Protein Synthesis Assay Research Articles

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: activation of aminoacyl-tRNA synthetase.

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in alpha-minimal essential medium containing either vehicle, genistein (l0(-7) - 10(-5) M) or daidzein (10(-7) - 10(-5) M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [3H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein ( 10(-5) M) or daidzein ( 10(-5) M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10(-7) M) or cycloheximide (10(-6) M) in the absence or presence of isoflavones. Moreover, when genistein (10(-7) 10(-5) M) or daidzein (10(-6) and 10(-5) M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10(-7) M). In addition, [3H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10(-6) and 10(-5) M) or daidzein (10(-5) M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P< 0.01). Dexamethasone caused a quadratic decline (P< 0.05) in polysome percentage, while insulin yielded a quadratic increase (P< 0.05). Protein synthesis assays measuring3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.

Therapeutic ultrasound for osteoradionecrosis: an in vitro comparison between 1 MHz and 45 kHz machines

Mandibular osteoradionecrosis is a serious chronic complication which may follow radiotherapy. Therapeutic ultrasound is a highly effective, inexpensive and readily available means of promoting revascularisation and healing. ‘Long wave’ ultrasound increases penetration depth and, therefore, seems to be more appropriate than traditional high frequency ultrasound. The aim of this study was to compare a new treatment using 45 kHz with the current standard 1 MHz machine. A traditional 1 MHz machine, pulsed 1:4, at intensities of 0.1, 0.4, 0.7 and 1.0 W/cm 2 (SAPA) was compared with a long wave machine, 45 kHz, at intensities of 5, 15, 30 and 50 mW/cm 2 (SA). The ultrasound was applied to human gingival fibroblasts and mandibular osteoblasts in vitro. Cell proliferation (DNA synthesis) and collagen and non-collagenous protein synthesis assays were performed, using radiolabelled thymidine and proline, respectively. Controls were sham-insonated and all readings were given as a percentage of controls. Fibroblast proliferation increased by 47% at 0.7 W/cm 2 (1 MHz) and by 43% at 50 mW/cm 2 (45 kHz), and osteoblast proliferation increased by 52% at 1.0 W/cm 2 (1 MHz), and by 35% at 30 mW/cm 2 (45 kHz). Fibroblast collagen production increased by 48% at 0.1 W/cm 2 (1 MHz), and by 44% at 15 mW/cm 2 (45 kHz) and osteoblast collagen production increased by 55% at 0.1 W/cm 2 (1 MHz) and by 112% at 30 mW/cm 2 (45 kHz). Long wave ultrasound was, therefore, capable of inducing a comparable or even higher enhancement of bone formation compared with traditional ultrasound, which, with its greater penetration, may accelerate the healing effect of ultrasound on osteoradionecrosis. The suggested intensity for 45 kHz ultrasound is 30 mW/cm 2.

Metabotropic glutamate receptors prevent nitric oxide-induced programmed cell death.

Activation of metabotropic glutamate receptor (mGluR) subtypes can prevent neuronal injury through the signal transduction pathways of nitric oxide (NO). It is this link to NO free radical injury and subsequent DNA damage that is the most intriguing. We therefore examined whether neuronal protection through mGluR activation was dependent on the molecular mechanisms of programmed cell death (PCD). The NO generators sodium nitroprusside and 3-morpholino-sydnonimine were administered to induce NO toxicity in primary hippocampal neurons. PCD was documented by hematoxylin and eosin nuclear staining, DNA gel electrophoresis, transmission electron microscopy, and protein synthesis assays. Following NO exposure, PCD induction was rapid and robust in approximately 70% of the neuronal population. Activation of specific mGluR subtypes with 1S,3R-ACPD and L-AP4, agents that are neuroprotective against NO, significantly limited the progression of PCD. In contrast, antagonism of mGluRs with L-AP3 did not prevent the development of PCD. Induction of new protein synthesis, a common requisite for PCD, was evident following NO exposure, but did not appear to represent a principal pathway of modulation by the mGluR agonists. Our studies suggest that mGluR modulation of NO-induced PCD represents a primary molecular pathway responsible for neuronal survival. Further elucidation of the molecular mGluR signaling pathways may yield new insight into specific genetic regulatory mechanisms responsible for neuronal injury.

Metabotropic glutamate receptors prevent nitric oxide‐induced programmed cell death

Activation of metabotropic glutamate receptor (mGluR) subtypes can prevent neuronal injury through the signal transduction pathways of nitric oxide (NO). It is this link to NO free radical injury and subsequent DNA damage that is the most intriguing. We therefore examined whether neuronal protection through mGluR activation was dependent on the molecular mechanisms of programmed cell death (PCD). The NO generators sodium nitroprusside and 3-morpholino-sydnonimine were administered to induce NO toxicity in primary hippocampal neurons. PCD was documented by hematoxylin and eosin nuclear staining, DNA gel electrophoresis, transmission electron microscopy, and protein synthesis assays. Following NO exposure, PCD induction was rapid and robust in approximately 70% of the neuronal population. Activation of specific mGluR subtypes with 1S,3R-ACPD and L-AP4, agents that are neuroprotective against NO, significantly limited the progression of PCD. In contrast, antagonism of mGluRs with L-AP3 did not prevent the development of PCD. Induction of new protein synthesis, a common requisite for PCD, was evident following NO exposure, but did not appear to represent a principal pathway of modulation by the mGluR agonists. Our studies suggest that mGluR modulation of NO-induced PCD represents a primary molecular pathway responsible for neuronal survival. Further elucidation of the molecular mGluR signaling pathways may yield new insight into specific genetic regulatory mechanisms responsible for neuronal injury. J. Neurosci. Res. 50: 549–564, 1997. © 1997 Wiley-Liss, Inc.

Oxygen, pHi and arrest of biosynthesis in brine shrimp embryos.

Embryos of the brine shrimp Artemia franciscana are able to withstand bouts of environmental anoxia for several years by entering a quiescent state, during which time metabolism is greatly depressed. Within minutes of oxygen removal, intracellular pH (pHi) drops at least 1.0 unit. This acidification has been strongly implicated in the arrest of both catabolic and anabolic processes in the cytoplasm. A global arrest of cytoplasmic translation accompanies the transition into anoxia or into aerobic acidosis (artificial quiescence imposed by intracellular acidification with CO2 in the presence of oxygen). Similarly, protein synthesis in isolated mitochondria from these embryos is also reduced markedly in response to acidic pH (80% reduction) or anoxia (79% reduction). The constancy of mRNA levels during quiescence indicates that protein synthesis is likely to be controlled at the translational level. Mitochondrial matrix pH is 8.2 during protein synthesis assays performed at the extramitochondrial pH optimum of 7.5. When this proton gradient is abolished with the K+/H+ ionophore nigericin, the extramitochondrial pH optimum for protein synthesis displays an alkaline shift of approximately 0.7 pH unit. These data suggest the presence of proton-sensitive translational components within the mitochondrion. The oxygen dependency of mitochondrial protein synthesis is not explained simply by blockage of the electron transport chain or by the increased redox state. Whereas oxygen deprivation substantially depresses protein synthesis by 77% after 1 h, normoxic incubations with saturating concentrations of cyanide or antimycin A have only a modest effect (36% reduction, cyanide; 20%, antimycin A). This cyanide- and antimycin-insensitive, but hypoxia-sensitive, inhibitory signature for the arrest of protein synthesis suggests the presence of a molecular oxygen sensor within the mitochondrion.

Responses of insect cells to baculovirus infection: protein synthesis shutdown and apoptosis.

Protein synthesis is globally shut down at late times postinfection in the baculovirus Autographa californica M nuclear polyhedrosis virus (AcMNPV)-infected gypsy moth cell line Ld652Y. A single gene, hrf-1, from another baculovirus, Lymantria dispar M nucleopolyhedrovirus, is able to preclude protein synthesis shutdown and ensure production of AcMNPV progeny in Ld652Y cells (S. M. Thiem, X. Du, M. E. Quentin, and M. M. Berner, J. Virol. 70:2221-2229, 1996; X. Du and S. M. Thiem, Virology 227:420-430, 1997). AcMNPV contains a potent antiapoptotic gene, p35, and protein synthesis arrest was reported in apoptotic insect cells induced by infection with AcMNPV lacking p35. In exploring the function of host range factor 1 (HRF-1) and the possible connection between protein synthesis shutdown and apoptosis, a series of recombinant AcMNPVs with different complements of p35 and hrf-1 were employed in apoptosis and protein synthesis assays. We found that the apoptotic suppressor AcMNPV P35 was translated prior to protein synthesis shutdown and functioned to prevent apoptosis. HRF-1 prevented protein synthesis shutdown even when the cells were undergoing apoptosis, but HRF-1 could not functionally substitute for P35. The DNA synthesis inhibitor aphidicolin could block both apoptosis and protein synthesis shutdown in Ld652Y cells infected with p35 mutant AcMNPVs but not the protein synthesis shutdown in wild-type AcMNPV-infected Ld652Y cells. These data suggest that protein synthesis shutdown and apoptosis are separate responses of Ld652Y cells to AcMNPV infection and that P35 is involved in inducing a protein synthesis shutdown response in the absence of late viral gene expression in Ld652Y cells. A model was developed for these responses of Ld652Y cells to AcMNPV infection.

Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro.

Strains of Bacteroides fragilis associated with diarrhea in children (termed enterotoxigenic B. fragilis, or ETBF) produce a heat-labile ca. 20-kDa protein toxin (BFT). The purpose of this study was to examine the activity of BFT on polarized monolayers of human intestinal epithelial cells (T84 cells). In Ussing chambers, BFT had two effects. First, BFT applied to either the apical or basolateral surfaces of T84 monolayers diminished monolayer resistance. However, the time course, magnitude, and concentration dependency differed when BFT was applied to the apical versus basolateral membranes. Second, only basolateral BFT stimulated a concentration-dependent and short-lived increase in short circuit current (Isc; indicative of C1- secretion). Time course experiments indicated that Isc returned to baseline as resistance continued to decrease, indicating that these two electrophysiologic responses to BFT are distinct. Light microscopic studies of BFT-treated monolayers revealed only localized cellular changes after apical BFT, whereas basolateral BFT rapidly altered the morphology of nearly every cell in the monolayer. Transmission and scanning electron microscopy after basolateral BFT confirmed a striking loss of cellular microvilli and complete dissolution of some tight junctions (zonula occludens) and zonula adherens without loss of desmosomes. The F-actin structure of BFT-treated monolayers (stained with rhodamine-phalloidin) revealed diminished and flocculated staining at the apical tight junctional ring and thickening of F-actin microfilaments in focal contacts at the basolateral monolayer surface compared to those in similarly stained control monolayers. BFT did not injure T84 monolayers, as assessed by lactic dehydrogenase release and protein synthesis assays. These studies indicate that BFT is a nonlethal toxin which acts in a polar manner on T84 monolayers to stimulate C1- secretion and to diminish monolayer resistance by altering the apical F-actin structure of these cells. BFT may contribute to diarrheal disease associated with ETBF infection by altering epithelial barrier function and stimulating C1- secretion.

Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species.

Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses.

Changes in Na+/K+-ATPase expression during adaptive cell differentiation in avian nasal salt gland

Chronic salt stress in ducklings (Anas platyrhynchos) resulted in a sustained accumulation of cyclic AMP in the secretory cells of the nasal salt glands. Adaptive increases in the activity of the Na+/K+-ATPase, measured as ATP hydrolysis rates in freshly isolated tissue, were observed after 12 h of salt stress. This change in enzyme activity was associated with increases in protein abundance in the - as well as in the ss-subunit of Na+/K+-ATPase and an increase in ss-subunit glycosylation. We investigated whether the increase in the cytosolic cyclic AMP concentration and the adaptive changes in Na+/K+-ATPase activity were causally related. Using an organotypic tissue culture system for salt gland slices from unstressed (naive) ducklings, we produced similar changes in Na+/K+-ATPase activity and subunit abundance by treating cultured tissue with drugs that elevate cytosolic cyclic AMP levels (forskolin, 8-CPT-cAMP) during a 15 h culture period. Protein synthesis assays using cultured tissue revealed that elevations in cytosolic cyclic AMP level mediate increases in Na+/K+-ATPase subunit abundance by slowing down the degradation of ATPase subunits. This increase in the amount of enzyme protein was associated with a significant increase in Na+/K+-ATPase activity in tissue homogenates. The time course of these changes in cyclic-AMP-treated cultured tissue resembled that observed in salt-stressed intact animals, indicating that the elevation in cyclic AMP level in salt gland tissue may constitute a portion of the signalling events ultimately leading to the adaptive increase in Na+/K+-ATPase activity in vivo.

Effects of TNFα on verocytotoxin cytotoxicity in purified human glomerular microvascular endothelial cells

In the pathogenesis of the hemolytic uremic syndrome (HUS), endothelial damage of glomeruli and arterioles of the kidney appears to play a central role. Previous studies have shown that verocytotoxin-1 (VT-1) cytotoxicity on human vein endothelial cells require additional stimuli, in particular the inflammatory mediator tumor necrosis factor alpha (TNF alpha). In this study the effects of VT on human glomerular microvascular endothelial cells (GMVEC) were examined. A reproducible method was developed for the isolation and purification of large numbers of highly purified GMVEC. The obtained GMVEC were over 99% pure; their endothelial origin was demonstrated by the expression of the endothelial antigens von Willebrand factor, EN-4, PECAM-1 and V,E-cadherin. Upon stimulation with TNF alpha the cells expressed the endothelial-specific adhesion molecule E-selectin. A limited number of fenestral structures was observed by scanning electron microscopy (SEM), suggesting glomerular origin of the endothelial cells. Cytotoxicity of VT-1 to GMVEC was evaluated by determination of the number of viable adherent cells and by assay of overall protein synthesis after exposure to varying concentrations of VT-1. In non-stimulated GMVEC, cytotoxicity of VT-1 was inversely related to the degree and duration of confluence, subconfluent cells being the most sensitive. In highly confluent GMVEC, VT cytotoxicity required pre-exposure of the cells to the inflammatory mediator TNF alpha, which induced an increase in the number of VT receptors on GMVEC. Thin layer chromatography of extracted glycolipids from the GMVEC showed binding of VT-1 to globotriaosylceramide (Gb3), known to be the functional receptor for VT. There were no major differences in protein synthesis inhibition with equal concentrations VT-1 and VT-2. In conclusion, in this study we provide a reproducible method to isolate, purify and culture well characterized human GMVEC on a routine basis. In vitro studies with these GMVEC demonstrate that VT cytotoxicity depends on the degree of confluence and the additional preexposure to the inflammatory mediator TNF alpha. These observations provide further insight into the complex events that may occur in glomeruli in the pathogenesis of HUS.

Structural analysis of ricin and implications for inhibitor design

Ricin is a potent cytotoxin with experimental and clinical uses; it has also been used as a poison. There is considerable interest in identifying or designing inhibitors of the toxin that could be administered as antidotes. The X-ray structure of ricin A-chain is known and a plausible mechanism of action has been proposed. This provides a structural and chemical framework around which inhibitors could be designed; such a structure-based project is underway. Computer programs such as DOCK, GRID, SYBYL, and CHEMX have been used to map the ricin A-chain binding site and to search for potential inhibitors. Inhibitor candidates can be assayed kinetically in a protein synthesis assay and binding can be observed crystallographically. Taken together, a workable search algorithm has been developed and initial tests indicate that at least one ricin A-chain inhibitor, pteroic acid, has been identified.

Acute Depression of Mitochondrial Protein Synthesis during Anoxia: CONTRIBUTIONS OF OXYGEN SENSING, MATRIX ACIDIFICATION, AND REDOX STATE

Mitochondrial protein synthesis is acutely depressed during anoxia-induced quiescence in embryos of Artemia franciscana. Oxygen deprivation is accompanied in vivo by a dramatic drop in extramitochondrial pH, and both of these alterations strongly inhibit protein synthesis in isolated mitochondria. Here we show that the oxygen dependence is not explained simply by blockage of the electron transport chain or by the increased redox state. Whereas oxygen deprivation substantially depressed protein synthesis within 5 min and resulted in a 77% reduction after 1 h, aerobic incubations with saturating concentrations of cyanide or antimycin A had little effect during the first 20 min and only a modest effect after 1 h (36 and 20% reductions, respectively). Yet the mitochondrial NAD(P)H pools were fully reduced after 2-3 min with all three treatments. This cyanide- and antimycin-insensitive but hypoxia-sensitive pattern of protein synthesis depression suggests the presence of a molecular oxygen sensor within the mitochondrion. Second, we show for the first time that acidification of extramitochondrial pH exerts inhibition on protein synthesis specifically through changes in matrix pH. Matrix pH was 8.2 during protein synthesis assays performed at the extramitochondrial pH optimum of 7.5. When this proton gradient was abolished with nigericin, the extramitochondrial pH optimum for protein synthesis displayed an alkaline shift of approximately 0.7 pH unit. These data suggest the presence of proton-sensitive translational components within the mitochondrion.

Murine Cardiotrophin-1 Stimulates the Acute-Phase Response in Rat Hepatocytes and H35 Hepatoma Cells

Mouse cardiotrophin-1 (CT-1) is a hypertrophy-inducing factor for cardiac myocytes and interacts with cell surface receptors that incorporate the signaling molecule gp130. Because other cytokines utilizing this receptor subunit stimulate acute-phase protein synthesis, we tested cardiotrophin-1 in in vitro assays of protein synthesis by primary rat hepatocytes, rat hepatoma cells (H35), and human hepatoma cells (HepG2). CT-1 showed a dose-dependent induction of protein synthesis by primary rat hepatocytes, with effective concentrations ranging from 0.1 to 100 ng/ml. Production of a number of acute-phase proteins, including alpha 1-cysteine proteinase inhibitor ( alpha 1-CPI), alpha 1-proteinase inhibitor (alpha 1-Pi), alpha 2-macroglobulin, and alpha 1-acid glycoprotein, was markedly increased at 48 and 72 h of cytokine stimulation. In rat H35 cells, CT-1 stimulated alpha 1-Pi and alpha 1-CPI protein production and upregulated alpha 1-CPI mRNA levels with similar potency. Compared with other IL-6-type human cytokines at optimal concentrations in parallel assays, CT-1 induced similar levels of acute-phase proteins as human oncostatin M (OM) and leukemia inhibitory factor (LIF), whereas human IL-6 induced the greatest levels of alpha 1-CPI or alpha 1-Pi production by H35 cells. When tested on human HepG2 cells, murine CT-1 was far less effective, in that it stimulated alpha 1-antichymotrypsin production only at very high concentrations (100 ng/ml) but did not alter haptoglobin or alpha 1-Pi. Human OM and IL-6 were effective at lower concentrations and induced much higher levels of acute-phase protein synthesis, whereas LIF activity was similar to that to CT-1. These results show that murine CT-1 is a strong acute-phase mediator for rat hepatocytes in vitro and its activity is similar to LIF on rat hepatocytes, H35 cells, and HepG2 cells.

Attenuated familial adenomatous polyposis (AFAP). A phenotypically and genotypically distinctive variant of FAP.

The usual manifestation of familial adenomatous polyposis (FAP) is hundreds or thousands of colonic adenomas. The authors previously described a colon cancer-prone syndrome characterized by fewer adenomas (1-100), most located in the proximal colon, and upper gastrointestinal lesions, particularly fundic gland polyps and duodenal adenomas. The colonic adenomas are often flat rather than polypoid, a feature emphasized in earlier reports with the term "hereditary flat adenoma syndrome." The syndrome has an autosomal dominant pattern of inheritance and is linked to the adenomatous polyposis coli (APC) locus at 5q. This is a descriptive study based on one family that was followed for more than a decade. Total cell RNA was isolated from cultured lymphoblasts, and an in vitro protein synthesis assay was used to detect APC mutations. Sixteen individuals whose APC mutation status was known had sequential endoscopic evaluations. Five patients were given one or more courses of sulindac. There was perfect concordance between clinical affected status and an APC mutation. All affected members generated a 16-kDa polypeptide from the mutant allele, consistent with a 2-base pair deletion at the extreme 5' end of the APC gene. Sixteen mutation-positive individuals underwent upper gastrointestinal endoscopy and colonoscopy; 13 had colonic adenomas, with the number visualized at any one examination ranging from 1 to greater than 50. Upper gastrointestinal examination revealed fundic gland polyps in 15, gastric or duodenal adenomas in 4, and periampullary carcinoma in 1. AFAP is a phenotypically distinctive syndrome, differing from classic FAP by having fewer colonic adenomas that tend to be proximally distributed and flat rather than polypoid. The position of the APC germline mutation appears to allow for the molecular differentiation between FAP and the attenuated variant in that the extreme 5' APC mutations are associated with the latter.

Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently.

Drugs that show protective effects from ricin toxicity in in vitro protein synthesis assays.

We used an in-vitro, inhibition of protein synthesis assay (PSI) to test a wide variety of drugs for possible therapeutic use against ricin, a toxic glycoprotein that causes death in animals by inhibiting protein synthesis. Selection of test drugs was based on possible interference with ricin activity at different stages of the toxic process. Most of the drugs tested had no effect on ricin-induced PSI, were toxic when tested alone, or enhanced the toxicity of ricin. The only ones showing protection were galactose, lactose, and several derivatives of these sugars, Brefeldin A (BFA), 3'-azido-3'-deoxythymidine (AZT), and a purine derivative (BM33203). THe sugar derivatives provided 50% protection against a PSI ED99 of ricin (0.1 micrograms/ml). Concentrations of BFA greater than 0.5 micro M caused about 50% PSI by itself, but blocked any further inhibitory effects of ricin. AZT, at optimum concentrations, reached a maximum protection level of about 40% in the presence of an ED99 dose of ricin, while the nucleoside derivative, BM33203 and AZT appeared to have an additive effect, showing up to 80% protection from an ED99 dose of ricin. Drugs showing protection in the PSI cell assay showed no protection from ricin in a cell-free translation assay used to determine if they would block ricin at the protein synthesis site.

Mutations in the B-domain of insulin-like growth factor-I influence the oxidative folding to yield products with modified biological properties.

The oxidative folding of human insulin-like growth factor (IGF)-I yields two major disulphide folding isomers. In the present study, B-domain analogues of IGF-I were used to investigate the effect of mutations on the folding reaction and to investigate the functional implications of misfolding. The analogues used were substitutions of the native Glu3 by Gly or Arg, or the native Glu9 by Lys. IGF-I and these analogues were also prepared attached to a hydrophobic 13-amino-acid N-terminal extension, Met-Phe-Pro-Ala-Met-Pro-Leu-Ser-Ser-Leu-Phe-Val-Asn, referred to as 'Long-IGF-I' analogues. Each IGF was fully reduced and refolded to yield native and misfolded isomers, which were subsequently purified for biological characterization. Analysis of the folding reaction at equilibrium revealed a distribution of folding isomers characteristic for each peptide. The yield of the native disulphide folding isomer was increased for the Glu3 substitutions, but not for the Glu9 substitution. The main alternative folding isomer was present in the IGF-I analogues in reduced proportions. Except for [Gly3]IGF-I the N-terminal extension increased the yield of the native isomer which was maximal for the analogue Long-[Arg3]IGF-I. A folding intermediate for the latter analogue was isolated and partially characterized. The biological assays showed that all the main alternative isomers bound poorly to IGF-binding proteins (IGFBPs) secreted by L6 myoblasts. Moreover, these isomers bound to the type 1 IGF receptor with 0.5-25% the affinity of the native isomer. In a rat L6 myoblast protein-synthesis assay, the observed biological activity of the native and main alternative isomers was explained by their modified IGFBP- and receptor-binding properties. We propose that the N-terminal extension imparts a steric constraint at a crucial point in folding, thus allowing native disulphide bonds to form efficiently.

The genetic diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) using in vitro protein synthesis (IVPS) assays for hMSH2 and hMLH1

The genetic diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) using in vitro protein synthesis (IVPS) assays for hMSH2 and hMLH1

Activity-dependent regulation of a ribosomal RNA epitope in the chick cochlear nucleus

Elimination of auditory nerve activity results in rapid metabolic changes, cell atrophy, and cell death in nucleus magnocellularis (NM), the cochlear nucleus of the chick. The transneuronal signals involved in the activity-dependent regulation of NM neurons are not well understood. One of the most rapid transneuronal effects is alteration in protein synthesis by NM neurons. Previous studies using an in vitro preparation of the brain stem auditory system suggested that up-regulation of protein synthesis in NM neurons requires the action of some trophic substance released by active auditory nerve fibers. Here, similar results were obtained when measuring changes in immunoreactivity using a monoclonal antibody (Y10B) that recognizes ribosomal RNA. This immunolabeling assay has advantages over the global protein synthesis assay in that it is not sensitive to possible changes in specific activity of the precursor pool or possible differences in the uptake of the labeled amino acids. Unilateral stimulation of the auditory nerve for 1 h resulted in greater immunolabeling of NM neurons on the stimulated side of the slice. This is consistent with previous in vivo results after unilateral deafferentation. Blockade of synaptic transmission by maintaining the slice in a low-Ca 2+/high Mg 2+ medium prevented the stimulation-induced difference in immunolabeling. Electrical stimulation of the postsynaptic NM neurons alone (antidromic stimulation, via electrical stimulation of NM neuron axons) did not result in greater immunolabeling. Rather, antidromically stimulated neurons tended to show lighter labeling. Thus, the transneuronal regulation of ribosomes in NM neurons appears to require some substance released from the active auditory nerve. Further, blockade of protein synthesis using cycloheximide did not prevent the activity-dependent difference in immunolabeling. Thus, the activity-dependent regulation of ribosomes does not appear to require new protein synthesis during the period of differential activity.