Mechanism of aminoacylation of tRNA : Influence of spermine on the kinetics of aminoacyl-tRNA synthesis by isoleucyl- and valyl-tRNA synthetases from Mycobacterium smegmatis

Isoleucyl-tRNA synthetase has been purified to homogeneity from Mycobacterium smegmatis. The influence of spermine on the kinetics of valyl-tRNA and isoleucyl-tRNA formation has been investigated by Cleland's method (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104–137, 173–187, 188–196). The results suggest that in the presence of spermine and suboptimal concentration of Mg2+, the formation of valyl-tRNA and isoleucyl-tRNA follows a sequential* mechanism. In the presence of an optimal concentration of Mg2+, both valyl-tRNA and isoleucyl-tRNA formation proceeds by a ping-pong mechanism. However, in the presence of spermine and optimal concentrations of Mg2+, valyl-tRNA formation follows the ping-pong mechanism while isoleucyl-tRNA formation follows the sequential mechanism.

Immediate and residual effects of heat stress and restricted intake on milk protein and casein composition and energy metabolism

The effects of heat stress on dairy production can be separated into 2 distinct causes: those effects that are mediated by the reduced voluntary feed intake associated with heat stress, and the direct physiological and metabolic effects of heat stress. To distinguish between these, and identify their effect on milk protein and casein concentration, mid-lactation Holstein-Friesian cows (n = 24) were housed in temperature-controlled chambers and either subjected to heat stress HS; temperature-humidity index (THI) ~78 or kept in a THI < 70 environment and pair-fed with heat-stressed cows (TN-R) for 7 d. A control group of cows was kept in a THI < 70 environment with ad libitum feeding (TN-AL). A subsequent recovery period (7 d), with THI < 70 and ad libitum feeding followed. Intake accounted for only part of the effects of heat stress. Heat stress reduced the milk protein concentration, casein number, and casein concentration and increased the urea concentration in milk beyond the effects of restriction of intake. Under HS, the proportion in total casein of αS1-casein increased and the proportion of αS2-casein decreased. Because no effect of HS on milk fat or lactose concentration was found, these effects appeared to be the result of specific downregulation of mammary protein synthesis, and not a general reduction in mammary activity. No residual effects were found of HS or TN-R on milk production or composition after THI < 70 and ad libitum intake were restored. Heat-stressed cows had elevated blood concentrations of urea and Ca, compared with TN-R and TN-AL. Cows in TN-R had higher serum nonesterified fatty acid concentrations than cows in HS. It was proposed that HS and TN-R cows may mobilize different tissues as endogenous sources of energy.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

Macromolecular synthesis in mycobacteriophage I3 infected cells

DNA-, RNA- and protein synthesis have been studied inMycobacterium smegmatis cells infected with phage 13. The macromolecular synthesis continued until the end of latent period. Early RNA and protein synthesis were necessary prior to the commencement of DNA replication. The infecting phage DNA sedimented as larger than unit length of genome, after initiation of DNA synthesis. Although the host DNA was not degraded, 90 percent of the RNA synthesized after phage infection hybridized to phage DNA.

The viral coat protein is regulated by HSP70 and HSP40 in Potato virus A infection

Plus-stranded (plus) RNA viruses multiply within a cellular environment as tightly integrated units and rely on the genetic information carried within their genomes for multiplication and, hence, persistence. The minimal genomes of plus RNA viruses are unable to encode the molecular machineries that are required for virus multiplication. This sets requisites for the virus, which must form compatible interactions with host components during multiplication to successfully utilize primary metabolites as building blocks or metabolic energy, and to divert the protein synthesis machinery for production of viral proteins. In fact, the emerging picture of a virus-infected cell displays tight integration with the virus, from simple host and virus protein interactions through to major changes in the physiological state of the host cell. This study set out to develop a method for the identification of host components, mainly host proteins, that interact with proteins of Potato virus A (PVA; Potyvirus) during infection. This goal was approached by developing affinity-tag based methods for the purification of viral proteins complexed with associated host proteins from infected plants. Using this method, host membrane-associated viral ribonucleoprotein (RNP) complexes were obtained, and several host and viral proteins could be identified as components of these complexes. One of the host proteins identified using this strategy was a member of the heat shock protein 70 (HSP70) family, and this protein was chosen for further analysis. To enable the analysis of viral gene expression, a second method was developed based on Agrobacterium-mediated virus genome delivery into plant cells, and detection of virally expressed Renilla luciferase (RLUC) as a quantitative measure of viral gene expression. Using this method, it was observed that down-regulation of HSP70 caused a PVA coat protein (CP)-mediated defect associated with replication. Further experimentation suggested that CP can inhibit viral gene expression and that a distinct translational activity coupled to replication, referred to as replication-associated translation (RAT), exists. Unlike translation of replication-deficient viral RNA, RAT was dependent on HSP70 and its co-chaperone CPIP. HSP70 and CPIP together regulated CP turnover by promoting its modification by ubiquitin. Based on these results, an HSP70 and CPIP-driven mechanism that functions to regulate CP during viral RNA replication and/or translation is proposed, possibly to prevent premature particle assembly caused by CP association with viral RNA.

Impact of resistance exercise on ribosome biogenesis is acutely regulated by post-exercise recovery strategies

Muscle hypertrophy occurs following increased protein synthesis, which requires activation of the ribosomal complex. Additionally, increased translational capacity via elevated ribosomal RNA (rRNA) synthesis has also been implicated in resistance training-induced skeletal muscle hypertrophy. The time course of ribosome biogenesis following resistance exercise (RE) and the impact exerted by differing recovery strategies remains unknown. In the present study, the activation of transcriptional regulators, the expression levels of pre-rRNA, and mature rRNA components were measured through 48 h after a single-bout RE. In addition, the effects of either low-intensity cycling (active recovery, ACT) or a cold-water immersion (CWI) recovery strategy were compared. Nine male subjects performed two bouts of high-load RE randomized to be followed by 10 min of either ACT or CWI. Muscle biopsies were collected before RE and at 2, 24, and 48 h after RE. RE increased the phosphorylation of the p38-MNK1-eIF4E axis, an effect only evident with ACT recovery. Downstream, cyclin D1 protein, total eIF4E, upstream binding factor 1 (UBF1), and c-Myc proteins were all increased only after RE with ACT. This corresponded with elevated abundance of the pre-rRNAs (45S, ITS-28S, ITS-5.8S, and ETS-18S) from 24 h after RE with ACT. In conclusion, coordinated upstream signaling and activation of transcriptional factors stimulated pre-rRNA expression after RE. CWI, as a recovery strategy, markedly blunted these events, suggesting that suppressed ribosome biogenesis may be one factor contributing to the impaired hypertrophic response observed when CWI is used regularly after exercise.

The variants of the protein toxins abrin and ricin A useful guide to understanding the processing events in the toxin transport

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by k1-versus-concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin-I (4.13 × 105 M−1 s−1) association with putative receptor (4-methylumbelliferyl-α-D-galactoside) is nearly two times more often than abrin III (2.6 × 105 M−1 s−1) at 20°C. Equillibrium binding constants of abrin I and II to thymocyte at 37°C were 2.26 × 107 M−1 and 2.8 × 107 M−1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41–42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 1 I-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.

Pleiotropic Roles of a Ribosomal Protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over-or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over-or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect - specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.

Studies on rabbit reticulocyte ribosomes and polyribosomes and their relation to hemoglobin synthesis

This dissertation is divided into three parts.

The first section is concerned with protein synthesis in cellfree systems from reticulocytes. The sub-cellular reticulocyte fractions, reagents, etc. have been examined for the presence of traces of ribonuclease, using. an assay based upon the loss of infectivity of RNA fran bacteriophage MS2. This assay is sensitive to 5 x 10^-7 γ RNase/ml. In addition, the loss of synthetic capacity of an 80S ribosome on dissociation has been studied, and can be attributed to loss of messenger RNA when the monomer is separated into subunits. The presence of ribonuclease has been shown to be a major cause of polyribosome disintegration during cell-free protein synthesis.

The second section concerns the changes in ribosomes and polyribosomes which occur during the maturation of a reticulocyte into an erythrocyte. With increasing age, the cells lose a large proportion of the ribonucleoprotein, but the percentage of ribosomes present as polyribosomes is only slightly altered. The loss of hemoglobin synthesis on maturation is probably due to both the loss of total ribosomes and to the lessened specific activity of the polyribosomes.

The third section contains analytical ultracentrifugation data on 80S ribosomes, polyribosomes, and ribosomal RNA from reticulocytes. The 60s and 40s subunits, obtained by dissociation of the 80s particle with inorganic pyrophosphate, were also studied. The RNA from reticulocyte ribosomes has been examined under a variety of denaturing conditions, including dimethyl sulfoxide treatment, formaldehyde reaction and thermal denaturation. From these studies we can conclude that the 28S and 16S RNA's are single polynucleotide chains and are not made up of smaller RNA subunits hydrogen-bonded together.

Part I. Regulation of development in the cellular slime mold Dictyostelium discoideum. Part II. Polysomes and RNA synthesis during early development of the surf clam Spisula solidissima

Part I. The cellular slime mold Dictyostelium discoideum is a simple eukaryote which undergoes a multi-cellular developmental process. Single cell myxamoebae divide vegetatively in the presence of a food source. When the food is depleted or removed, the cells aggregate, forming a migrating pseudoplasmodium which differentiates into a fruiting body containing stalk and spore cells. I have shown that during the developmental cycle glycogen phosphorylase, aminopeptidase, and alanine transaminase are developmentally regulated, that is their specific activities increased at a specific time in the developmental cycle. Phosphorylase activity is undetectable in developing cells until mid-aggregation whereupon it increases and reaches a maximum at mid-culmination. Thereafter the enzyme disappears. Actinomycin D and cycloheximide studies as well as studies with morphologically aberrant and temporally deranged mutants indicate that prior RNA and concomitant protein synthesis are necessary for the rise and decrease in activity and support the view that the appearance of the enzyme is regulated at the transcriptional level. Aminopeptidase and alanine transaminase increase 3 fold starting at starvation and reach maximum activity at 18 and 5 hours respectively.

The cellular DNA s of D. discoideum were characterized by CsC1 buoyant density gradient centrifugation and by renaturation kinetics. Whole cell DNA exhibits three bands in CsCl: ρ = 1.676 g/cc (nuclear main band), 1.687 (nuclear satellite), and 1.682 (mitochondrial). Reassociation kinetics at a criterion of Tm -23°C indicates that the nuclear reiterated sequences make up 30% of the genome (Cot_1/2 (pure) 0.28) and the single-copy DNA 70% (Cot_1/2(pure) 70). The complexity of the nuclear genome is 30 x 10⁹ daltons and that of the mitochondrial DNA is 35-40 x 10⁶ daltons (Cot_1/2 0.15). rRNA cistrons constitute 2.2% of nuclear DNA and have a ρ = 1.682.

RNA extracted from 4 stages during developmental cycle of Dictyostelium was hybridized with purified single-copy nuclear DNA. The hybrids had properties indicative of single-copy DNA-RNA hybrids. These studies indicate that there are, during development, qualitative and quantitative changes in the portion of the single-copy of the genome transcribed. Overall, 56% of the genome is represented by transcripts between the amoeba and mid-culmination stages. Some 19% are sequences which are represented at all stages while 37% of the genome consists of stage specific sequences.

Part II. RNA and protein synthesis and polysome formation were studied during early development of the surf clam Spisula solidissima embryos. The oocyte has a small number of polysomes and a low but measurable rate of protein synthesis (leucine-³H incorporation). After fertilization, there is a continual increase in the percentage of ribosomes sedimenting in the polysome region. Newly synthesized RNA (uridine-5-³H incorporation) was found in polysomes as early as the 2-cell stage. During cleavage, the newly formed RNA is associated mainly with the light polysomes.

RNA extracted from polysomes labeled at the 4-cell stage is polydisperse, nonribosomal, and non-4 S. Actinomycin D causes a reduction of about 30% of the polysomes formed between fertilization and the 16-cell stage.

In the early cleavage stages the light polysomes are mostly affected by actinomycin.

Effect of fertilizer carbamide on proteolytic enzymes of fish Labeo rohita

Specific activities of acid, alkaline and neutral proteases in liver, muscle, brain, and gill of fish exposed to 50 ppm ambient carbamide for 15, 30 and 60 days and in control were estimated. It was observed that carbamide even at low concentration of 50 ppm inhibited proteolysis and favoured protein synthesis.

Suitability of sodium lactate as cryoprotectant and its effect on gel forming ability and protein denaturation of croaker fish surimi during frozen storage

The effect of sodium lactate is compared with sucrose + sorbitol + sodium tri-poly phosphate as cryoprotectant on gel forming ability & protein denaturation of croaker surimi during frozen storage at -20±2°C for 90 days was evaluated. The quality of Croaker surimi with 6% (w/v) sodium lactate was examined in terms of biochemical parameters of muscle protein, thaw drip, gel strength and calcium ATPase activity :.omparing with those of surimi added with sucrose/sorbitol & without additive as control. Both the cryoprotectants minimized the negative effects of frozen storage on physico-chemical traits of myofibrillar proteins which was evident from the biochemical and sensory parameters. The residual Ca2+ ATPase activity and gel strength of surimi with sodium lactate were higher than those of control throughout 90 days of storage. Ca2+ A TPase activity and gel strength found a high positive correlation. From the results, it was found that sodium lactate was equally effective in preservation of croaker muscle protein native structure during frozen storage as the sucrose/ sorbitol and also less sweet without any risk of maillard browning.

Rana grylio virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK-GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells.

Functional domains and the antiviral effect of the double-stranded RNA-dependent protein kinase PKR from Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2 alpha phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2 alpha. The interaction between PoPKR and eIF2 alpha is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2 alpha phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.