Block In Initiation Research Articles

82-OR: Exosomes from Human ß-Cells Incorporate Stress Granule Components and May Serve as Biomarkers of ß-Cell Stress in Type 1 Diabetes

In early T1D, β cells are subjected to stresses such as inflammation, putative viral infections and increased ER protein load. Under these conditions, β cells activate the integrated stress response, which is highlighted by a block in the translation of mRNAs. These translationally-inhibited mRNAs are compartmentalized into non-membranous inclusions known as stress granules (SGs). During chronic stress, SGs may be sequestered into endosomes and released as components of extracellular vesicles (EVs) into circulation. We hypothesized, the RNA content of EVs may serve as a reflection of ongoing β cell stress. To test this possibility, we measured EV-associated preproinsulin (PPI) mRNA from supernatant of a cultured human β cell line and human islets treated with and without cytokines (IL-1β and IFN-γ) to mimic T1D conditions. Cytokine treatment resulted in a block in mRNA translation initiation, decreased protein synthesis, and increased formation of SGs. Studies using single molecule FISH confirmed that in stressed human β cells there is significant colocalization of PPI mRNA with G3BP1, a marker of SGs. Moreover, stressed human β cells expressing G3BP1-RFP and CD63-GFP (a marker of EVs) exhibited co-localization of SGs with EVs, suggesting that SG contents incorporate into EVs. PPI mRNA was recoverable almost exclusively in EVs isolated from supernatants of stressed human islet cultures. Pharmacological inhibition of EV release decreased PPI mRNA content in the culture media, implying that PPI mRNA is released from β cells within EVs. Droplet digital PCR analysis demonstrated significantly higher levels of PPI mRNA in circulation of new-onset T1D subjects compared to healthy, age-matched controls. Taken together, our data demonstrate that PPI mRNA is released from stressed β cells as a consequence of translational blockade, SG formation, and EV release, and indicate that circulating PPI mRNA might serve as a biomarker reflective of β cell stress. Disclosure F. Syed: None. B.F. Maier: None. E. Anderson-Baucum: None. T.L. Mastracci: None. C. Evans-Molina: Consultant; Self; Bristol-Myers Squibb. R. Mirmira: Advisory Panel; Self; Hibercell, Sigilon Therapeutics, Veralox Therapeutics. Employee; Spouse/Partner; Eli Lilly and Company. Funding National Institute of Diabetes and Digestive and Kidney Diseases (UC4DK104166 to R.M., C.E-M.), (R01DK60581 to R.M.), (R01DK093954 to C.E-M.); U.S. Department of Veterans Affairs (I01BX001733 to C.E-M.); JDRF (3-SRA-2014-41-Q-R to C.E-M, R.M.), (3-PDF-2016-199-A-N to F.S.); Sigma Beta Sorority; Ball Brothers Foundation; George and Frances Ball Foundation; Holiday Management Foundation (to C.E-M., R.M.)

Rheb1-Independent Activation of mTORC1 in Mammary Tumors Occurs through Activating Mutations in mTOR

Mechanistic target of rapamycin complex 1 (mTORC1) is a master modulator of cellular growth, and its aberrant regulation is recurrently documented within breast cancer. While the small GTPase Rheb1 is the canonical activator of mTORC1, Rheb1-independent mechanisms of mTORC1 activation have also been reported but have not been fully understood. Employing multiple transgenic mouse models of breast cancer, we report that ablation of Rheb1 significantly impedes mammary tumorigenesis. In the absence of Rheb1, a block in tumor initiation can be overcome by multiple independent mutations in Mtor to allow Rheb1-independent reactivation of mTORC1. We further demonstrate that the mTOR kinase is indispensable for tumor initiation as the genetic ablation of mTOR abolishes mammary tumorigenesis. Collectively, our findings demonstrate that mTORC1 activation is indispensable for mammary tumor initiation and that tumors acquire alternative mechanisms of mTORC1 activation.

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

BackgroundWe previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5′ untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10–80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described.ResultsPolysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5′UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5′uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories.ConclusionsRegulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.

Defective lymphatic valve development and chylothorax in mice with a lymphatic-specific deletion of Connexin43

Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43-/- mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43∆LEC) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.

NPM and BRG1 Mediate Transcriptional Resistance to Retinoic Acid in Acute Promyelocytic Leukemia

Perturbation in the transcriptional control of genes driving differentiation is an established paradigm whereby oncogenic fusion proteins promote leukemia. From a retinoic acid (RA)-sensitive acute promyelocytic leukemia (APL) cell line, we derived an RA-resistant clone characterized by a block in transcription initiation, despite maintaining wild-type PML/RARA expression. We uncovered an aberrantinteraction among PML/RARA, nucleophosmin (NPM), and topoisomerase II beta (TOP2B). Surprisingly, RA stimulation in these cells results in enhanced chromatin association of the nucleosome remodeler BRG1. Inhibition of NPM or TOP2B abrogated BRG1 recruitment. Furthermore, NPM inhibition and targeting BRG1 restored differentiation when combined with RA. Here, we demonstrate a role for NPM and BRG1 in obstructing RA differentiation and implicate chromatin remodeling in mediating therapeutic resistance in malignancies. NPM mutations are the most common genetic change in patients with acute leukemia (AML); therefore, our model may be applicable to other more common leukemias driven by NPM.

Identification of post-transcriptional regulatory networks during myeloblast-to-monocyte differentiation transition

During hematopoiesis it is clearly emerging that microRNAs are integrated with target genes in regulatory circuitries involved in the decisions regarding the ability to self-renew and to generate a differentiated progeny in hematopoietic cells including myeloid cells (1). microRNAs are able to modulate genes expression mainly by tuning the rate of proteins’ synthesis inhibiting the initiation or later stages of translation. A change in the association of an mRNA with polysomes is indicative of changes in its translation state. For instance, a block in translational initiation would result in reduced ribo- some density on the affected mRNA and a shift toward the lower-density fractions of the gradient (2). Prediction algorithms usually provide hundreds of target genes for each microRNA and the identification of reliable target genes is feasible only through single-gene approaches. To identify microRNA-mRNA networks relevant for the transition from myeloblasts to monocytes, we here evaluated the localization of microRNAs and mRNAs in ribosomal/polysomal cell fractions obtained by sucrose density gradient centrifugation from the myeloblastic cell line HL60 induced or not to differentiate by 1,25-dihydroxyvitamin D3 treatment to induce monocyte/macrophage differentiation. The co-localization of miRNAs and predicted target mRNAs in low-density riboso- mal fractions is strongly indicative of their functional interaction. Intersection between mRNAs shifted across the fractions after treatment with putative target genes of modulated microRNAs showed a series of molecular net- works relevant for the monocyte cell fate determination, as for example the post-tran- scriptional regulation of the Polo-like kinase 1 (PLK1) by miR-22-3p and let-7e-5p. The disclosing of new molecular players involved in myeloid cell fate determina- tion paves the way for the identification of new potentially interesting molecular tar- gets for the treatment of acute myeloid leukemia cells.

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.

An interaction that gets things started

The nuclear envelope clearly helps regulate eukaryotic DNA replication, but many of the molecular pieces of this process are still unknown. On page 177, Martins et al. describe a novel, direct interaction between the nuclear envelope and the genome, mediated by the inner nuclear membrane protein LAP2b and the nuclear matrix–associated protein HA95, that is required for the initiation of replication. Figure Disrupting the chromatin–nuclear envelope link (bottom) prevents DNA replication initiation. Having cloned HA95 two years ago, the authors now find that it interacts with LAP2b through two distinct domains. Abolishing the HA95–LAP2b interaction leads to the proteasome-mediated breakdown of prereplication complex component Cdc6, and a block in replication initiation. The HA95–LAP2b interaction is not required for DNA replication elongation or nuclear envelope reassembly after mitosis.The results add to the growing list of functions for HA95, which is also required for normal nuclear envelope breakdown and chromatin condensation, and is believed to facilitate the export of unspliced viral RNA from the nucleus. In replication initiation, HA95 probably works by protecting the prereplication complex from degradation. Martins et al. propose that the global distribution of HA95 in the nucleus and its ability to bind multiple ligands may explain why it appears in several critical regulatory pathways. ▪

Deficiency of dietary EAA preferentially inhibits mRNA translation of ribosomal proteins in liver of meal-fed rats.

The goal of these studies was to investigate the mechanisms by which amino acid supply regulates global rates of protein synthesis as well as the translation of ribosomal protein (rp) mRNAs in liver. In the experiments conducted, male weanling rats were trained over a 2-wk period to consume their daily food intake within 3 h. On day 14, rats were fed the control diet or an isocaloric, isonitrogenous diet lacking glycine, tryptophan, leucine, or the branched-chain amino acids (BCAA) for 1 h. Feeding Trp-, Leu-, or BCAA-deficient diets resulted in significant reductions in serum insulin, hepatic protein synthesis, eukaryotic initiation factor 2B (eIF2B) activity, and phosphorylation of eIF4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase (S6K1). Phosphorylation of eIF2alpha was inversely related to eIF2B activity under all conditions. Alterations in the hepatic synthesis of rp were assessed by changes in the distribution of rp (S4, S8, L26) mRNAs across sucrose density gradients and compared with non-rp (beta-actin, albumin) mRNAs. In all dietary treatments, non-rp mRNAs were mostly polysome associated. Conversely, the proportion of rp mRNAs residing in polysomes was two- to fivefold less in rats fed diets lacking tryptophan, leucine, or BCAA compared with rats fed the control diet. Total hepatic abundance of all mRNAs examined did not differ among treatment groups. For all parameters examined, there were no differences between rats fed the glycine-deficient diet and rats fed the control diet. The data suggest that essential amino acid (EAA) deficiency inhibits global rates of liver protein synthesis via a block in translation initiation. Additionally, the translation of rp mRNAs is preferentially repressed in association with decreased S6K1 phosphorylation.

Synchronous block in DNA synthesis initiation with change in chromatin topology mediated by VP16.

The impact of chromatin topology on the DNA synthetic process was studied in the human squamous-cell carcinoma cell line SQ-20B. A 1-h exposure < or = 10 microM VP16 produced an increase in DNA supercoil tension, measured by recording laser light scatter from salt-extracted nuclei. This change was precisely paralleled by a decrease in DNA synthesis. The effects on both DNA supercoiling and DNA synthesis were suppressed at VP16 concentrations between 10 and 20 microM. The changes in DNA supercoiling and synthesis at VP16 concentrations -10 microM were eliminated by coincubation with mimosine, a DNA synthesis initiator poison. We conclude that brief exposure to low concentrations of VP16 disturbs the balance of torsional energy within discrete replicon domains by affecting normal topoisomerase II activity at sites of replication initiation. The resultant increase in negative supercoil tension mediates a topologic checkpoint, limiting the initiation of DNA synthesis. Such a checkpoint may be a common pathway for control, both during the normal replicative cycle and subsequent to DNA damage.

Availability of a second upstream AUG can completely overcome inhibition of protein synthesis initiation engendered by mRNA secondary structure encompassing the start codon

Secondary structure analysis of the mRNA from a nonproductive construct carrying the nonstructural gene 3 (NS3) of Japanese Encephalitis Virus revealed the presence of a potential 28 nucleotide long stem and loop beginning with the guanine of the initiation codon AUG that had a calculated stabilization energy of −13 kcal/mol ( ΔG 0 f). Provision of an additional AUG along with three codons upstream resulted in complete relief of inhibition. N-terminal amino acid sequence of the recombinant protein was consistent with initiation of protein synthesis having occurred from the upstream AUG. Similar levels of NS3 specific RNA in E. coli cells carrying the expressing and nonexpressing constructs and restoration of recombinant protein expression following deletion of segments beginning with the stem and loop confirmed the role of this structure in blocking expression at the level of translation initiation. Our approach exploits the ability of a ribosome in motion to open up downstream secondary structural elements of considerable stability and represents a novel and widely applicable strategy to overcome a block in translation initiation caused by mRNA secondary structure around the translation start site.

TOR controls translation initiation and early G1 progression in yeast.

Saccharomyces cerevisiae cells treated with the immunosuppressant rapamycin or depleted for the targets of rapamycin TOR1 and TOR2 arrest growth in the early G1 phase of the cell cycle. Loss of TOR function also causes an early inhibition of translation initiation and induces several other physiological changes characteristic of starved cells entering stationary phase (G0). A G1 cyclin mRNA whose translational control is altered by substitution of the UBI4 5' leader region (UBI4 is normally translated under starvation conditions) suppresses the rapamycin-induced G1 arrest and confers starvation sensitivity. These results suggest that the block in translation initiation is a direct consequence of loss of TOR function and the cause of the G1 arrest. We propose that the TORs, two related phosphatidylinositol kinase homologues, are part of a novel signaling pathway that activates eIF-4E-dependent protein synthesis and, thereby, G1 progression in response to nutrient availability. Such a pathway may constitute a checkpoint that prevents early G1 progression and growth in the absence of nutrients.

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.

Preparation of a Cell-Free Translation System with Minimal Loss of Initiation Factor eIF-2/eIF-2B Activity

Cell-free translation systems prepared from suspension-cultured HeLa S3 cells or mouse L cells by hypotonic shock followed by Dounce homogenization poorly initiated the translation of exogenous mRNA. In contrast, cell extracts prepared from cells exposed to the detergent lysolecithin translated exogenous mRNA readily. The block in initiation in the former lysates was localized to the ribosome fraction. During in vitro translation polysomes from homogenized cells disaggregated but the run-off ribosomes were unable to reinitiate translation. The block resulted from a decrease in eukaryotic initiation factor 2 (eIF-2) or the guanine nucleotide exchange factor (eIF-213) activity, since the addition of eIF-2 or eIF-2B to these latter extracts substantially improved the capacity of the extract to initiate translation of exogenous mRNA. Extracts from homogenized cells, but not from detergent-treated cells, showed enhanced ability to phosphorylate the α subunit of exogenous eIF-2. We show that the method of cell extract preparation greatly influences the state of eIF2/eIF-2B activity in the resulting extract and that extracts in which this activity is maintained can readily initiate translation on exogenous mRNA and reinitiate on endogenous mRNA.

Suppression of MHC class I RNA in highly oncogenic cells occurs at the level of transcription initiation.

The oncogenic potential of some transformed cell lines has been shown in animal tumor models to be the direct result of MHC class I mRNA suppression. Rodent cells established with the highly oncogenic human adenovirus type 12 exhibit greatly reduced levels of class I Ag compared with cells derived with the nononcogenic adenovirus type 5. By using seven independently derived adenovirus-transformed cell lines, we have investigated the mechanism responsible for down-regulation of class I RNA expression. In all adenovirus 12-transformed cells examined, class I RNA suppression was the result of a block in transcription initiation. Highly oncogenic cell lines derived from a spontaneous melanoma and a chemically induced sarcoma also showed greatly reduced transcription of class I genes. We conclude that suppression of MHC class I RNA in a variety of highly oncogenic cells results largely from a substantial decrease in transcription initiation.

Photodynamic effects of Photofrin II on cell division in human NHIK 3025 cells.

Human cervix carcinoma cells of the line NHIK 3025 were exposed to light after 18 h incubation with Photofrin II. After this photodynamic treatment cells in the interphase were retarded with respect to entry into mitosis for a period which increased with increasing light dose. Following the prolonged interphase, an increase in the mitotic index was observed, giving rise to a 3-fold higher level of mitotic cells compared to the control level. Staining of methanol-fixed cells with the DNA-specific dye mithramycin indicated that the increase in mitotic index was due to a prolongation of the metaphase. For all the light doses studied most of the metaphase cells could be characterized as three-group metaphases or c-metaphase-like structures for the first 8 h after treatment. An approximately 10-fold increase above the control level in the number of tripolar mitoses was also observed. A 2h incubation in a Photofrin II-free medium after the 18 h incubation with Photofrin II and before light exposure reduced the fluorescence of the cells by 30 per cent. However, this wash-out period had no effect on the increase in mitotic index after light exposure. A light dose corresponding to 80 per cent survival (as assayed on asynchronous cells) was given to cells in mitosis after Photofrin II incubation. This treatment delayed more than 90 per cent of the metaphase cells from entering the anaphase for at least 1 h. Cells photodynamically treated in the anaphase and telophase entered the interphase at a similar rate as control cells. These observations indicate a temporary block in the initiation of the anaphase and a prolongation of the metaphase. A microscopic study of cells immunologically stained for beta-tubulin 1 h after photodynamic treatment indicated that the organization of the spindle apparatus was disturbed by the photodynamic treatment. Such perturbations are suggested to be the cause of the observed accumulation of cells in mitosis.

Induction of proteins in response to low temperature in Escherichia coli.

When the growth temperature of an exponential culture of Escherichia coli is abruptly decreased from 37 to 10 degrees C, growth stops for several hours before a new rate of growth is established. During this growth lag the number of proteins synthesized is dramatically reduced, and at one point only about two dozen proteins are made; 13 of these are made at differential rates that are 3 to 300 times increased over the rates at 37 degrees C. The protein with the highest rate of synthesis during the lag is not detectably made at 37 degrees C. The identities of several of these cold shock proteins correlate with previous observations that indicate a block in translation initiation at low temperatures.

A block in initiation of simian virus 40 assembly results in the accumulation of minichromosomes containing an exposed regulatory region.

The initiation of simian virus 40 assembly is blocked at the nonpermissive temperature in cells infected with the viral capsid protein VP1 mutant tsC219. Greater than 95% of the minichromosomes isolated from these cells are accessible to cleavage by Bgl I and Sph I, which recognize the sequences near the viral replication origin and in the transcription enhancer elements, respectively. The accessibility of the Ori region to Bgl I is considerably reduced when virion assembly is allowed to proceed in tsC219-infected cells at the permissive temperature. A reduced accessibility to Bgl I is also observed for chromatin isolated from cells infected with wt776, the wild-type parental strain of tsC219. For wt776 chromatin, variability to Bgl I sensitivity is observed and this can be correlated to the relative virion-to-chromatin yield. A similar correlation is not apparent for restriction endonucleases that recognize sequences within the coding region of simian virus 40 chromatin. These results, considered together, indicate that, when virion assembly initiation is blocked, nucleosomes are nonrandomly arranged with respect to the viral regulatory sequences. It appears that the open regulatory region in minichromosomes is established during replication and that a protected regulatory region is generated with the onset of virion assembly.

Effects of noncarbohydrate substrates on protein synthesis in hearts from fed and fasted rats

An overnight fast reduced RNA content and resulted in lower rates and efficiency of protein synthesis when rat hearts were perfused in vitro and supplied glucose as oxidizable substrate. Decreased efficiency of synthesis was associated with development of a block in peptide chain initiation in hearts of both fed and fasted rats. Provision of pyruvate increased the rate and efficiency of protein synthesis in fasted but not fed tissue, and partially overcame the initiation block in both groups. A mixture of glucose, pyruvate and insulin increased the efficiency of protein synthesis and decreased ribosomal subunit content to similar values in both groups of hearts. Noncarbohydrate substrates, including pyruvate, lactate, acetoacetate and beta-hydroxybutyrate, supported higher rates of protein synthesis than glucose in hearts of fasted, but not fed rats. However, mixtures of glucose and either pyruvate, acetoacetate or beta-hydroxybutyrate increased the synthetic rate in fed tissue. Provision of noncarbohydrate substrates increased energy availability, as indicated by higher creatine-P/creatine ratios in both groups of hearts, but the synthetic rate increased as a function of creatine-P/creatine ratio only in the fasted tissue. Octanoate and leucine accelerated protein synthesis and increased energy availability in the fed tissue. The mixtures of glucose and noncarbohydrate substrates or octanoate elevated glucose-6-P content. These studies indicate that an overnight fast decreased the capacity for protein synthesis and modified the regulation of synthesis by noncarbohydrate substrates.

Liquid holding recovery in a DEB-Inactivated rad3 strain of Saccharomyces cerevisiae carrying a thermosensitive prt mutation

The mutation prt1-1 with a thermosensitive block in initiation of protein synthesis was introduced into a rad3 strain to study the effect of inhibition of protein synthesis on liquid holding recovery (LHR) from the lethal effects of diepoxybutane (DEB). Liquid holding of the prt1-1rad3 strain under restrictive conditions did not decrease the level of recovery as compared with the permissive temperature. Post-incubation of cells in growth medium under permissive conditions prior to LH resulted in the loss of capacity for LHR, while cells post-incubated under restrictive conditions were fully capable of LHR. The results are interpreted as indicating that protein synthesis during LH is not required for the increase in survival and that the occurrence of protein synthesis prior to liquid holding abolishes the capacity for LHR.