Response Of Mammalian Cells Research Articles

Gene‐specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of eIF-2 alpha. Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORF1 fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of eIF-2 that delivers charged initiator tRNA(iMet) to the ribosome. When the levels of eIF-2.GTP.Met-tRNA(iMet) ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of eIF-2 by the protein kinase GCN2 decreases the concentration of eIF-2.GTP.Met-tRNA(iMet) complexes by inhibiting the guanine nucleotide exchange factor for eIF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of eIF-2.

Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.

Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; 3 copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-alpha-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.

Mitogen-induced early increase in cytosolic free Mg2+ concentration in single Swiss 3T3 fibroblasts

Events related to the early mobilization of Mg2+ in mammalian cells in response to external stimuli are not well characterized. We examined changes in cytoplasmic free Mg2+ concentrations ([Mg2+]i) after mitogenic stimulation in single mouse Swiss 3T3 fibroblasts, using digital ratio imaging microscopy of the fluorescent probe mag-fura-2. Stimulation with bombesin or epidermal growth factor (EGF) in combination with insulin led to a significant increase in mean [Mg2+]i levels from basal 0.22 mM to 0.29-0.35 mM after 30-60 min. The response showed some heterogeneity among individual cells with respect to the extent of the increase; approximately 10% of the cells showed no [Mg2+]i response. Bombesin or EGF alone induced a significant increase in [Mg2+]i but was less effective than when combined with insulin. In medium without added Mg2+, the increase in [Mg2+]i was considerably decreased, either with bombesin plus insulin or EGF plus insulin. These results provide direct evidence for the mobilization of Mg2+ as an early cellular response to growth factors.

Intracellular Proteins Produced by Mammalian Cells in Response to Environmental Stress

AbstractA condensed review of the literature on mammalian cells focusing on aspects of particular interest to biochemical and biomedical engineers.

Enhancement of DNA repair capacity of mammalian cells by carcinogen treatment.

To determine whether DNA excision repair is enhanced in mammalian cells in response to DNA damage, as it is in bacteria as part of the SOS response, we used an expression vector-host cell reactivation assay to measure cellular DNA repair capacity. When UV-damaged chloramphenicol acetyltransferase (CAT) vector DNA was introduced into monkey cells (CV-1), the level of CAT activity was inversely related to the UV fluence due to inhibition of CAT gene expression by UV photoproducts. When CV-1 cells were treated with either UV radiation or mitomycin C, 24-48 h before transfection, CAT expression from the UV-irradiated plasmid was increased. This increase also occurred in a line of normal human cells, but not in repair-deficient human xeroderma pigmentosum cells. We confirmed that this increase in CAT expression was due to repair, and not to production of damage-free templates by recombination; the frequency of generation of supF+ recombinants after transfection with UV-irradiated pZ189 vectors carrying different point mutations in the supF gene did not significantly increase in carcinogen-treated CV-1 cells. From these results we conclude that carcinogen treatment enhances the excision-repair capacity of normal mammalian cells.

Molecular and Cellular Effects of Heat-shock and Related Treatments of Mammalian Tissue-culture Cells

Most, if not all, organisms, from prokaryotes to the most complex eukaryotes, exhibit specific homeostatic-type responses when their environmental circumstance is perturbed. Such responses range from temporary and reversible modifications of enzyme function, by which specific metabolic pathways are redirected or arrested, to more long-term changes, which may involve altered patterns of gene expression of a more permanent nature. The subject of this paper is the induction of a particular set of genes in cultured mammalian cells in response to growth at elevated temperatures or in medium containing amino acid analogs. These treatments (as well as a variety of others) give rise to what we refer to as the stress response, and for heat shock there is evidence that the response has a protective effect on cells (Gerner et al. 1976; McAllister and Finkelstein 1980; Petersen and Mitchell 1981).