengers have proliferated for 40 years. UCPs lower Δ μH+ and prevent ROS formation and resulting apoptosis. These results were applied to diabetology and gerontology. The pancreatic ρo cells did not secrete insulin, and mtDNA mutations caused diabetes, owing to the deficient Δ μH+. Insulin resistance was closely related to UCPs and other energy regulators. The resulting high-glucose environment caused glycation of proteins and ROS-mediated apoptosis in vascular cells involved in diabetic complications. Telomeres, oxphos, and ROS are determinants in cellular aging. Cell division and ROS shortened telomeres and accelerated aging. In aged cells, Δ μH+ was reduced by the slow respiration, and this change induced apoptosis. Cybrids made from aged cytoplasts and ρo cells showed that both decreased expression of nDNA, and somatic mutations of mtDNA are involved in the slowing of respiration in aged cells.

Iron-dependent changes in cellular energy metabolism: influence on citric acid cycle and oxidative phosphorylation

Iron modulates the expression of the critical citric acid cycle enzyme aconitase via a translational mechanism involving iron regulatory proteins. Thus, the present study was undertaken to investigate the consequences of iron perturbation on citric acid cycle activity, oxidative phosphorylation and mitochondrial respiration in the human cell line K-562. In agreement with previous data iron increases the activity of mitochondrial aconitase while it is reduced upon addition of the iron chelator desferrioxamine (DFO). Interestingly, iron also positively affects three other citric acid cycle enzymes, namely citrate synthase, isocitric dehydrogenase, and succinate dehydrogenase, while DFO decreases the activity of these enzymes. Consequently, iron supplementation results in increased formation of reducing equivalents (NADH) by the citric acid cycle, and thus in increased mitochondrial oxygen consumption and ATP formation via oxidative phosphorylation as shown herein. This in turn leads to downregulation of glucose utilization. In contrast, all these metabolic pathways are reduced upon iron depletion, and thus glycolysis and lactate formation are significantly increased in order to compensate for the decrease in ATP production via oxidative phosphorylation in the presence of DFO. Our results point to a complex interaction between iron homeostasis, oxygen supply and cellular energy metabolism in human cells.

The efflux of biotin from human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PMBCs) are readily available for sampling and are a useful model for studying biotin metabolism in human cells. To better understand biotin handling by PMBCs, we investigated the mechanism(s) and kinetics of biotin efflux from PMBCs. Human PMBCs were incubated with [ 3H]biotin at 475 pmol/L to load the cells. The [ 3H]biotin-loaded cells were then harvested and incubated in [ 3H]biotin-free media for up to 20 hours. At various intervals, aliquots of the PMBC suspensions were collected and analyzed for intracellular [ 3H]biotin. [ 3H]Biotin efflux from cells at 37°C was fast and triphasic; the half-lives for the three elimination phases were 0.2 ± 0.02 hours, 1.2 ± 0.1 hours, and 21.9 ± 13.6 hours. Such a triphasic [ 3H]biotin efflux could reflect (1) rapid efflux of free biotin, (2) slower release of biotin bound to intracellular molecules, and (3) even slower release from carboxylases in cellular organelles. Incubation at 4°C rather than 37°C increased the [ 3H]biotin retained at 20 hours from 27% to 85%. This observation is consistent with transporter-mediated efflux. When cellular glucose utilization was reduced by 2-deoxy- d-glucose and sodium fluoride, [ 3H]biotin efflux was similar to controls, suggesting that biotin efflux does not directly require metabolic energy. When [ 3H]biotin-loaded cells were incubated in external medium containing unlabeled biotin analogs, [ 3H]biotin efflux was accelerated approximately two times compared with incubation in a biotin-free medium. This observation suggests that biotin efflux is mediated by the same transporter that mediates biotin uptake from the extracellular medium (i.e., classic countertransport).

Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase.

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

Lipoxygenase in Human Tumor Cells.

Tumor cell proliferation and metastasis proceed via a network of interdependent molecular events with a vast array of molecular players and signal transduction mechanisms differing in various types of human tumors. In the sequence of events necessary for carcinogenesis, arachidonate metabolites have been documented to play a significant role at several steps. Arachidonate metabolism in human cells occurs via several enzymatic pathways, including enzymes such as cyclo-oxygenases and lipoxygenases. This review pays particular attention to one member of the lipoxygenase family of enzymes, namely 12-lipoxygenase, since an arachidonate metabolite generated via 12-lipoxygenase action, 12(S)-HETE, has been shown to elicit various prometastatic effects of tumor cells in vivo and in vitro. We focus especially on mechanisms of activation and modulation of 12-lipoxygenase expression in human tumor cells, since various tumor cells express 12-lipoxygenase or are responsive to metabolites derived from 12-lipoxygenase action, thus offering a potential for successful therapeutic intervention against such tumors.

Nitric Oxide-Donor Compounds Inhibit Lipoxygenase Activity

The nitric oxide (NO)-releasing agents sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) inhibit dioxygenase activity of lipoxygenase in human platelets and human CHP100 neuroblastoma cells, leading the latter to necrosis. The effect of both NO-donors on the dioxygenase reaction was investigated by using soybean lipoxygenase type II (LOX-2) as a model for the mammalian enzyme. SNP and SNAP were competitive inhibitors of LOX-2, with inhibition constants of 525 μM and 710 μM, respectively. Both compounds inactivated LOX-2 by reducing the catalytic iron to the inactive Fe(II) form and counteracted the H2O2-mediated activation of the eLOX-2-catalyzed dioxygenase reaction. Similarly, the co-oxidative and peroxidative activities of LOX-2 were also inhibited by the NO-releasing agents. These findings suggest that the biological role played by NO can be mediated, at least in part, by the inactivation of lipoxygenase, a key-enzyme for the arachidonic acid metabolism in human cells.

Melatonin Inhibits LDL Receptor Activity and Cholesterol-Synthesis in Freshly Isolated Human Mononuclear Leukocytes

There is some indirect evidence that the pineal hormone melatonin can suppress plasma levels of cholesterol in hypercholesterolemic rats. We have examined the effects of the hormone on cellular cholesterol metabolism in freshly isolated human mononuclear leukocytes. Incubation of cells for up to 20 h in a lipid-free medium resulted in an increase in the rate of cholesterol synthesis from [14C]acetate and the high affinity accumulation and degradation of [125I]labeled low density lipoprotein (LDL). Addition of melatonin in increasing concentrations to the incubation medium at zero time inhibited cholesterol synthesis and the specific accumulation and degradation of [125I]labeled LDL; at a concentration of 100 μM, the inhibitions were 38 %, 42 %, and 48 %, respectively. Similar results were obtained using [14C]mevalonate as precursor. Fatty acid synthesis was not altered under these conditions. In contrast to cholesterol the synthesis of the first cyclic compound lanosterol was not affected by the pineal hormone. These results implicate that melatonin inhibits this pathway between lanosterol and cholesterol. The action of melatonin on LDL receptor activity appeared to be mediated by a decrease in the number of LDL receptors and not by a change in binding affinity. Pharmacological characterization of the potential melatonin receptor site using several analogs hire tryptamine, 5-hydroxytryptamine, N-acetyl-5-hydroxtryptamine, 5-methoxytryptamine, and 6-chloromelatonin indicated that the 5-methoxy group is indispensible for the hormone action on cholesterol synthesis. The data provide evidence that melatonin can modulate cholesterol metabolism in human cells.

Inhibition of inositol 1,4,5-trisphosphate metabolism in permeabilised SH-SY5Y human neuroblastoma cells by a phosphorothioate-containing analogue of inositol 1,4,5-trisphosphate

Electrically permeabilised [3H]inositol-labelled SH-SY5Y human neuroblastoma cells were employed to examine the effects of two synthetic, phosphatase-resistant analogues of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on the metabolism of cell membrane-derived [3H]Ins(1,4,5)P3 or exogenous [5-32P]Ins(1,4,4)P3. Incubation of permeabilised SH-SY5Y cells for 5 min at 37 degrees C with carbachol and guanosine 5'-[gamma-thio]triphosphate caused a decrease in [3H]phosphoinositol phospholipid levels and an increase in [3H]inositol phosphate accumulation with inositol 4-phosphate, inositol 1,4-bisphosphate, Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate comprising approximately 79%, 16%, 3% and 2%, respectively, of the increase. Inositol 1-phosphate levels did not increase upon stimulation, nor was inositol 4-phosphate converted rapidly to inositol. In parallel incubations, the analogues, DL-inositol 1,4,5-trisphosphorothioate (DL-InsP3S3) and DL-inositol 1,4-bisphosphate 5-phosphorothioate (DL-InsP3S), and synthetic racemic Ins(1,4,5)P3 (DL-InsP3), altered the profile of the [3H]inositol phosphates recovered and led, at millimolar concentrations, to a 10-15-fold increase in [3H]Ins(1,4,5)P3. The extent of inhibition of [3H]Ins(1,4,5)P3 metabolism was, however, greatest in the presence of synthetic D-Ins(1,4,5)P3 (greater than or equal to 5 mM), when [3H]Ins(1,4,5)P3 comprised approximately 50% of the increase in total [3H]inositol phosphates. Thus, under these conditions, at least 50% of [3H]inositol phosphates were derived from [3H]phosphatidylinositol 4,5-bisphosphate. [32P]Pi release from exogenous [5-32P]Ins(1,4,5)P3 was also inhibited by DL-InsP3S3, DL-InsP3S and DL-InsP3, with half-maximal inhibition at approximately 50 microM, 160 microM and 240 microM respectively. These actions were approximately ten times more potent than the effects of these compounds on [3H]inositol phosphate accumulation, indicating that homogenous mixing of exogenous and cell-membrane-derived Ins(1,4,5)P3 does not occur. These findings indicate that DL-InsP3S3 and DL-InsP3S inhibit Ins(1,4,5)P3 5-phosphatase. In contrast, the effects of synthetic DL-InsP3 and D-Ins(1,4,5)P3 are due to isotopic dilution. Whilst DL-InsP3S3 was the most potent inhibitor of dephosphorylation of exogenous or cell-membrane-derived Ins(1,4,5)P3, it was the weakest inhibitor of 3-kinase-catalysed Ins(1,4,5)P3 phosphorylation. Similarly, although approximately 50 times less potent than DL-InsP3S3, 2,3-diphosphoglycerate inhibited Ins(1,4,5)P3 5-phosphatase activity and was apparently without effect of Ins(1,4,5)P3 3-kinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Characteristics of interferon induced tryptophan metabolism in human cells in vitro

Interferon-γ-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-γ. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthracilic acid and 3-hydroxyanthranilic acid). Interferon-γ regulated only the activity of indoleamine 2,3-dioxygenase. All other enzyme activities detected were independent of interferon-γ, as shown by the capacity of the cells to metabolize l-kynurenine or N-formyl- l-kynurenine. Increasing the extracellular l-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular l-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK-HEP-1. The activity was unaffected by extracellular l-tryptophan with dermal fibroblasts and A 498. Though interferon-γ was the most potent inducer of tryptophan metabolism, interferon-α and/or -β showed small but distinct action on some of the cells. In all cells which reacted to interferon-γ by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-γ action, that the extent of this induction is influenced by extracellular l-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation of 3-hydroxyanthranilic acid from tryptophan which is regulated by interferon-γ.

Effects of benzaldehyde on protein metabolism of human cells cultivated in vitro

The mechanism by which the antitumour agent benzaldehyde inhibits cell growth has been investigated. Human NHIK 3025 cells were synchronized by selection of mitotic cells and the protein content at various stages of the cell cycle was recorded by use of flow cytometry. In the presence of benzaldehyde (concentrations above 0.5 mM, ∼50 μg/ ml) the rate of protein accumulation was reduced to the same extent throughout the cell cycle. The rates of protein synthesis and protein degradation were measured by incorporation and release, respectively, of radioactively labelled valine in exponentially growing cells. It was found that benzaldehyde primarily reduced the rate of protein synthesis, while it induced only a very small effect (reduction) on the rate of protein degradation. When comparing the rate of cell-cycle progression with the rate of protein accumulation, it was found that the median interphase duration was equal to the protein doubling time even for concentrations of benzaldehyde giving a marked reduction in the rate of protein accumulation. Similar results have been observed on these cells using the specific protein synthesis inhibitor cycloheximide. However, the two drugs have different effects during mitosis, since benzaldehyde but not cycloheximide induces a specific mitotic inhibition. It is, therefore, possible that benzaldehyde inhibits the protein synthesis by a mechanism different from that of cycloheximide, a mechanism which simultaneously results in a specific mitotic inhibition. A hypothesis is proposed on the mechanism of action of benzaldehyde: that the drug might inhibit a process in the cells which activates enzymes. Such an effect might possibly entail a reduced protein synthesis as well as a prolonged mitosis. In addition, it might also count for the reported de-transforming activity of benzaldehyde on malignant cells.

Effect of cycloheximide and dexamethasone phosphate on hyaluronic acid synthesis and secretion in cultured human skin fibroblasts.

We have studied some aspects of the regulation of hyaluronic acid synthesis in cultured human skin fibroblasts derived from normal individuals. In these cells 85% of radiolabeled glucosamine is incorporated into two types of macromolecules, hyaluronic acid and glycoproteins, characterized by district partitions between the cell and medium compartments. Treatment of cells with cycloheximide under conditions in which protein and glycoprotein synthesis is inhibited by at least 90% has no effect on hyaluronic acid synthesis. This result is consistent with the hypothesis that protein synthesis is not necessary for the synthesis of hyaluronic acid. Conversely, 5 X 10(-6) M dexamethasone phosphate inhibits hyaluronic acid by 60% but has no effect on protein or glycoprotein or glycoprotein synthesis. Concomitant treatment with dexamethasone phosphate and cycloheximide restores the synthesis of hyaluronic acid to normal values. The dexamethasone phosphate effect on hyaluronic acid synthesis is therefore not a primary induction response since it requires protein synthesis. The secretion of hyaluronic acid and glycoproteins from the cells into the medium is not affected by either of the two drugs. This experimental approach will be useful in the study of the regulation of glycosaminoglycan metabolism in human cells especially with respect to disorders of connective tissue.

Characteristics of benzo[ a]pyrene and A-ring reduced 7,12-dimethyl benz[ a]anthracene induced neoplastic transformation of human cells in vitro

The polynuclear aromatic hydrocarbons (PAH) benzo[ a]pyrene (BP) and the A-ring reduced analogue of 7,12-dimethylbenz[ a]anthracene (DMBA), 1,2,3,4-tetrahydro-7,12-dimethylbenz[ a]anthracene (TH-DMBA) are carcinogenic to human cells. The unsaturated PAH, DMBA exhibits no carcinogenic activity on human cells as measured by growth in soft agar. The TH-DMBA and BP treated cells exhibit a colony frequency in soft agar of 84 and 86, respectively. These anchorage independent cells, when seeded on the chick embryonic skin (CES) organ cultures, are invasive and form a fibrosarcoma. It is highly unlikely that TH-DMBA, which does not contain an aromatic A-ring, can undergo metabolism in human cells in culture to form a bay region 3,4-dihydrodiol-1,2-epoxide. These results suggest that an alternate mechanism for the induction of carcinogenesis is appropriate to explain the absence of bay region diol-epoxide metabolite as the ultimate form of the carcinogen in TH-DMBA induced carcinogenesis in human diploid cells.

Lipoprotein receptors and the genetic control of cholesterol metabolism in cultured human cells

Biochemical analysis of cultured fibroblasts obtained from human subjects with the autosomal dominant disorder familial hypercholesterolemia has led to the identification of a hitherto unrecognized cell-surface receptor for serum lipoproteins. This receptor plays a critical role in the physiologic regulation of cholesterol metabolism in human cells, and its absence leads to elevation of serum-cholesterol levels and severe atheroslerosis.