Presence Of Puromycin Research Articles

Structural Basis for the Antiproliferative Activity of the Tob-hCaf1 Complex

The Tob/BTG family is a group of antiproliferative proteins containing two highly homologous regions, Box A and Box B. These proteins all associate with CCR4-associated factor 1 (Caf1), which belongs to the ribonuclease D (RNase D) family of deadenylases and is a component of the CCR4-Not deadenylase complex. Here we determined the crystal structure of the complex of the N-terminal region of Tob and human Caf1 (hCaf1). Tob exhibited a novel fold, whereas hCaf1 most closely resembled the catalytic domain of yeast Pop2 and human poly(A)-specific ribonuclease. Interestingly, the association of hCaf1 was mediated by both Box A and Box B of Tob. Cell growth assays using both wild-type and mutant proteins revealed that deadenylase activity of Caf1 is not critical but complex formation is crucial to cell growth inhibition. Caf1 tethers Tob to the CCR4-Not deadenylase complex, and thereby Tob gathers several factors at its C-terminal region, such as poly(A)-binding proteins, to exert antiproliferative activity.

HMGA2 Maintains Oncogenic RAS-Induced Epithelial-Mesenchymal Transition in Human Pancreatic Cancer Cells

Pancreatic cancer is a highly aggressive malignancy due to elevated mitotic activities and epithelial-mesenchymal transition (EMT). Oncogenic RAS and transforming growth factor-beta signaling are implicated in these malignant features. The mechanisms that underlie EMT need to be addressed since it promotes tissue invasion and metastasis. The high-mobility group A protein 2 (HMGA2) is a non-histone chromatin factor that is primarily expressed in undifferentiated tissues and tumors of mesenchymal origin. However, its role in EMT in pancreatic cancer is largely unknown. Here we report that HMGA2 is involved in EMT maintenance in human pancreatic cancer cells. Specific knockdown of HMGA2 inhibited cell proliferation, leading to an epithelial-state transition that restores cell-cell contact due to E-cadherin up-regulation. Consistently, an inverse correlation between HMGA2-positive cells and E-cadherin-positive cells was found in cancer tissues. Inhibition of the RAS/MEK pathway also induced an epithelial transition, together with HMGA2 down-regulation. Transcriptional repressors of the E-cadherin gene, such as SNAIL, decreased after HMGA2 knockdown since HMGA2 directly activated the SNAlL gene promoter. The decrease of SNAIL after RAS/MEK inhibition was suppressed by HMGA2 overexpression. Further, let-7 microRNA-mediated HMGA2 down-regulation had no effect on the prevention of the transformed phenotype in these cells. These data shed light on the importance of HMGA2 in reversibly maintaining EMT, suggesting that HMGA2 is a potential therapeutic target for the treatment of pancreatic cancer.

Conditional deletion of Nbs1 in murine cells reveals its role in branching repair pathways of DNA double-strand breaks

NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Here, we have constructed mouse Nbs1-null embryonic fibroblasts and embryonic stem cells, through the Cre-loxP and sequential gene targeting techniques. We show that cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways.

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5'-(beta,gamma-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.

Staphylococcus aureus ClpYQ plays a minor role in stress survival

Although bacteria lack the proteasome-ubiquitin proteolytic pathway, the homologue of the beta-type proteasome subunit, ClpQ, is highly conserved among bacterial species. ClpQ associates with its ATPase partner, ClpY, to form a two-component protease, which also structurally resembles the 26S proteasome. Here we have disrupted clpQ and clpY of the versatile pathogen Staphylococcus aureus in order to examine the significance of the ClpYQ protease for growth under stress conditions. We found that the mutant, in contrast to the wild type, was unable to form colonies at very high temperatures. To our knowledge, this is the first-described phenotype of ClpYQ in Gram-positive bacteria. However, in the presence of puromycin and under all other stress conditions, tested growth of the clpYQ mutant cells was similar to growth of the wild type. Additionally, the absence of ClpYQ did not affect virulence as measured by a murine skin abscess model. Transcriptional analysis revealed that clpQ and clpY are expressed as part of a four-cistronic operon encompassing xerC and codY, and that expression is modestly induced by heat. In conclusion, our data indicates that ClpYQ plays only a secondary role in the degradation of non-native proteins in S. aureus.

Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.

Degradation of Antizyme Inhibitor, an Ornithine Decarboxylase Homologous Protein, Is Ubiquitin-dependent and Is Inhibited by Antizyme

Ornithine decarboxylase (ODC) is the most notable example of a protein degraded by the 26 S proteasome without ubiquitination. Instead, ODC is targeted to degradation by direct binding to a polyamine-induced protein termed antizyme (Az). Antizyme inhibitor (AzI) is an ODC-related protein that does not retain enzymatic activity yet binds Az with higher affinity than ODC. We show here that like ODC, AzI is also a short-lived protein that undergoes proteasomal degradation. However, in contrast to ODC degradation, the degradation of AzI is ubiquitin-dependent and does not require interaction with Az. Moreover, Az binding actually stabilizes AzI by inhibiting its ubiquitination. Substituting the C terminus of AzI with that of ODC, which together with Az constitutes the complete degradation signal of ODC, does not subvert AzI degradation from the ubiquitin-dependent mode to the Az-dependent mode, suggesting dominance of the ubiquitination signal. Our results suggest opposing roles of Az in regulating the degradation of AzI and ODC.

Establishment of stable melanoma cell line expressing a novel gene, jpk, using a tetracycline-controlled gene expression system.

Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli (1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.

DNA damage-processing in E. coli: on-going protein synthesis is required for fixation of UV-induced lethality and mutation

UV irradiation of E. coli produces photoproducts in the DNA genome. In consequence, some bacteria lose viability (colony-forming ability) or remain viable as mutant cells. However, the end-points of viability inactivation (lethality) or mutation are determined by cellular processes that act on the UV-damaged DNA. We have investigated the in vivo time course for processes that deal with cyclobutane pyrimidine dimers (CPD) which can be specifically removed by photoreactivation (PR). At different times during post-UV incubation, samples were challenged with PR and assayed for viability or mutation. We used excision-defective E. coli B/r cells and worked under yellow light to avoid background PR. During post-UV incubation (0–100 min) in fully supplemented defined medium, inactivation and mutation were initially significantly reversed by PR but the extent of this reversal decreased during continued incubation defining “fixation” of lethality or mutation, respectively. In contrast, if protein synthesis was restricted during the post-UV incubation, no fixation developed. When chloramphenicol was added to inhibit protein synthesis after 30 min of supplemented post-UV incubation, at a time sufficient for expression of UV-induced protein(s), fixation of lethality or mutation was still annulled (no change in the effectiveness of PR developed). Lethality fixation did progress when protein synthesis was restricted and the cells were incubated in the presence of puromycin or were either clpP or clpX defective. We discuss these and related results to suggest (1) on-going protein synthesis is required in the fixation process for lethality and mutation to sustain an effective level of a hypothetical protein sensitive to ClpXP proteolysis and (2) this protein plays a critical role in the process leading to exchange between Pol III activity and alternative polymerase activities required as each cell deals with damage in template DNA.

Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.

We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.

Recovery of Escherichia coli IFO3301 Injured by Glycine and Ethanol.

The recovery of Escherichia coli IFO3301 injured by glycine (Gly) and/or ethanol (Et) was investigated. After treatment with 0.39 M Gly (Gly0.39) and 3 M Et (Et3), cells recovered in VSHINSKY broth (V-broth) but were unable to grow in glycerol. Further, the recovery was delayed in the presence of 0.75 M Gly and 1 M Et (Et1). In Et1-injured cells, ammonium lactate was required to restore respiratory activity. The organic ingredients of V-broth rescued the respiratory activity of the injured cells in the presence of puromycin, chloramphenicol and sodium azide. Treatment with Et at 3 M stopped the respiratory activity completely in V-broth. The β-Galactosidase (β-Gal) activity of the cells decreased slightly after treatment with 0.26 M Gly or Et1. Not only 2 M Et (Et2) combined with Gly0.26 but also Et2 alone inhibited the β-Gal activity strongly. This indicated that Et inhibited the β-Gal induction more than Gly. The results suggested that Et plays a role in the prevention of protein synthesis and respiratory electron transport to inhibit bacterial growth.

Time sequence of the inhibition of endothelial adhesion molecule expression by reconstituted high density lipoproteins

We have used discoidal reconstituted high density lipoproteins (rHDL) containing apolipoprotein (apo) A-I and dimyristoyl phosphatidylcholine (DMPC) as a tool to investigate the time sequence of the HDL-mediated inhibition of vascular cell adhesion molecule (VCAM)-1 and E-selectin expression in cytokine-activated human umbilical vein endothelial cells (HUVECs). Specifically, we have asked a few questions — (i) how long do the cells need to be exposed to the rHDL before adhesion molecule expression is inhibited and (ii) how long does the inhibition persist after removing the rHDL from the cells. When the cells were not pre-incubated with the rHDL, there was no inhibition. The magnitude of the inhibition increased progressively with increasing duration of pre-incubation up to 16 h. Inhibition did not require the rHDL to be physically present during the activation of adhesion molecule expression by tumour necrosis factor(TNF)-α, excluding the possibility that the rHDL was merely interfering with the interaction between TNF-α and the cells. When HUVECs were pre-incubated for 16 h with rHDL, the inhibition remained substantial even if the rHDL were removed from the medium up to 8 h prior to addition of TNF-α. The HDL-mediated inhibition of VCAM-1 in HUVECs was unaffected by the presence of puromycin, an inhibitor of protein synthesis, excluding the possibility that HDL may have acted by stimulating the synthesis of a cell protein that itself inhibits adhesion molecule expression. These results have important implications in terms of understanding the mechanism(s) of the HDL-mediated inhibition of endothelial adhesion molecule expression.

Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'.

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A 'primary' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational 'cleavage' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of 'cleavage' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and beta-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal 'skip' from one codon to the next without the formation of a peptide bond.

Puromycin resistance (pac) gene as a selectable marker in vaccinia virus

The antibiotic puromycin, an inhibitor of protein synthesis, was shown to inhibit vaccinia virus (VV) replication. We evaluated the use of puromycin-resistance ( pac) gene as a selectable marker in VV. A recombinant vaccinia virus expressing pac (VV-pac) under the control of a viral early/late promoter was constructed and characterized. VV-pac grew in the presence of puromycin at concentrations that were inhibitory for the parental VV and toxic for the cells. Isolation of recombinant VV usually relies on plaque purification under selective conditions. Because virus plaquing was not feasible under inhibitory puromycin concentration, a protocol based on serial passage of virus was devised. The usefulness of this procedure in selecting pac expressing viruses was tested by isolating a recombinant VV.

Puromycin-rRNA interaction sites at the peptidyl transferase center.

The binding site of puromycin was probed chemically in the peptidyl-transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439, G2505, and G2553 for E. coli, and G2058, A2503, G2505, and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E. coli 23S rRNA. In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P' site of E. coli ribosomes. Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m2A2503/psi2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin, along with other peptidyl-transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the peptidyl transferase loop region. This streptogramin motif is also likely to provide binding sites for the 3' termini of the acceptor and donor tRNAs. In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.

Development of novel lipophilic derivatives of DADLE (leucine enkephalin analogue): intestinal permeability characateristics of DADLE derivatives in rats.

The objective of this study is to examine the intestinal permeability of novel lipophilic derivatives of DADLE (Tyr-D-Ala-Gly-Phe-D-Leu), an enkephalin analogue, using isolated rat intestinal membranes. The novel lipophilic derivatives of DADLE were synthesized by chemical modification with various fatty acids at the C terminus. The pharmacological activities of these DADLE derivatives were assessed by a hot plate test. The intestinal permeability of these derivatives was estimated by the in vitro Ussing chamber method. We obtained four different DADLE derivatives including acetyl-DADLE (DADLE-C2), butyryl-DADLE (DADLE-C4), caproyl-DADLE (DADLE-C6), and caprylyl-DADLE (DADLE-C8). All the derivatives of DADLE had at least 75% of the activity of native DADLE, suggesting that chemical modification of DADLE at the C terminus did not markedly affect its pharmacological activity. These DADLE derivatives were more stable than native DADLE in jejunal and colonic homogenates. A "bell-shaped" profile was observed between the apparent permeability coefficients (Papp) of DADLE derivatives and lipophilicity. In particular, DADLE-C4 had the greatest permeability characteristics across the intestinal membrane of the acyl derivatives studied in this experiment. The permeability of DADLE-C4 across the jejunal membrane was further improved in the presence of puromycin, amastatin, and sodium glycocholate (NaGC), all at a concentration of 0.5 mM. We suggest that the combination of chemical modification with butyric acid and the application of a protease inhibitor are effective for improving the absorption of DADLE across the intestinal membrane.

Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size

Studies of truncated apoB peptides in human subjects with familial hypobetalipoproteinemia, as well as of puromycin-generated spectra of nascent apoB peptides in rat and hamster liver, suggest that a minimum size is required for N-terminal fragments of apoB to be efficiently assembled into full-sized VLDL. We report here results of experiments undertaken to examine this phenomenon in greater detail by expressing individual carboxyl-truncated human apoB constructs in McArdle cells. Thus, apoB-29, -32, -37, -42, -47, -53, -70 and full length apoB-100 were transiently expressed in rat McA-RH7777 hepatoma cells, or human apoB-31 and apoB-53 were stably expressed in the same cells, and the secreted VLDL particles were characterized by kinetic gradient ultracentrifugal flotation. Calibration with rat plasma VLDL subfractions showed that about 90 and 50%, respectively, of lipoprotein particles containing endogenous rat B-100 and B-48 floated between fractions 2–8 of the 11-fraction gradient. This corresponds to the normal VLDL diameter range of about 47 to 28 nm, with the remaining half of rat B-48 recovered as HDL particles in the 1.1 g/ml range. In contrast, regardless of their size, only 2–5% of any of the truncated human apoB peptides expressed in these cells was recovered in the VLDL region of the gradient. The remaining 95+% of the lipoproteins were found as high density particles; as previously found in other systems the densities of the latter were inversely related to their peptide chain-length. Furthermore, transiently expressed full-length human apoB-100 was inefficiently secreted as VLDL by these cells, with the remainder appearing as LDL-sized particles. Thus, although we showed that McA-RH7777 cells secreted endogenous rat apoB as normal-sized VLDL, we found them unsuitable for our original purpose of using human apoB fragments to further define effects of apoB size on VLDL assembly. These cells appeared unable to efficiently use any size of human apoB for that process. Pulse-labeled untransfected McA-RH7777 cells chased in the presence of puromycin did, however, show a sharp decline in VLDL assembly efficiency for endogenous nascent rat apoB peptides shorter than B-48, similar to that originally found in normal rat liver. —Xiao, Q., J. Elovson, and V. N. Schumaker. Rat McA-RH7777 cells efficiently assemble rat apolipoprotein B-48 or larger fragments into VLDL but not human apolipoprotein B of any size.

Alphaviruses to the rescue?

The ability to genetically engineer animal viruses enables their application to medical research. The development of non-retroviral RNA viruses as vectors to deliver foreign genes for vaccination and gene therapy is particularly interesting, especially the domestication of the alphavirus group. Two papers in this field have recently been published1xTwo-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou, C. and Liljestrom, P. J. Virol. 1999; 73: 1092–1098PubMedSee all References, 2xNoncytopathic Sindbis virus RNA vectors for heterologous gene expression. Agapov, E.V. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12689–12994Crossref | PubMed | Scopus (155)See all References.Alphaviruses offer several advantages as expression vectors: they have a broad host range, high levels of expression and a small genome that is easy to manipulate. Vectors based on Semliki Forest virus (SFV), Sindbis virus and Venezuelan equine encephalitis have been constructed and deliver and express their cargo efficiently. However, the generation of wild-type virus, owing to recombination between the artificial genome and a helper genome, remains a problem in many recombinant viral systems. This biosafety issue is a concern, especially if vectors are to be used in vaccine delivery, and methods have been developed to reduce this risk. In the SFV system, Smerdou and Liljestrom1xTwo-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou, C. and Liljestrom, P. J. Virol. 1999; 73: 1092–1098PubMedSee all References1 have prevented the production of wild-type virus by using two, independent helper RNAs; this system also circumvents the requirement of a viral protease and this portion of the genome has been mutated, conferring additional biosafety. Thus, at least two recombination events and one reversion are required to generate replication-competent virus particles. Extensive analysis has failed to demonstrate the presence of wild-type viruses, emphasizing the biosafety of the two-helper system.The expression strategy of alphaviruses imposes a severe burden on normal cellular biochemistry and infected cells die quickly, restricting the use of these vectors when long-term gene expression is required. However, Agapov et al.2xNoncytopathic Sindbis virus RNA vectors for heterologous gene expression. Agapov, E.V. et al. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12689–12994Crossref | PubMed | Scopus (155)See all References2 have developed noncytotoxic Sindbis virus vectors, which produce large amounts of foreign protein, by transfecting Sindbis virus replicons that express a puromycin N-acetyltransferase into cells grown in the presence of puromycin. Cells that survive in the presence of the drug carry replicons that are noncytotoxic and express the puromycin-inactivating enzyme. Such replicons can then be engineered to express other foreign genes for long periods in a noncytotoxic manner, which could be useful in both vaccinology and in the development of gene therapy.

Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.

Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.

Release of α-melanocyte-stimulating hormone from rat splenocytes in vitro is dependent on protein synthesis

The pro-opiomelanocortin (POMC) peptide product α-melanocyte-stimulating hormone (α-MSH) has a variety of anti-inflammatory effects within the immune system. Using an antiserum specific for α-MSH, we have now demonstrated for the first time that this peptide is secreted from splenocytes. Basal secretion of α-MSH from the spleen in vitro was some 300-fold less than that detected from the neurointermediate lobe. Secretion of α-MSH from the spleen was increased 5-fold in the presence of puromycin, indicating that splenocyte α-MSH secretion is under the regulation of a product of endogenous protein synthesis. ACTH and β-endorphin were also released from splenocytes in vitro, but their release was not affected by incubation with puromycin. Secretion of the POMC peptides from splenocytes or NIL was not affected by corticosterone. These data show that the many anti-inflammatory effects within the immune system ascribed to α-MSH may actually be mediated through paracrine effects of immune α-MSH.