Cessation Of Protein Synthesis Research Articles

Differential toxicity of abrin in human cell lines of different organ origin

Abrus precatorius is a highly toxic seed containing the poison abrin. Similar in properties to ricin, this toxin binds to ribosomes causing cessation of protein synthesis and cell death. With an estimated human lethal dose of 0.1–1 μg/kg, it has been the cause of fatalities due to accidental and intentional ingestion. In present study, we profiled seven human cell lines of different organ origin, for their sensitivity against abrin toxicity. These cell lines are, A549, COLO 205, HEK 293, HeLa, Hep G2, Jurkat, SH-SY5Y and derived from lung, intestine, kidney, cervix, liver, immune and nervous system respectively. MTT, NR, CVDE and LDH assays have been used to determine their response against abrin toxin. Among these cell lines A549 was the most sensitive cell line while Hep G2 was found least sensitive cell lines. Hep G2 cells are shown to have mitochondrial resistance and delayed generation of oxidative stress compared to A549 cells. Remarkable variation in sensitivity against abrin toxicity prompted the evaluation of Bcl2, Bax and downstream caspases in both cells. Difference in Bcl2 level has been shown to play important role in variable sensitivity. Findings of present study are helpful for selection of suitable cellular model for toxicity assessment and antidote screening.

Abstract A67: The mTOR inhibitor, everolimus, enhances the cytotoxicity of pseudomonas-based immunotoxins: Mechanistic insights

Abstract Pseudomonas exotoxin (PE)-based immunotoxins (antibody-toxin fusion proteins) have produced frequent complete remissions in patients with hairy cell leukemia but far fewer responses in other cancers. PE and PE-immunotoxins kill cells via a pathway that includes endocytosis, proteolytic processing, transport to the ER and translocation of an enzymatically active toxin fragment from the ER to the cytosol. In the cytosol the toxin ADP-ribosylates EF2 which results in the cessation of protein synthesis. Using two cell lines (KB or Nalm6) that exhibit moderate resistance to immunotoxin killing, we screened 500 compounds from the MIPE-3 library seeking compounds that enhanced the cytotoxicity of immunotoxins targeting either mesothelin or CD22. Results indicated that as few as 20 compounds could enhance cytotoxicity and only a few appeared potentially useful in a clinical setting. One compound of particular interest was the mammalian target of rapamycin (mTOR) inhibitor, everolimus. The mTOR pathway plays a pivotal role in cell signaling and proliferation and ultimately tumorigenesis. Moreover, protein synthesis is the best characterized process controlled by the mTORC1 pathway, making everolimus an excellent candidate to enhance the cytotoxic activity of PE-immunotoxins. We tested combinations of PE-immunotoxins and everolimus on various tumor cell lines (Nalm-6, Daudi, MD-MBA-468 and KB) using different concentration of both compounds. In all cell lines, the cytotoxicity of the combination was greater than either compound alone. As could be anticipated, the increased cytotoxicity was associated with enhanced reductions in cellular protein synthesis: confirming synergy between the immunotoxin and everolimus. To define the mechanism for the observed synergy, we evaluated changes in the levels of the principal constituents of the mTORC1 pathway. We noted that in 24 hours the immunotoxin drastically reduced the level of the p70S6 kinase in a dose dependent manner. No significant changes were seen in the levels of the TSC1, TSC2, FKBP12, mTOR, 4EBP1 or S6 proteins. Reduced levels of p70S6 kinase resulted in the absence of phospho-S6 protein from cells treated with everolimus and immunotoxin compared to the reduced phosphorylation observed with everolimus alone. We suggest that the lack of the p70S6K caused by the immunotoxin-mediated inhibition of protein synthesis is an essential element of the synergy with everolimus-mediated inhibition of mTORC1. Further, results of synergy in vitro were replicated in xenograft models where combinations exhibited enhanced anti-tumor action compared to either agent alone. Citation Format: Antonella Antignani, Lesley Mathews-Griner, Mark Ferrer, Nathan Simon, Evan Angelus, Manjie Huang, Ira Pastan, Craig Thomas, David J. FitzGerald. The mTOR inhibitor, everolimus, enhances the cytotoxicity of pseudomonas-based immunotoxins: Mechanistic insights. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A67.

Abstract 2907: Everolimus enhances the cytotoxic action of the immunotoxin, HA22, directed to CD22 on B-cell malignancies

Abstract Recombinant immunotoxins, antibody-toxin fusion proteins, are therapeutic proteins designed as targeted anti-cancer agents. Clinical trials of immunotoxins directed to surface CD22 or mesothelin are ongoing with a 50% complete response rate in patients with refractory Hairy Cell Leukemia as well as dramatic responses in some patients with advanced mesothelioma. To identify agents that could improve clinical efficacy further, a screen of 500 approved or near-approved compounds (MIPE-4) was conducted, revealing everolimus as a potential enhancer of Pseudomonas exotoxin-based recombinant immunotoxins. When the immunotoxin HA22 (moxetumomab pasudotox), which targets CD22, was added to various B-cell lines, everolimus enhanced cytotoxicity by 2-10-fold. Kinetic analyses indicated that combinations of HA22 and everolimus (0.25 uM) accelerated the cessation of protein synthesis in Nalm-6 and CA46 cells compared to either agent alone. Likewise, in CellTiterGlo assays of cell viability, combinations of HA22 and everolimus resulted in a greater loss of ATP from cells. Western blot analysis of key survival proteins indicated an early loss of Mcl-1 in combination treatments. Additional experiments are ongoing to extend these observations with the goal of uncovering insights in the mechanism of synergy seen with these two compounds. Citation Format: Antonella Antignani, Matteo Pasetto, Chris Choo, Evan Angelus, Lesley Mathews, Rajarshi Guha, Paul Shinn, Marc Ferrer, Craig J. Thomas, Ira Pastan, David J. Fitzgerald. Everolimus enhances the cytotoxic action of the immunotoxin, HA22, directed to CD22 on B-cell malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2907. doi:10.1158/1538-7445.AM2014-2907

Deregulation of poly(A) polymerase I in Escherichia coli inhibits protein synthesis and leads to cell death.

Polyadenylation plays important roles in RNA metabolism in both prokaryotes and eukaryotes. Surprisingly, deregulation of polyadenylation by poly(A) polymerase I (PAP I) in Escherichia coli leads to toxicity and cell death. We show here that mature tRNAs, which are normally not substrates for PAP I in wild-type cells, are rapidly polyadenylated as PAP I levels increase, leading to dramatic reductions in the fraction of aminoacylated tRNAs, cessation of protein synthesis and cell death. The toxicity associated with PAP I is exacerbated by the absence of either RNase T and/or RNase PH, the two major 3′ → 5′ exonucleases involved in the final step of tRNA 3′-end maturation, confirming their role in the regulation of tRNA polyadenylation. Furthermore, our data demonstrate that regulation of PAP I is critical not for preventing the decay of mRNAs, but rather for maintaining normal levels of functional tRNAs and protein synthesis in E. coli, a function for polyadenylation that has not been observed previously in any organism.

Heme-Regulated eIF2α Kinase in Erythropoiesis and Oxidative Stress

Abstract SCI-23 Besides serving as a prosthetic group for hemoglobin, heme also regulates translation by inhibiting the kinase activity of heme-regulated translational inhibitor (HRI). HRI is the only known protein kinase that senses the intracellular heme concentration and does so through its two heme-binding domains. HRI is activated in heme deficiency by autophosphorylation, and phosphorylates the α-subunit of eukaryotic initiation factor 2 (eIF2α), which impairs the recycling of eIF2 for further translational initiation and results in cessation of protein synthesis. In this manner, HRI serves as a feedback inhibitor and coordinates the translation of globin mRNAs with the intracellular heme concentration to ensure that globin proteins are not made in excess of the heme available for the formation of hemoglobin. Excess of globin chains or heme is cytotoxic. In the absence of HRI, uncontrolled protein synthesis in heme deficiency results in globin aggregation and precipitation in red blood cells and their precursors. HRI is responsible for the physiological adaptation that produces hypochromic, microcytic erythrocytes in iron deficiency. The expression of HRI was increased during late stages of erythropoiesis with higher expression in Ter119high erythroblasts and reticulocytes, correlating with the active synthesis and regulation of globins at these stages of erythroid differentiation. In addition to inhibiting global protein synthesis, the second important function of eIF2α phosphorylation is to reprogram translation and the subsequent transcription of genes required for stress response. In mammalian cells, translation of the transcription factor ATF4 mRNA is upregulated specifically by eIF2α phosphorylation via upstream open reading frames in the 5’UTR. Activation of the HRI-ATF4 stress response pathway in nucleated erythroid precursors is required for adaptation to acute and chronic oxidative stress. Furthermore, this HRI-dependent ATF4 pathway is also operative and necessary for erythroid differentiation, especially under stress conditions. In chronic iron deficiency, HRI is necessary for adaptive gene expression for erythroid differentiation as well as for iron heme and redox homeostasis. Beyond heme deficiency and oxidative stress, HRI is also activated by osmotic shock and heat shock. HRI deficiency in mice exacerbates erythropoietic protoporphyria and renders β-thalassemia intermedia embryonically lethal. Hri−/− mice also develop ineffective erythropoiesis during iron/heme deficiency and are severely compromised upon phenylhydrazine-induced acute hemolytic anemia. Thus, translational regulation by HRI plays a critical role in the manifestation of red cell diseases in mice and may be a significant modifier of such diseases in humans. Disclosures: No relevant conflicts of interest to declare.

704 Cold-Agglutinin Hemolytic Anemia: A Harbinger of Recurrent Lymphoma

704 Cold-Agglutinin Hemolytic Anemia: A Harbinger of Recurrent Lymphoma

Perturbing the ubiquitin pathway reveals how mitosis is hijacked to denucleate and regulate cell proliferation and differentiation in vivo.

BackgroundThe eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.Methodology and Principal FindingsWe replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.Conclusions and SignificanceK6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

Characterization of pokeweed antiviral protein binding to mRNA cap analogs: Competition with nucleotides and enhancement by translation initiation factor iso4G

Pokeweed antiviral protein (PAP) is a type I ribosomal inactivating protein (RIP). PAP binds to and depurinates the sarcin/ricin loop (SRL) of ribosomal RNA resulting in the cessation of protein synthesis. PAP has also been shown to bind to mRNA cap analogs and depurinate mRNA downstream of the cap structure. The biological role of cap binding and its possible role in PAP activity are not known. Here we show the first direct quantitative evidence for PAP binding to the cap analog m(7)GTP. We report a binding affinity of 43.3+/-0.1 nM at 25 degrees C as determined by fluorescence quenching experiments. This is similar to the values reported for wheat cap-binding proteins eIFiso4E and eIFiso4F. van't Hoff analysis of m(7)GTP-PAP equilibrium reveals a binding reaction that is enthalpy driven and entropy favored with TDeltaS degrees contributing 15% to the overall value of DeltaG degrees . This is in contrast to the wheat cap-binding proteins which are enthalpically driven in the DeltaG degrees for binding. Competition experiments indicate that ATP and GTP compete for the cap-binding site on PAP with slightly different affinities. Fluorescence studies of PAP-eIFiso4G binding reveal a protein-protein interaction with a K(d) of 108.4+/-0.3 nM. eIFiso4G was shown to enhance the interaction of PAP with m(7)GTP cap analog by 2.4-fold. These results suggest the involvement of PAP-translation initiation factor complexes in RNA selection and depurination.

Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm

Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.

Calcium-Mediated Responses of Maize to Oxygen Deprivation

Oxygen limitation dramatically alters the patterns of gene expression as well as development of plants. Complete removal of O2 leads to an immediate cessation of protein synthesis followed by a selective synthesis of about twenty anaerobic proteins in maize (Zea mays L.) seedlings. Among these are enzymes involved in glycolysis and related processes. However, inducible genes that have different functions were also found; they may function in other, perhaps more long-term, processes of adaptations to flooding, such as aerenchyma formation and root-tip death. Our recent research has addressed two questions: how these gene expression changes are initiated and how do these responses culminate in the overall adaptation of plants to flooding-stress. The results obtained indicate that an early rise in cytosolic Ca2+ as well as a quick establishment of ionic homeostasis may be essential for the induction of adaptive changes at the cellular as well as organismal level.

Distinct Regulation of Internal Ribosome Entry Site-mediated Translation following Cellular Stress Is Mediated by Apoptotic Fragments of eIF4G Translation Initiation Factor Family Members eIF4GI and p97/DAP5/NAT1

Many cellular stresses lead to the inhibition of protein synthesis. Despite this, some cellular mRNAs are selectively translated under these conditions. It was suggested that the presence of internal ribosome entry site (IRES) sequences in the 5'-untranslated regions allow these mRNAs to be actively translated despite the overall cessation of protein synthesis. Here we tested the hypothesis that the IRES elements of genes that are involved in the control of cell survival are distinctly regulated by cellular stresses. We show that the transient conditions of cellular stress favor the translation of pro-survival IRES, while the severe apoptotic conditions support translation of pro-death IRES elements. Furthermore, activation of pro-death IRES during the etoposide-induced apoptosis is caspase-dependent and correlates with the expression of apoptotic fragments of two members of the eIF4G translation initiation factor family, p97/DAP5/NAT1 and eIF4GI. Our results suggest that the regulation of IRES translation during stress contributes to the fine-tuning of cell fate.

Substrate specificity of a maize ribosome-inactivating protein differs across diverse taxa.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.

Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells.

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.

Id2 Expression Increases With Differentiation of Human Myeloid Cells

Id proteins are helix-loop-helix (HLH) transcriptional factors that lack the basic DNA binding domain. The Id proteins have been reported generally to function as inhibitors of cell differentiation, and their gene expression is often downregulated during cell differentiation. We examined the expression of human Id mRNAs by Northern hybridization in 11 human myeloid cell lines, several myeloid cell lines induced to differentiate, fresh myeloid leukemia samples, and normal human myeloid cells. Id2 mRNA was expressed in myelomonoblastic and monoblastic leukemic cells (PLB-985, THP-1, and U-937) but was weakly expressed in myeloblastic leukemic cells (KG-1 and HL-60). Id2 mRNA levels markedly increased with induction of differentiation of myeloid blasts (HL-60, PLB-985, THP-1, and U-937) toward either granulocytes or macrophages. Examination of fresh acute myeloid leukemic samples from 22 individuals also showed prominent Id2 mRNA expression in those samples having more differentiated blasts. Using the French-American-British classification, only 2 of 8 M0/M1 samples expressed Id2 mRNA; however, 10 of 13 M2/M3/M4 samples expressed it. In normal human myeloid cells, Id2 mRNA was expressed in cultured macrophages from bone marrow and in mature granulocytes and monocytes from peripheral blood. The half-life of Id2 mRNA was short (1 hour), and its expression was inducible by cessation of protein synthesis. Id3 mRNA was moderately expressed in monoblastic cell lines (THP-1 and U-937), and levels decreased with their differentiation. Almost no Id3 expression was detectable in either other myeloid leukemia lines, fresh leukemic samples, or normal human myeloid cells by Northern analyses. Id1 mRNA was not detected by polymerase chain reaction in either leukemic or normal myeloid cells except in K562 myeloid/erythroid cells. These results showed that Id2 mRNA was constitutively expressed in more mature myeloid blast cells and level markedly increased with terminal myeloid differentiation, suggesting that Id2 protein may inhibit an HLH transcriptional complex that normally represses myeloid differentiation.

A novel translational regulation function for the simian virus 40 large-T antigen gene.

Cells use the interferon-induced, double-stranded-RNA-dependent protein kinase PKR as a defense against virus infections. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2, resulting in the cessation of protein synthesis. Viruses have evolved various strategies to counteract this cellular defense. In this paper, we show that simian virus 40 (SV40) large-T antigen can antagonize the translational inhibitory effect resulting from the activation of PKR in virus-infected cells. Unlike the situation with other virus-host cell interactions, SV40 large-T antigen does not block the activation of PKR, suggesting that SV40 counteracts the cellular antiviral response mediated by PKR at a step downstream of PKR activation. Mutational analysis of large-T antigen indicates that a domain located between amino acids 400 and 600 of large-T antigen is responsible for this function. These results define a novel translational regulatory function for the SV40 large-T antigen.

Requirement for Protein Synthesis in Antigen Processing by B Cells

The role de novo protein synthesis plays in Ag processing by B cells was investigated. Cycloheximide (CHX) inhibited Ag processing in normal and transformed B cells. B lymphoblastoid cells required a 2-6 hr longer CHX pretreatment period than splenic B cells to inhibit Ag processing function. Immunoprecipitation experiments demonstrated that the half-life of class II/invariant chain (Ii) complexes was similar in normal and transformed B cells. B lymphoblastoid cells differed from splenic B cells in that a significant fraction of total class II-associated p31 Ii was modified with sialic acid (Ip). The kinetics of loss of class II-associated Ip in CHX-treated cells correlated with loss of Ag processing function. In addition, the half-life of a subpopulation of class II molecules that are unstable in sodium dodecyl sulfate at room temperature was greater in transformed cells. Our results suggest that B lymphoblastoid cells, but not splenic B cells, contain a long-lived pool of class II/Ii complexes that can bind and present peptides generated in endosomal compartments for a significant time period after cessation of protein synthesis.

A biphasic change in ribosomal conformation during transneuronal degeneration is altered by inhibition of mitochondrial, but not cytoplasmic protein synthesis

Following loss of eighth nerve input, 20-40% of neurons in the neonatal chick cochlear nucleus, nucleus magnocellularis (NM), undergo cell death. Intracellular changes that precede the death of NM neurons include increased oxidative metabolism and mitochondrial volume, decreased cytoplasmic protein synthesis, and destruction of ribosomes. Six hours following afferent deprivation, dying NM neurons demonstrate complete loss of ribosomes and cessation of protein synthesis, suggesting that the rapid destruction of ribosomes leads to neuronal death. Increased NM neuron death occurs when mitochondrial upregulation is prevented by chloramphenicol, a mitochondrial protein synthesis inhibitor. This finding suggests that increased oxidative capacity is required for neuronal survival following loss of afferent input. To study changes in the ribosomes of afferent-deprived NM neurons, we obtained a monoclonal antibody to ribosomal RNA. This monoclonal antibody, Y10B, labels ribosomes of all NM neurons receiving normal synaptic activity. Following removal of afferent input, NM neurons demonstrate a biphasic change in their pattern of Y10B label. During the initial phase, there is a uniform decrease in the density of Y10B label. In the second phase, some NM neurons recover the capacity to bind the Y10B antibody while others remain unlabeled. During this second phase, NM neurons putatively destined to die, based on their failure to synthesize protein, are unlabeled by the Y10B antibody. New gene expression is not necessary to initiate the change in ribosomal immunoreactivity that leads deafferented NM neurons toward cell death. Blocking cytoplasmic protein synthesis with cycloheximide had no effect on the biphasic change in Y10B labeling of afferent-deprived NM neurons. Treating chicks with chloramphenicol, however, prevented the recovery of Y10B immunoreactivity in NM neurons during the second phase of the response to afferent deprivation.

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

ABSTRACTThe effect of hydroxyurea and 5‐fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L−1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached.FdUrd (25 mg·L−1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L−1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2‐h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.

Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect.

The PKC1 gene of Saccharomyces cerevisiae encodes a homologue of the Ca(2+)-dependent isozymes of mammalian protein kinase C (Levin, D.cE., F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner. 1990. Cell. 62:213-224). Cells depleted of the PKC1 gene product display a uniform phenotype, a behavior indicating a defect in the cell division cycle (cdc). These cells arrest division after DNA replication, but before mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrest growth with small buds. We created conditional alleles of PKC1 to explore the nature of this unusual cdc defect. In contrast to PKC1-depleted cells, all of the conditional pkc1 mutants isolated were suppressed by the addition of CaCl2 to the medium, suggesting that the mutant enzymes could be activated by Ca2+. Arrest of growth and cell division in the conditional mutants was accompanied by cessation of protein synthesis, rapid loss of viability, and release of cellular material into the medium, suggesting cell lysis. This conclusion was supported by the observation that a pkc1 deletion mutant was capable of proliferation in osmotically stabilized medium, but underwent rapid cell lysis when shifted to hypo-osmotic medium. We have incorporated these observations into a model to explain the cdc-specific arrest of pkc1 mutants.

Inhibition of protein synthesis and heat protection: Histidinol‐resistant mutant cell lines

The mechanism of histidinol (HST)-induced heat protection was investigated to test the hypothesis that the cessation of protein synthesis itself is one of the events involved in heat protection. For this study, we isolated three HST-resistant mutant strains. HST (5 mM), which inhibited protein synthesis by 88% in the wild type, caused only 0, 9, and 25% inhibition in three mutants, respectively. The drug, which afforded heat protection, (i.e., a 125-fold increase in survival from 4 x 10(-3) to 5 x 10(-1) after 2 hr at 43 degrees C in wild type), did not protect mutant cells from heat killing. In contrast, cycloheximide (10 micrograms/ml) which inhibited protein synthesis by 95% in both wild type and mutant cell types, protected both cell types from heat killing. Therefore, these results suggest that the cessation of protein synthesis, per se, preventing synthesis of nascent polypeptides, is a major event leading to heat protection.