Targeting Protein Synthesis Research Articles

Resistance to drugs targeting protein synthesis in mycobacteria

Many antibiotics exert their effects by interfering with protein synthesis. Studies of the molecular mechanisms of antibiotic resistance in clinical strains of mycobacteria have revealed mutations in ribosomal RNAs. This type of acquired resistance was previously unknown in bacterial pathogens and was made possible because mycobacteria have only a single set of rRNA genes.

Translation and the cytoskeleton: a mechanism for targeted protein synthesis

This review describes the critical evidence that in eukaryotic cells polyribosomes, mRNAs and components of the protein synthetic machinery are associated with the cytoskeleton. The role of microtubules, intermediate filaments and microfilaments are discussed; at present most evidence suggests that polyribosomes interact with the actin filaments. The use of non-ionic detergent/deoxycholate treatment in the isolation of cytoskeletal-bound polysomes is described and the conclusion reached that at low salt concentrations this leads to mixed preparations of polysomes derived from both the cytoskeleton and the endoplasmic reticulum. At present the best approach for isolation of cytoskeletal-bound polysomes appears to involve extraction with salt concentrations greater than 130 mM after an initial non-ionic detergent treatment. Such polysomes appear to be enriched in certain mRNAs and thus it is suggested that they are involved in translation of a unique set of proteins. The evidence for mRNA localisation is presented and the role of the cytoskeleton in transport and localisation of RNA discussed. Recent data on the role of the 3' untranslated region in the targeting of mRNAs both to particular regions of the cell and for translation on cytoskeletal-bound polysomes is described. The hypothesis is developed that the association of polysomes with the cytoskeleton is the basis of a mechanism for the targeting of mRNAs and the compartmentalization of protein synthesis.

Overexpression of cloned genes using recombinant escherichia coli regulated by a T7 promoter: II. Two‐stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures.

Changes in architecture of the Golgi complex and other subcellular organelles during myogenesis.

Myogenesis involves changes in both gene expression and cellular architecture. Little is known of the organization, in muscle in vivo, of the subcellular organelles involved in protein synthesis despite the potential importance of targeted protein synthesis for formation and maintenance of functional domains such as the neuromuscular junction. A panel of antibodies to markers of the ER, the Golgi complex, and the centrosome were used to localize these organelles by immunofluorescence in myoblasts and myotubes of the mouse muscle cell line C2 in vitro, and in intact single muscle fibers from the rat flexor digitorum brevis. Antibodies to the ER stained structures throughout the cytoplasm of both C2 myoblasts and myotubes. In contrast, the spatial relationship between nucleus, centrosome, and Golgi complex was dramatically altered. These changes could also be observed in a low-calcium medium that allowed differentiation while preventing myoblast fusion. Muscle fibers in vivo resembled myotubes except that the ER occupied a smaller volume of cytoplasm and no staining was found for one of the Golgi complex markers, the enzyme alpha-mannosidase II. Electron microscopy, however, clearly showed the presence of stacks of Golgi cisternae in both junctional and extrajunctional regions of muscle fibers. The perinuclear distribution of the Golgi complex was also observed in live muscle fibers stained with a fluorescent lipid. Thus, the distribution of subcellular organelles of the secretory pathway was found to be similar in myotubes and muscle fibers, and all organelles were found in both junctional and extrajunctional areas of muscle.

Effects of plasmid copy number and runaway plasmid replication on overproduction and excretion of beta-lactamase from Escherichia coli.

Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of beta-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of beta-lactamase. Runaway plasmid replication results from readthrough of tac promoter transcripts into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary to achieve the level of overproduction necessary for excretion of beta-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lacI gene to the plasmid.

Accumulation of c-fos mRNA in rat hippocampus during acquisition of a brightness discrimination

Training rats to attain a foot-shock-motivated brightness discrimination in a Y-maze results in an early and transient increase of hippocampal c-fos mRNA levels. Maximal accumulation was observed immediately after training, returning to basal levels during the following 2 h. A similar increase was obtained when rats were subjected to a pseudotraining with an equal number of runs, but with random pairing of the choice of bright and dark alleys with foot shock. It is suggested that induction of hippocampal c-fos mRNA expression is a necessary, but not sufficient, prerequisite for the formation of long-term memory trace. This early gene expression seems rather to correspond to an initial stage induced by complex stimulus presentation of both the training and the pseudotraining procedure. The subsequent late synthesis or processing of target proteins finally contributing to the formation of a permanent trace requires the action of further convergent signals to principal cells, probably mediating reward or emotional influences.

Susceptibility of Giardia lamblia to aminoglycoside protein synthesis inhibitors: correlation with rRNA structure.

The very limited development of antiparasitic agents targeting protein synthesis stems in part from the belief that parasite and host ribosomes are sufficiently similar to preclude selective toxicity. However, recent studies have revealed that Giardia lamblia rRNA has an unusual size and sequence; consequently, this organism and its homogeneous rRNA provide a useful model for the development of protein synthesis inhibitors with antiparasitic activity. In this study, I determined the sequence and secondary structure of the 3' end of the small-subunit RNA, the target for aminoglycoside inhibitory activity. The primary structure of these 140 nucleotides includes two blocks of sequence highly conserved among other organisms; the remaining sequence, although not conserved, can be folded into a secondary structure common to all rRNAs. The presence of U-1495 within one of the conserved blocks predicts hygromycin susceptibility. Also, a specific base pair (C-1409.G-1491) implicated in paromomycin susceptibility is present; whereas all procaryotes have this base pair, it is absent in many eucaryotes (including mammals). Conversely, kanamycin and apramycin resistance can be predicted from substitution of A-1408 with G. A growth inhibition assay was used to test the susceptibility of G. lamblia to a variety of aminoglycosides. After 48 h, 8 of 11 aminoglycosides tested failed to inhibit growth at a concentration of 200 micrograms/ml. Paromomycin and hygromycin, however, inhibited growth of three strains tested by 50% at 50 to 60 micrograms/ml and by close to 90% at 120 micrograms/ml. These results correlate well with the sequence and secondary-structure analyses. Paromomycin may be clinically useful when the toxicity of standard antigiardial drugs is of concern.

Characterization of Plasmodium falciparum sexual stage antigens and their biosynthesis in synchronised gametocyte cultures

Synchronised gametocyte cultures were used to study the biosynthesis of the sexual stage target antigens ( M r 230 000, 48 000 and 25 000) for anti gamete/zygote antibodies. These antigens were shown to be synthesized during gametocyte development from day 2–3 onwards until gametogenesis occurred. After gametogenesis a 25 kDa protein was predominantly synthesized, whereas synthesis of the other target proteins was hardly detectable. The 48, 45, and 25 kDa proteins appeared to be glycosylated, in addition the 25 kDa was also acylated in that it bound [ 3H]palmitic acid covalently. The iso-electric point (p I) of these proteins was assessed as being 6.0 ± 0.1 (for both 48 and 45 kDa) and 5.6 ± 0.1 (for 25 kDa).

Effect of Human Monocyte‐Released Cytostatic Factors on Target Cell DNA Synthesis, Protein Synthesis, and ATP Content

Cultured human monocytes activated in vitro with lymphokines and lipopolysaccharide release cytostatic protein factors that may be separated by ion-exchange chromatography into two populations, termed cytostatic factors I and II (CF I and II). The effect of CF I and II on target cell DNA synthesis, protein synthesis, and ATP content and the possible presence of a CF-associated protease activity were investigated. Inhibition of DNA synthesis was detected 4 h after addition of either CF I or CF II, whereas inhibition of protein synthesis was first detected after 10-15 h. Moreover, 20 h after addition of either CF I or CF II the average DNA synthesis per cell was inhibited by 25-35%, as compared with 10-15% inhibition of protein synthesis. No significant alteration in cellular ATP content was observed in cells culture up to 25 h with either CF I or CF II. Thus, neither protein synthesis nor generation of ATP appeared to be primary targets of either CF I or CF II, but the factors may act more directly on DNA synthesis. Protease activity was not associated with either CF I or CF II, and the factors thus differed from a previously reported cytolytic protease released from activated murine macrophages.