Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells.

The rate of protein synthesis is rapidly down-regulated in mammalian cells following the induction of apoptosis. Inhibition occurs at the level of polypeptide chain initiation and is accompanied by the phosphorylation of the alpha subunit of initiation factor eIF2 and the caspase-dependent cleavage of initiation factors eIF4G, eIF4B, eIF2alpha and the p35 subunit of eIF3. Proteolytic cleavage of these proteins yields characteristic products which may exert regulatory effects on the translational machinery. Inhibition of caspase activity protects protein synthesis from long-term inhibition in cells treated with some, but not all, inducers of apoptosis. This review describes the initiation factor modifications and the possible signalling pathways by which translation may be regulated during apoptosis. We discuss the significance of the initiation factor cleavages and other changes for protein synthesis, and the implications of these events for our understanding of the cellular changes associated with apoptosis.

Translational control by the La antigen. Structure requirements for rescue of the double-stranded RNA-mediated inhibition of protein synthesis.

The La antigen is a protein which can bind both single-stranded and double-stranded forms of RNA and has regulatory effects on gene expression at the levels of transcription and translation. It was previously shown to inhibit the activation of the dsRNA-dependent protein kinase PKR by sequestering and/or unwinding double-stranded RNA. Here, we demonstrate that, as predicted by these properties, the La antigen can rescue protein synthesis in the reticulocyte lysate system from inhibition by low concentrations of dsRNA. This effect is reversed by higher concentrations of dsRNA. Using a series of deletion mutants we have investigated the structural features of the La antigen that are required for these effects. The ability to bind dsRNA is influenced by regions within both the previously characterized N-terminal RNP motif and the C-terminal half of the protein. La mutants with either N-terminal or C-terminal deletions retain the ability to inhibit the protein kinase activity of PKR and to rescue protein synthesis from inhibition by dsRNA. It is notable that sequences in the C-terminal half of the La antigen, including a phosphorylation site at Ser366, which are needed for other regulatory effects of the protein on gene expression are dispensable for the effects of La on PKR. We suggest that La regulates PKR activity solely as a result of its ability to act as an RNA-binding protein that can compete with PKR for limiting amounts of dsRNA.

Inhibition of the double-stranded RNA-dependent protein kinase PKR by mammalian ribosomes.

Previous evidence has shown that the majority of the interferon-inducible, double-stranded RNA-dependent protein kinase PKR is associated with ribosomes in vivo. Here we show that ribosomes are inhibitory for PKR activity since they compete with dsRNA for binding to PKR, inhibit the activation of the protein kinase by dsRNA, and prevent the phosphorylation of the PKR substrate eIF2alpha. We suggest that ribosomes constitute a reservoir of inactive PKR and that the protein kinase must be displaced from the ribosome by dsRNA in order to become activated.

Nuclear localization of the interferon-inducible protein kinase PKR in human cells and transfected mouse cells.

The levels and subcellular distribution of the interferon-inducible double-stranded RNA-dependent protein kinase PKR have been measured in human Daudi cells and stably transfected mouse NIH 3T3 cells expressing the human protein kinase. Immunofluorescence of intact cells and quantitative immunoblotting of cell extracts indicate that PKR occurs in both the cytoplasm and the cell nucleus, with staining specifically in the nucleolus. The ratio of cytoplasmic to nuclear PKR is approximately 5:1 in control cells; in response to interferon treatment the protein kinase is induced severalfold in the cytoplasm whereas the level in the nucleus does not increase significantly. Analysis of individual transfected cells by confocal microscopy reveals a pattern of distribution of PKR similar to that in Daudi cells, with immunostaining of cytoplasm and nucleoli. Similar results are observed whether cells expressing wild-type PKR or a catalytically inactive mutant form of the kinase are analyzed, but untransfected 3T3 cells are not stained by the antibody used. Two-dimensional isoelectric focusing analysis of PKR in whole cell extracts reveals the presence of multiple forms with different pI values whereas similar analysis of the nuclear fraction indicates only one predominant species with a relatively basic pI. These results suggest that PKR may have a role in the cell nucleus as well as the cytoplasm and that the subcellular distribution of the protein kinase may be related to post-translational modifications.

The La antigen inhibits the activation of the interferon-inducible protein kinase PKR by sequestering and unwinding double-stranded RNA.

The La (SS-B) autoimmune antigen is an RNA-binding protein that is present in both nucleus and cytoplasm of eukaryotic cells. The spectrum of RNAs that interact with the La antigen includes species which also bind to the interferon-inducible protein kinase PKR. We have investigated whether the La antigen can regulate the activity of PKR and have observed that both the autophosphorylation of the protein kinase that accompanies its activation by dsRNA and the dsRNA-dependent phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF-2 by PKR are inhibited in the presence of recombinant La antigen. This inhibition is partially relieved at higher concentrations of dsRNA. Once activated by dsRNA the protein kinase activity of PKR is insensitive to the La antigen. We have demonstrated by a filter binding assay that La is a dsRNA binding protein. Furthermore, when recombinant La is incubated with a 900 bp synthetic dsRNA or with naturally occurring reovirus dsRNA it converts these substrates to single-stranded forms. We conclude that the La antigen inhibits the dsRNA-dependent activation of PKR by binding and unwinding dsRNA and that it may therefore play a role in the regulation of this protein kinase in interferon-treated or virus-infected cells.

Regulation of the interferon-inducible eIF-2 alpha protein kinase by small RNAs.

This review describes the structure and function of the double-stranded RNA-dependent protein kinase (PKR) and its interaction with RNA activators and inhibitors. The abilities of small virally-encoded RNAs such as VAI RNA of adenovirus, the Epstein-Barr virus encoded (EBER) RNAs and the Tat-responsive region RNA of HIV-1 to bind to and regulate PKR are reviewed, and the physiological implications of such regulation for the control of viral replication and cell growth are discussed. The potential effects on the activity of PKR of other proteins that bind double-stranded RNA and/or small viral and cellular RNAs are also considered.

Stimulation by insulin of protein synthesis in cultured chick embryo fibroblasts.

Insulin stimulates protein synthesis in skeletal muscle of young animals and has been reported to exert similar effects on a variety of mammalian cell types in culture. However, with chick embryo fibroblasts we found that the extent of this effect was very sensitive to the culture conditions of the cells prior to the insulin treatment. The most reproducible results were obtained with cells that had been sub-cultured into medium in which fetal calf serum was replaced with 2% horse serum. Insulin only stimulated protein synthesis when added at supra-physiological concentrations. Insulin-like growth factor 1 was effective at much lower concentrations. Since chick embryo fibroblasts can be obtained in good yield, they offer, if treated under appropriate conditions, a suitable system for study of the mechanisms by which insulin and IGF-1 promote the initiation of protein synthesis in eukaryotic cells.

Time involvement in journal reading and a suggested facilitation.

In the field of continuing postgraduate education, the reading of professional journals is an accepted method of keeping informed in regard to new developments and contemporary thought on different philosophies of treatment. Not only is such reading realized as being an efficient educational vehicle, but there is now a growing awareness of the actual necessity for any clinician to set aside specific time to the exercise. It is not always readily apparent, however, how great the time commitment can be, even when the reading is limited to selected articles. This paper sets out to indicate in quantitative terms the time that could be expected to be utilized by both general dental practitioners and academic teachers if a thorough and conscientious perusal of the journals were to be undertaken. A practical strategy for facilitating at least part of the problem is put forward in the hope of reaching a helpful compromise.

The integration of restorations with existent partial dentures.

The relationship between an occlusal rest or clasp arm of a partial denture and the respective supporting tooth is critical and should be exact. Because of the necessity on occasion to remove the natural supportive enamel due to underlying caries, it becomes important that any restoration replacing the enamel should have the same configuration as before if the function of the rest or clasp is to be restored. This intricacy is often casually dealt with in practice. A simple but more exact technique to overcome this problem is described.

An evaluation of three methods of polishing porcelain by comparison of surface topography with the original glaze.

The value of a procedure for polishing porcelain restorations that would avoid the necessity of glazing in a furnace following minor chairside adjustments is discussed. The efficacy of three polishing systems--a diamond paste, a pumice and water slurry followed by whiting, and a proprietary method--were tested in the laboratory using surface profile recording. The results showed that both the diamond paste and the pumice/whiting gave surface finishes similar to the original glazed surface. The smoothest surface was produced by the diamond paste. The proprietary porcelain finishing kit created the least smooth surface.

Effect of starvation and diabetes on the activity of the eukaryotic initiation factor eIF-2 in rat skeletal muscle.

The ability of the initiation factor eIF-2 in skeletal muscle extracts to form ternary initiation complexes ([Met-tRNA(f).eIF-2.GDP]) is decreased by either starvation or diabetes. These conditions also impair the ability of muscle extracts to dissociate [eIF-2.GDP], suggesting inhibition of the guanine nucleotide exchange reaction essential for eIF-2 recycling. We could not, however, detect any change in the phosphorylation state of the alpha subunit of eIF-2. This suggests that eIF-2 activity may be regulated in this system by a mechanism not involving its phosphorylation.

Dentinal temperature transients caused by exposure to CO2 laser irradiation and possible pulpal damage.

An investigation is described that attempts to establish, in vitro, the characteristics of heat transference following laser irradiation of bovine dentinal tissue and the relationship with the periodicity of radiation. The results of this study appear to indicate that at depths of overlying dentine of up to 3 mm, laser-induced thermal injury to the pulp is a definite possibility. Fail-safe facilities to prevent build up of heat must be incorporated into the design of dental lasers to allow their beneficial effects to be utilized without the risk of iatrogenic damage.

CO2 laser application to the mineralized dental tissues--the possibility of iatrogenic sequelae.

From the many types of laser that are available commercially, the CO2 laser is presently thought to have the greatest potential for use in dentistry. An outline of the generation of emitted radiation from such a laser is given together with a review of work previously carried out and reported in the literature regarding application to the teeth. The main points of interest are the effects of radiation on the enamel and dentine, but concern is felt towards the possible iatrogenic damage of thermal origin that may occur within the pulp following irradiation of the dental tissues.

Finishing of amalgam restorations: to what degree is it necessary?

This paper attempts to elicit from the literature evidence to guide the clinician in deciding what degree of finishing and polishing is appropriate for amalgam restorations. Much of the justification for the polishing of amalgams has, in the past, been empirically based. Comprehensive scientific investigation into the validity of this practice does, however, present a number of methodological problems which have yet to be successfully overcome. An attempt is made to define what is meant by the term 'polished' (in a clinical context) while the benefits of polishing, as far as they are known, are discussed. The roles that cavity preparation, carving and burnishing play in the finishing of a restoration are reviewed, as is the conflicting evidence regarding their respective advantages and disadvantages. The importance of marginal adaptation, the relationship of polishing to secondary caries, and the dilemma in choosing (in appropriate cases) between the replacement of a restoration or its improvement by polishing are also considered.

An investigation of possible iatrogenic damage caused by metal rubber dam clamps.

Rubber dam may be held in place over a tooth by metal clamps. It has been shown that a mismatch of contact between the clamp gripping edge and the tooth surface may be reduced to a point contact, thereby concentrating the gripping force generated by the bow of the clamp. Experiments were conducted to measure the force. Clinically realistic loading of the tooth surface by sections of the gripping edge of clamps was carried out using special apparatus. Examination by scanning electron microscopy showed that iatrogenic damage to the tooth could occur. Therefore a plea is made for clamps to be redesigned to reduce any harm.

Insulin processing and action in adipocytes: evidence for generation of insulin-containing vesicles by leupeptin and monensin.

Incubation of adipocytes with 125I-insulin plus leupeptin or monensin, but not chloroquine, resulted in the appearance of a novel peak of 125I-insulin (modal density about 1.20 g/ml) on density gradient centrifugation; the appearance of the peak depended on the presence of specific insulin receptors on the cell surface. The fractions comprising this peak contained vesicles, probably originating from the Golgi apparatus, and dit not appear to be contaminated with lysosomes, mitochondria or plasma membrane. Entrapment of insulin in these vesicles per se did not prevent the activation of glucose transport, acetyl-CoA carboxylase or pyruvate dehydrogenase by insulin.