Disrupt Protein Synthesis Research Articles

ПРЕНАТАЛЬНОЕ ПИТАНИЕ ДОМАШНЕЙ ПТИЦЫ И ЕГО ПОСТНАТАЛЬНЫЕ ЭФФЕКТЫ (обзор)

Fast growth rate in modern meat-type poultry is accompanied by several metabolic disorders resulting from the discrepancy between embryonic and postembryonic growth and development. Prenatal period of avian ontogenesis is characterized by abrupt physiologic and metabolic alterations and hence any disturbance at this stage can affect the hatch efficiency and subsequent postnatal growth and productivity (E.T. Moran, 2007; V.L. Christensen et al., 2004). The embryonic development in eggs can be supported by the in ovo nutrition using natural nutrients (amino acids, carbohydrates, vitamins) as well as growth stimulators and hormones; this approach can also prepare the poults for the intense postnatal growth (P.R. Ferket, 2016). From the nutrigenomic point of view, the nutrients and bioactive substances can affect gene expression (V.I. Fisinin et al., 2006; L. Bordoni et al., 2019). The experiments with the in ovo nutrition proved that the injections of nutrients can affect physiological status of broiler embryos and hatched broiler chicks. E.g. the injections of carbohydrates enlarge the pool of energy available for the embryo and decreases the catabolism of proteins and lipids during the final stage of incubation, resulting in the increases in the weight of the hatched chicks and in postnatal growth rate, supported by better development of the gastrointestinal tract (R. Kornasio et al., 2011; R. Jhaet al., 2019). All amino acids are necessary for the developing embryo; the absence of any of the amino acids can disrupt protein synthesis and homeostasis in the embryo, resulting in poorer postnatal growth and development. A bulk of studies were published which demonstrated the positive effects of the in ovo injections of individual or mixed amino acids on postnatal growth rate (Y. Ohta et al., 2001; T.M. Shafey et al., 2014; L.L. Yu et al., 2018). Ca. 94 % of total metabolizable energy in the embryo is generated via the oxidation of fatty acids. These oxidative processes, in turn, generate substantial amounts of free radicals which can result in vast cellular damage (P. Surai and V.I. Fisinin, 2013; A. Yigit et al., 2014). The administration of vitamins with antioxidative activity (like С or E) during the embryonic period positively affected the postnatal development of the immune system in chicks (S.A. Selim et al., 2012; S. Nowaczewski et al., 2012). The administration of L-carnitine into the embryos was shown to enhance pre-hatch glucose utilization in the anaerobic conditions and postnatal growth in the chicks (T.M. Shafey et al., 2010; A.M. Dolgorukova, 2017). The in ovo nutrition can therefore be an instrument of significant improvement of the hatchability of the injected eggs and subsequent growth efficiency in hatched chicks, resulting in explicit economic effect (E.D. Peebles, 2018). It should, however, be noted that this technique has not still found application in the commercial poultry production and that for wider knowledge on the stimulating effects of different nutrients on the development of avian embryo further research is required.

PRENATAL NUTRITION OF POULTRY AND ITS POSTNATAL EFFECTS (review)

Fast growth rate in modern meat-type poultry is accompanied by several metabolic disorders resulting from the discrepancy between embryonic and postembryonic growth and development. Prenatal period of avian ontogenesis is characterized by abrupt physiologic and metabolic alterations and hence any disturbance at this stage can affect the hatch efficiency and subsequent postnatal growth and productivity (E.T. Moran, 2007; V.L. Christensen et al., 2004). The embryonic development in eggs can be supported by the in ovo nutrition using natural nutrients (amino acids, carbohydrates, vitamins) as well as growth stimulators and hormones; this approach can also prepare the poults for the intense postnatal growth (P.R. Ferket, 2016). From the nutrigenomic point of view, the nutrients and bioactive substances can affect gene expression (V.I. Fisinin et al., 2006; L. Bordoni et al., 2019). The experiments with the in ovo nutrition proved that the injections of nutrients can affect physiological status of broiler embryos and hatched broiler chicks. E.g. the injections of carbohydrates enlarge the pool of energy available for the embryo and decreases the catabolism of proteins and lipids during the final stage of incubation, resulting in the increases in the weight of the hatched chicks and in postnatal growth rate, supported by better development of the gastrointestinal tract (R. Kornasio et al., 2011; R. Jhaet al., 2019). All amino acids are necessary for the developing embryo; the absence of any of the amino acids can disrupt protein synthesis and homeostasis in the embryo, resulting in poorer postnatal growth and development. A bulk of studies were published which demonstrated the positive effects of the in ovo injections of individual or mixed amino acids on postnatal growth rate (Y. Ohta et al., 2001; T.M. Shafey et al., 2014; L.L. Yu et al., 2018). Ca. 94 % of total metabolizable energy in the embryo is generated via the oxidation of fatty acids. These oxidative processes, in turn, generate substantial amounts of free radicals which can result in vast cellular damage (P. Surai and V.I. Fisinin, 2013; A. Yigit et al., 2014). The administration of vitamins with antioxidative activity (like С or E) during the embryonic period positively affected the postnatal development of the immune system in chicks (S.A. Selim et al., 2012; S. Nowaczewski et al., 2012). The administration of L-carnitine into the embryos was shown to enhance pre-hatch glucose utilization in the anaerobic conditions and postnatal growth in the chicks (T.M. Shafey et al., 2010; A.M. Dolgorukova, 2017). The in ovo nutrition can therefore be an instrument of significant improvement of the hatchability of the injected eggs and subsequent growth efficiency in hatched chicks, resulting in explicit economic effect (E.D. Peebles, 2018). It should, however, be noted that this technique has not still found application in the commercial poultry production and that for wider knowledge on the stimulating effects of different nutrients on the development of avian embryo further research is required.

Hereditary angioedema: Investigational therapies and future research.

The future therapies for hereditary angioedema will likely involve the development of oral agents as alternatives to parenteral administration of drugs, specific targeting of proteins and/or enzymes that are not yet possible (e.g., factor XIIa), new agents that target the β₂ receptor with sustained action properties, testing of products to determine whether the β1 receptor contributes significantly to attacks of angioedema, disrupting protein synthesis by using RNA technology as an alternative to enzyme inhibition, and, finally, gene therapy to attempt to cure the disease. Complete inhibition of attacks may well require sustained blood levels of C1 inhibitor that exceed 85% of normal, and it may be possible to delete the prekallikrein gene (analogous to familial prekallikrein deficiency), which is the one factor that might alleviate bradykinin formation, even by factor XII-independent initiating mechanisms, with the possible exception of Mannose Associated Serine Protease 1 (MASP-1) cleavage of high molecular weight kininogen (HK). Deletion of the light chain of high-molecular-weight kininogen would eliminate all possibilities for bradykinin formation, except tissue kallikrein cleavage of low-molecular-weight kininogen to support normal physiologic function to at least 50%.

Ribosomal stress-surveillance: three pathways is a magic number.

Cells rely on stress response pathways to uphold cellular homeostasis and limit the negative effects of harmful environmental stimuli. The stress- and mitogen-activated protein (MAP) kinases, p38 and JNK, are at the nexus of numerous stress responses, among these the ribotoxic stress response (RSR). Ribosomal impairment is detrimental to cell function as it disrupts protein synthesis, increase inflammatory signaling and, if unresolved, lead to cell death. In this review, we offer a general overview of the three main translation surveillance pathways; the RSR, Ribosome-associated Quality Control (RQC) and the Integrated Stress Response (ISR). We highlight recent advances made in defining activation mechanisms for these pathways and discuss their commonalities and differences. Finally, we reflect on the physiological role of the RSR and consider the therapeutic potential of targeting the sensing kinase ZAKα for treatment of ribotoxin exposure.

Thiopeptides Induce Proteasome-Independent Activation of Cellular Mitophagy.

Thiopeptide antibiotics are emerging clinical candidates that exhibit potent antibacterial activity against a variety of intracellular pathogens, including Mycobacterium tuberculosis (Mtb). Many thiopeptides directly inhibit bacterial growth by disrupting protein synthesis. However, recent work has shown that one thiopeptide, thiostrepton (TSR), can also induce autophagy in infected macrophages, which has the potential to be exploited for host-directed therapies against intracellular pathogens, such as Mtb. To better define the therapeutic potential of this class of antibiotics, we studied the host-directed effects of a suite of natural thiopeptides that spans five structurally diverse thiopeptide classes, as well as several analogs. We discovered that thiopeptides as a class induce selective autophagic removal of mitochondria, known as mitophagy. This activity is independent of other biological activities, such as proteasome inhibition or antibiotic activity. We also find that many thiopeptides exhibit potent activity against intracellular Mtb in macrophage infection models. However, the thiopeptide-induced mitophagy occurs outside of pathogen-containing autophagosomes and does not appear to contribute to thiopeptide control of intracellular Mtb. These results expand basic understanding of thiopeptide biology and provide key guidance for the development of new thiopeptide antibiotics and host-directed therapeutics.

Kanamycin-induced production of 2′,3′-cyclic AMP in Escherichia coli

In contrast to the well-characterized second messenger adenosine 3′,5′-cyclic monophosphate (3′,5′-cAMP), the biological roles of its isomer 2′,3′-cAMP remain largely unknown, especially in bacteria. Recent work reported that RNase I-dependent elevation of 2′,3′-cNMP levels in Escherichia coli correlated with reduced biofilm production, and separate studies demonstrated E. coli ribonuclease activation in response to aminoglycoside antibiotics. Here we report that E. coli produced 2′,3′-cAMP in response to kanamycin at sub-inhibitory levels. Surprisingly, other aminoglycosides like streptomycin or gentamicin did not generate levels of 2′,3′-cAMP detectable by 31P NMR. Interestingly, because 2′,3′-cAMP is also produced in E. coli strains expressing a plasmid-encoded kanamycin resistance gene but not by other ribosome-targeting antibiotics, this kanamycin-specific production may not reflect disrupted protein synthesis. Overall, this finding provides a link between aminoglycoside-induced ribonuclease activity and 2′,3′-cAMP production in E. coli.

Effect of IAPP on the proteome of cultured Rin-5F cells

BackgroundIslet amyloid polypeptide (IAPP) or amylin deposits can be found in the islets of type 2 diabetes patients. The peptide is suggested to be involved in the etiology of the disease through formation of amyloid deposits and destruction of β islet cells, though the underlying molecular events leading from IAPP deposition to β cell death are still largely unknown.ResultsWe used OFFGEL™ proteomics to study how IAPP exposure affects the proteome of rat pancreatic insulinoma Rin-5F cells. The OFFGEL™ methodology is highly effective at generating quantitative data on hundreds of proteins affected by IAPP, with its accuracy confirmed by In Cell Western and Quantitative Real Time PCR results. Combining data on individual proteins identifies pathways and protein complexes affected by IAPP. IAPP disrupts protein synthesis and degradation, and induces oxidative stress. It causes decreases in protein transport and localization. IAPP disrupts the regulation of ubiquitin-dependent protein degradation and increases catabolic processes. IAPP causes decreases in protein transport and localization, and affects the cytoskeleton, DNA repair and oxidative stress.ConclusionsResults are consistent with a model where IAPP aggregates overwhelm the ability of a cell to degrade proteins via the ubiquitin system. Ultimately this leads to apoptosis. IAPP aggregates may be also toxic to the cell by causing oxidative stress, leading to DNA damage or by decreasing protein transport. The reversal of any of these effects, perhaps by targeting proteins which alter in response to IAPP, may be beneficial for type II diabetes.

Tryptophan-Rich and Proline-Rich Antimicrobial Peptides.

Due to the increasing emergence of drug-resistant pathogenic microorganisms, there is a world-wide quest to develop new-generation antibiotics. Antimicrobial peptides (AMPs) are small peptides with a broad spectrum of antibiotic activities against bacteria, fungi, protozoa, viruses and sometimes exhibit cytotoxic activity toward cancer cells. As a part of the native host defense system, most AMPs target the membrane integrity of the microorganism, leading to cell death by lysis. These membrane lytic effects are often toxic to mammalian cells and restrict their systemic application. However, AMPs containing predominantly either tryptophan or proline can kill microorganisms by targeting intracellular pathways and are therefore a promising source of next-generation antibiotics. A minimum length of six amino acids is required for high antimicrobial activity in tryptophan-rich AMPs and the position of these residues also affects their antimicrobial activity. The aromatic side chain of tryptophan is able to rapidly form hydrogen bonds with membrane bilayer components. Proline-rich AMPs interact with the 70S ribosome and disrupt protein synthesis. In addition, they can also target the heat shock protein in target pathogens, and consequently lead to protein misfolding. In this review, we will focus on describing the structures, sources, and mechanisms of action of the aforementioned AMPs.

Glyphosate Substitution for Glycine During Protein Synthesis as a Causal Factor in Mesoamerican Nephropathy

Mesoamerican Nephropathy (MeN), also known as Chronic Kidney Disease of unknown etiology (CKDu), is an unusual form of kidney disease affecting agricultural workers in Central America. Its prevalence is alarmingly high among young male sugarcane workers in Nicaragua and El Salvador. The absence of known etiologies for CKD, such as hypertension and diabetes, has led researchers to explore a number of potential risk factors, though none adequately explain the timing and epidemic nature of the disease. In this paper, we explore the idea that glyphosate, an herbicide routinely used on sugarcane, could play a significant causal role in MeN, mediated by its property as an analogue of the coding amino acid glycine. Glyphosate is a glycine molecule with a methyl phosphonyl group attached to its nitrogen atom. Its substitution in place of glycine could disrupt multiple proteins critical for kidney health. Here, we first present prior evidence from the research literature that glyphosate may be substituting erroneously for glycine. In particular, multiple species of both bacteria and plants have mutated to remove a highly conserved glycine residue in the enzyme in the shikimate pathway that is disrupted by glyphosate, and this mutation has caused the enzyme to be completely insensitive to glyphosate. We have identified multiple proteins with key roles related to kidney function, whose disruption by glyphosate substitution for critical glycine residues could explain most of the unique features of MeN. Specifically, glycine substitution in aquaporin, chloride channels, cytochrome C oxidase and collagen, among others, could contribute to dehydration, increased urinary acidification, renal fibrosis, rhabdomyolysis and mitochondrial dysfunction. While the hypothesis that glyphosate could be disrupting protein synthesis is not yet proven, it is remarkable how well it explains multiple features of MeN. Investigations to verify whether glyphosate is in fact disrupting protein synthesis are urgently needed.

Low-functional programming of the CREB/BDNF/TrkB pathway mediates cognitive impairment in male offspring after prenatal dexamethasone exposure

Adverse intrauterine environments can increase susceptibility to neuropsychiatric diseases that are related to cognitive impairment. In this study, we observed the cognitive impairment of male offspring rats after prenatal dexamethasone exposure (PDE) and explored the associated intrauterine programming mechanism. Pregnant Wistar rats were subcutaneously injected with 0.2mg/kgd dexamethasone from gestational day 9 (GD9) to GD20. A cohort of the pregnant rat group was sacrificed on GD20, and the male fetal rats were collected. Another group of pregnant rats delivered their offspring naturally, and the male adult offspring rats were subjected to behavioural tests postnatally at 26 weeks and then sacrificed. The adult PDE male offspring rats exhibited cognitive impairment, decreased cell proliferation and increased cell apoptosis in the hippocampus, along with damaged synaptic plasticity and disrupted protein synthesis. Meanwhile, activation of GR and downregulation of the cAMP responsive element binding protein (CREB)/brain-derived neurotrophic factor (BDNF)/tropomyosin receptor tyrosine B (TrkB) signalling pathway were found in the adult PDE offspring rats. Further examinations indicated consistent alterations to the fetal hippocampus by PDE. We concluded that PDE can cause cognitive impairment in adult male offspring rats. The mechanism may be associated with low-functional programming of the hippocampal CREB/BDNF/TrkB signalling pathway.

MELAS: Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-Like Episodes

Mitochondrial disease is caused by a deficiency in the energy supply to cells due to mitochondrial dysfunction. Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a mitochondrial disease that presents with stroke-like episodes such as acute onset of neurological deficits and characteristic imaging findings. Stroke-like episodes in MELAS have the following features: 1) neurological deficits due to localization of lesions in the brain, 2) episodes often accompany epilepsy, 3) lesions do not follow the vascular supply area, 4) lesions are more often seen in the posterior brain than in the anterior brain, 5) lesions spread to an adjacent area in the brain, and 6) neurological symptoms often disappear together with imaging findings, but later relapse. About 80% of patients with MELAS have an A-to-G transition mutation at the nucleotide pair 3243 in the dihydrouridine loop of mitochondrial tRNA^Leu(UUR), which causes the absence of posttranscriptional taurine modification at the wobble nucleotide of mitochondrial tRNA^Leu(UUR) and disrupts protein synthesis. However, the precise pathophysiology of stroke-like episodes is under investigation, with possible hypotheses for these episodes including mitochondrial angiopathy, mitochondrial cytopathy, and neuron-astrocyte uncoupling. With regard to treatment, L-arginine and taurine have recently been suggested for relief of clinical symptoms.

Insights into the Molecular Antiviral Mechanism of Pokeweed Protein from Phytolacca americana

Agriculture is an indispensable part of every person’s life, ensuring that nutritious and inexpensive food is readily available. As any other organisms, plants are subject to numerous parasitic infections. Biological evolution has allowed plants to produce a variety of toxic compounds to deal with their pathogens. American pokeweed plant (Phytolacca americana) manufactures pokeweed antiviral protein, a ribosome inactivating protein that disrupts protein synthesis and lowers infectivity of many plant and animal viruses. The intricate mechanism of PAP antiviral activity entails a delicate coordination and interplay of several factors, allowing the plant to battle its invaders. Here, we examine the molecular mechanism of this plant peptide, and describe a molecular model of pokeweed’s antiviral activity.

Neurodegenerative disease: The plot thickens.

Pathological variants of FUS that are associated with amyotrophic lateral sclerosis or frontotemporal dementia can form irreversible gel assemblies that trap and impair the function of ribonucleoproteins and, thereby, disrupt protein synthesis.

Clindamycin

Clindamycin

Liver inflammation and metabolic signaling in ApcMin/+ mice: the role of cachexia progression.

The ApcMin/+ mouse exhibits an intestinal tumor associated loss of muscle and fat that is accompanied by chronic inflammation, insulin resistance and hyperlipidemia. Since the liver governs systemic energy demands through regulation of glucose and lipid metabolism, it is likely that the liver is a pathological target of cachexia progression in the ApcMin/+ mouse. The purpose of this study was to determine if cancer and the progression of cachexia affected liver endoplasmic reticulum (ER)-stress, inflammation, metabolism, and protein synthesis signaling. The effect of cancer (without cachexia) was examined in wild-type and weight-stable ApcMin/+ mice. Cachexia progression was examined in weight-stable, pre-cachectic, and severely-cachectic ApcMin/+ mice. Livers were analyzed for morphology, glycogen content, ER-stress, inflammation, and metabolic changes. Cancer induced hepatic expression of ER-stress markers BiP (binding immunoglobulin protein), IRE-1α (endoplasmic reticulum to nucleus signaling 1), and inflammatory intermediate STAT-3 (signal transducer and activator of transcription 3). While gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression was suppressed by cancer, glycogen content or protein synthesis signaling remained unaffected. Cachexia progression depleted liver glycogen content and increased mRNA expression of glycolytic enzyme PFK (phosphofrucktokinase) and gluconeogenic enzyme PEPCK. Cachexia progression further increased pSTAT-3 but suppressed p-65 and JNK (c-Jun NH2-terminal kinase) activation. Interestingly, progression of cachexia suppressed upstream ER-stress markers BiP and IRE-1α, while inducing its downstream target CHOP (DNA-damage inducible transcript 3). Cachectic mice exhibited a dysregulation of protein synthesis signaling, with an induction of p-mTOR (mechanistic target of rapamycin), despite a suppression of Akt (thymoma viral proto-oncogene 1) and S6 (ribosomal protein S6) phosphorylation. Thus, cancer induced ER-stress markers in the liver, however cachexia progression further deteriorated liver ER-stress, disrupted protein synthesis regulation and caused a differential inflammatory response related to STAT-3 and NF-κB (Nuclear factor—κB) signaling.

Abstract 536: Elucidating the role of miRNA-31 in retinoblastoma

Abstract Retinoblastoma is the most common pediatric ocular cancer. Although early detection of the disease may have a favorable outcome for the patient, retinoblastoma can aggressively develop and become fatal. Current treatment strategies include focal therapies, chemotherapy and in more severe cases, enucleation (removal of the eye) is necessary. This cancer may initiate from acquired or inherited mutations in the RB1 gene. As a result, the mutant retinal cell is conferred an unrestrained proliferative ability that is facilitated by multiple genetic events. Determining the mechanism(s) responsible for tumor growth, invasion, and metastasis may provide an alternative treatment strategy that may delay enucleation, prevent toxic side effects from chemotherapy, or even prevent metastatic spread altogether. Genetic factors that are highly involved in both carcinogenesis and homeostasis are often regulated by miRNAs. miRNAs are small, non-coding molecules that negatively regulate gene expression by mediating mRNA cleavage and/or disrupting protein synthesis. Our rationale for this study is that investigating aberrant miRNA regulation in retinoblastoma will enable us to identify key genetic factors that can be targeted therapeutically. Our miRNA array suggests that there is a significant number of miRNAs whose expression is significantly downregulated between retinoblastoma tumors and normal human retinae. From this data, we have identified candidate miRNAs, such as miRNA-31, that have been previously reported to have an important anti-metastatic role in different cancer types. We are currently investigating the role of these miRNAs in regulating retinoblastoma progression. These functional studies will allow us to determine how increasing miRNA expression in retinoblastoma models can disrupt important signaling pathways associated with retinoblastoma progression and invasion. Citation Format: Vanessa Montoya, Paul J. Bryar, Marilyn Mets, Joanna Weinstein, Nikia Laurie. Elucidating the role of miRNA-31 in retinoblastoma. [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 536. doi:10.1158/1538-7445.AM2014-536

Crystal structure of active site mutant of antileukemic L-asparaginase reveals conserved zinc-binding site.

The atomic coordinates of the monoclinic, orthorhombic and trigonal forms of the D90E mutant of Escherichia coli type II asparaginase have been deposited with the RCSB PDB with accession codes 1JAZ, 1JJA and 1IHD, respectively. EC 3.5.1.1, l-asparagine amidohydrolase, l-asparaginase; EC 3.5.1.38, l-glutamine (l-asparagine) amidohydrolase, glutaminase-asparaginase; EC 6.3.5.6, l-aspartyl-tRNA(Asn) : l-glutamine amido-ligase (ADP-forming), asparaginyl-tRNA synthase (glutamine-hydrolysing).

CdiA from Enterobacter cloacae Delivers a Toxic Ribosomal RNase into Target Bacteria

Contact-dependent growth inhibition (CDI) is one mechanism of inter-bacterial competition. CDI(+) cells export large CdiA effector proteins, which carry a variety of C-terminal toxin domains (CdiA-CT). CdiA-CT toxins are specifically neutralized by cognate CdiI immunity proteins to protect toxin-producingcells from autoinhibition. Here, we use structure determination to elucidate the activity of a CDI toxin from Enterobacter cloacae (ECL). The structure of CdiA-CT(ECL) resembles the C-terminal nuclease domain of colicin E3, which cleaves 16S ribosomal RNA to disrupt protein synthesis. In accord with this structural homology, we show that CdiA-CT(ECL) uses the same nuclease activity to inhibit bacterial growth. Surprisingly, although colicin E3 and CdiA(ECL) carry equivalent toxin domains, the corresponding immunity proteins are unrelated in sequence, structure, and toxin-binding site. Together, these findings reveal unexpected diversity among 16S rRNases and suggest that these nucleases are robust and versatile payloads for a variety of toxin-delivery platforms.

Restrictive Streptomycin Resistance Mutations Decrease the Formation of Attaching and Effacing Lesions in Escherichia coli O157:H7 Strains

Streptomycin binds to the bacterial ribosome and disrupts protein synthesis by promoting misreading of mRNA. Restrictive mutations on the ribosomal subunit protein S12 confer a streptomycin resistance (Strr) phenotype and concomitantly increase the accuracy of the decoding process and decrease the rate of translation. Spontaneous Strr mutants of Escherichia coli O157:H7 have been generated for in vivo studies to promote colonization and to provide a selective marker for this pathogen. The locus of enterocyte effacement (LEE) of E. coli O157:H7 encodes a type III secretion system (T3SS), which is required for attaching and effacing to the intestinal epithelium. In this study, we observed decreases in both the expression and secretion levels of the T3SS translocated proteins EspA and EspB in E. coli O157:H7 Strr restrictive mutants, which have K42T or K42I mutations in S12. However, mildly restrictive (K87R) and nonrestrictive (K42R) mutants showed slight or indistinguishable changes in EspA and EspB secretion. Adherence and actin staining assays indicated that restrictive mutations compromised the formation of attaching and effacing lesions in E. coli O157:H7. Therefore, we suggest that E. coli O157:H7 strains selected for Strr should be thoroughly characterized before in vivo and in vitro experiments that assay for LEE-directed phenotypes and that strains carrying nonrestrictive mutations such as K42R make better surrogates of wild-type strains than those carrying restrictive mutations.

Heat Shock Protein Gp96 as Potential Regulator of Morphostasis after Partial Hepatectomy in Mice

Gp96 (also known as glucose-regulated protein 94, endoplasmin) is the endoplasmic reticulum (ER)-resident protein, which belongs to the heat shock protein HSP90 family. It is upregulated in response to glucose starvation and other stressful stimuli that disrupt protein synthesis in the ER. There, it is acting as a molecular chaperon involved in the correction of unfolded proteins, in the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, and in activation of protein translations that modulate the polypeptide traffic into the ER. In addition, it has been implicated in antigen presentation and MHC class I and II upregulation, in the activation and maturation of dendritic cells and proinflammatory cytokine secretion, as well as in chaperoning of integrins and Toll-like receptors, acting as a "danger signal" to the innate and adaptive immunity. Moreover, owing to its specific function in Ca2+ homeostasis and in the insulin- IGF/signaling pathways, it has been proposed that gp96 might participate in mechanisms that are critical for cell growth, differentiation, and responses to ER stress. Emphasizing that gp96, as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, may also be involved in the maintenance of morphostasis and self tolerance, in this survey we show that high levels of upregulation of gp96 in regenerating liver and thymus are followed by signs of transient autoimmunity, augmented apoptosis in thymus, and the presence of autoreactive NKT and regulatory T cells that might be involved in the control of rapid liver growth induced by partial hepatectomy.