RESUMO
Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.
Assuntos
Emetina , Ribossomos , Puromicina/farmacologia , Microscopia Crioeletrônica , Emetina/análise , Emetina/metabolismo , Ribossomos/metabolismo , Biossíntese de Proteínas , Peptídeos/metabolismo , Neurônios/metabolismoRESUMO
Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.
Assuntos
Escherichia coli , Biossíntese de Proteínas , Ribossomos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Peptídeos/metabolismo , Puromicina , Ribossomos/metabolismoRESUMO
Gemcitabine is a nucleoside analog widely used as an anticancer agent against several types of cancer. Although gemcitabine sometimes shows excellent effectiveness, cancer cells are often poorly responsive to or resistant to the drug. Recently, specific strains or dysbiosis of the human microbiome were correlated with drug reactivity and resistance acquisition. Therefore, we aimed to identify antibiotic compounds that can modulate the microbiome to enhance the responsiveness to gemcitabine. To achieve this, we confirmed the gemcitabine responsiveness based on public data and conducted drug screening on a set of 250 antibiotics compounds. Subsequently, we performed experiments to investigate whether the selected compounds could enhance the responsiveness to gemcitabine. First, we grouped a total of seven tumor cell lines into resistant and sensitive group based on the IC50 value (1 µM) of gemcitabine obtained from the public data. Second, we performed high-throughput screening with compound treatments, identifying seven compounds from the resistant group and five from the sensitive group based on dose dependency. Finally, the combination of the selected compound, puromycin dihydrochloride, with gemcitabine in gemcitabine-resistant cell lines resulted in extensive cell death and a significant increase in cytotoxic efficacy. Additionally, mRNA levels associated with cell viability and stemness were reduced. Through this study, we screened antibiotics to further improve the efficacy of existing anticancer drugs and overcome resistance. By combining existing anticancer agents and antibiotic substances, we hope to establish various drug combination therapies and ultimately improve cancer treatment efficacy.
Assuntos
Antibacterianos , Desoxicitidina , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Gencitabina , Ensaios de Triagem em Larga Escala , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Humanos , Ensaios de Triagem em Larga Escala/métodos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linhagem Celular Tumoral , Antibacterianos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Puromicina/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Sinergismo Farmacológico , Antineoplásicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
OBJECTIVES: To compare, in vitro, resin cement excess removal techniques at the veneer-tooth interface. MATERIALS AND METHODS: Anterior human teeth were restored with ceramic veneers and randomly divided according to the following techniques (n = 10): removal of excess resin cement with brush and dental floss, followed by light-curing with Valo (Group 1) or Elipar (Group 2) for 1 min and 40 s; tack-curing with Valo (Group 3) or Elipar (Group 4) for 1 s; and tack-curing with Valo (Group 5) or Elipar (Group 6) for 5 s. The tack-curing was followed by removal of excess with probe and dental floss and light-curing for 1 min and 40 s. The area of excess resin cement (mm2) was measured in micro-CT images using AutoCAD program. The failures at the cervical margin in the X, Y, and Z axes (µm) of greater value were measured using the DataViewer program. The specimens were submitted to microleakage with 2% basic fuchsin. RESULTS: According to the Kruskal-Wallis and multiple comparison test, the highest area of excess resin cement was found in Group 1 (5.06 mm2), which did not differ statistically from Groups 2 (3.70 mm2) and 5 (2.19 mm2). Groups 2, 3 (1.73 mm2), 4 (1.14 mm2), and 5 (2.18 mm2) did not differ statistically. Group 6 (0.77 mm2) obtained the lowest value, which did not differ statistically from Groups 3 and 4. According to the Kruskal-Wallis and Dunn test, there was no significant difference in failures in X (p = 0.981), Y (p = 0.860), and Z (p = 0.638) axes and no significant difference in microleakage (p = 0.203) among the groups. CONCLUSIONS: Tack-curing for 1 s or 5 s, followed by removal of excess resin cement using a probe and a dental floss, tended to result in a lower amount of excess material around the margin. CLINICAL RELEVANCE: The technique used for resin cement excess removal influences the amount of excess leaved at the veneer-tooth interface. Tack-curing for 1 s or 5 s is recommended to mitigate the excess resin cement.
Assuntos
Cerâmica , Cimentos de Resina , Humanos , Pescoço , Puromicina , Microtomografia por Raio-XRESUMO
Prosthetic infections are associated with high morbidity, mortality, and relapse rates, making them still a serious problem for implantology. Staphylococcus aureus is one of the most common bacterial pathogens causing prosthetic infections. In response to the increasing rate of bacterial resistance to commonly used antibiotics, this work proposes a method for combating pathogenic microorganisms by modifying the surfaces of synthetic polymeric biomaterials using proteolytic enzyme inhibitors (serine protease inhibitors-4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and puromycin). While using techniques based on the immobilization of biologically active molecules, it is important to monitor the changes occurring on the surface of the modified biomaterial, where spectroscopic techniques (e.g., FTIR) are ideal. ATR-FTIR measurements demonstrated that the immobilization of both inhibitors caused large structural changes on the surface of the tested vascular prostheses (polyester or polytetrafluoroethylene) and showed that they were covalently bonded to the surfaces of the biomaterials. Next, the bactericidal and antibiofilm activities of the tested serine protease inhibitors were determined using the CLSM microscopic technique with fluorescent staining. During LIVE/DEAD analyses, a significant decrease in the formation of Staphylococcus aureus biofilm after exposure to selected concentrations of native inhibitors (0.02-0.06 mg/mL for puromycin and 0.2-1 mg/mL for 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) was demonstrated.
Assuntos
Anti-Infecciosos , Infecções Estafilocócicas , Sulfonas , Humanos , Prótese Vascular , Antibacterianos/farmacologia , Biofilmes , Inibidores de Serina Proteinase/farmacologia , Staphylococcus aureus , Materiais Biocompatíveis , Puromicina , Peptídeo HidrolasesRESUMO
Prime editing is a programmable genetic method that can precisely generate any desired small-scale variations in cells without requiring double-strand breaks and DNA donors. However, higher editing efficiency is greatly desirable for wide practical applications. In this study, we developed a target-specific prime editing reporter (tsPER) and a universal prime editing reporter (UPER) to facilitate rapid selection of desired edited cells through puromycin screening. The modification efficiency of HEK3_i1CTT_d5G in HEK293T cells improved from 36.37 % to 64.84 % with the incorporation of tsPER. The target sequence of interested genes could be custom inserted into a selection cassette in tsPER to establish personalized reporters. The UPER demonstrated PE3 editing efficiency up to 74.49 % on HEK3_i1CTT_d5G and 73.52 % on HEK3_i1His6, achieved through co-selection with an additional pegRNA (puro) to repair the mutant PuroR cassette. Overall, tsPER and UPER robustly improved the efficiency of prime editing. Both of these approaches expand enrichment strategies for genomically modified cells and accelerate the generation of genetically modified models.
Assuntos
Edição de Genes , Humanos , Edição de Genes/métodos , Células HEK293 , Genes Reporter , Sistemas CRISPR-Cas , Puromicina/farmacologiaRESUMO
ABSTRACT: Mechanisms of proteostasis in anucleate circulating platelets are unknown and may regulate platelet function. We investigated the hypothesis that plasma-borne growth factors/hormones (GFHs) maintain constitutive translation in circulating platelets to facilitate reactivity. Bio-orthogonal noncanonical amino acid tagging (BONCAT) coupled with liquid chromatography-tandem mass spectrometry analysis revealed constitutive translation of a broad-spectrum translatome in human platelets dependent upon plasma or GFH exposure, and in murine circulation. Freshly isolated platelets from plasma showed homeostatic activation of translation-initiation signaling pathways: phosphorylation of p38/ERK upstream kinases, essential intermediate MNK1/2, and effectors eIF4E/4E-BP1. Plasma starvation led to loss of pathway phosphorylation, but it was fully restored with 5-minute stimulation by plasma or GFHs. Cycloheximide or puromycin infusion suppressed ex vivo platelet GpIIb/IIIa activation and P-selectin exposure with low thrombin concentrations and low-to-saturating concentrations of adenosine 5'-diphosphate (ADP) or thromboxane analog but not convulxin. ADP-induced thromboxane generation was blunted by translation inhibition, and secondary-wave aggregation was inhibited in a thromboxane-dependent manner. Intravenously administered puromycin reduced injury-induced clot size in cremaster muscle arterioles, and delayed primary hemostasis after tail tip amputation but did not delay neither final hemostasis after subsequent rebleeds, nor final hemostasis after jugular vein puncture. In contrast, these mice were protected from injury-induced arterial thrombosis and thrombin-induced pulmonary thromboembolism (PE), and adoptive transfer of translation-inhibited platelets into untreated mice inhibited arterial thrombosis and PE. Thus, constitutive plasma GFH-driven translation regulates platelet G protein-coupled receptor reactivity to balance hemostasis and thrombotic potential.
Assuntos
Agregação Plaquetária , Trombose , Camundongos , Humanos , Animais , Trombina/metabolismo , Tromboxanos , Puromicina/efeitos adversosRESUMO
To expand the scope of genomic editing, a C-to-G transversion-based editor called CGBE has been developed for precise single-nucleotide genomic editing. However, limited editing efficiency and product purity have hindered the development and application of CGBE. In this study, we introduced the Puromycin-Resistance Screening System, referred to as CGBE/ABE-PRSS, to select genetically modified cells via the CGBE or ABE editors. The CGBE/ABE-PRSS system significantly improves the enrichment efficiency of CGBE- or ABE-modified cells, showing enhancements of up to 59.6 % compared with the controls. Our findings indicate that the CGBE/ABE-PRSS, when driven by the CMV promoter, results in a higher enrichment of edited cells compared to the CAG and EF1α promoters. Furthermore, we demonstrate that this system is compatible with different versions of both CGBE and ABE, enabling various cell species and simultaneous multiplexed genome editing without any detectable random off-targets. In conclusion, our developed CGBE/ABE-PRSS system facilitates the selection of edited cells and holds promise in both basic engineering and gene therapy applications.
Assuntos
Resistência Microbiana a Medicamentos , Edição de Genes , Edição de Genes/métodos , Humanos , Resistência Microbiana a Medicamentos/genética , Sistemas CRISPR-Cas , Células HEK293 , Regiões Promotoras Genéticas , Puromicina/farmacologia , AnimaisRESUMO
The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5'-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5'-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management.
Assuntos
Transdução de Sinais , Animais , Relação Estrutura-Atividade , Transdução de Sinais/efeitos dos fármacos , Masculino , Camundongos , Estrutura Molecular , Relação Dose-Resposta a Droga , Humanos , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/metabolismo , Encefalinas/química , Encefalinas/metabolismo , Encefalinas/farmacologia , Puromicina/farmacologia , Puromicina/metabolismo , Puromicina/química , Analgésicos/farmacologia , Analgésicos/química , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/metabolismo , RatosRESUMO
The modification of the surgical polypropylene mesh and the polytetrafluoroethylene vascular prosthesis with cecropin A (small peptide) and puromycin (aminonucleoside) yielded very stable preparations of modified biomaterials. The main emphasis was placed on analyses of their antimicrobial activity and potential immunomodulatory and non-cytotoxic properties towards the CCD841 CoTr model cell line. Cecropin A did not significantly affect the viability or proliferation of the CCD 841 CoTr cells, regardless of its soluble or immobilized form. In contrast, puromycin did not induce a significant decrease in the cell viability or proliferation in the immobilized form but significantly decreased cell viability and proliferation when administered in the soluble form. The covalent immobilization of these two molecules on the surface of biomaterials resulted in stable preparations that were able to inhibit the multiplication of Staphylococcus aureus and S. epidermidis strains. It was also found that the preparations induced the production of cytokines involved in antibacterial protection mechanisms and stimulated the immune response. The key regulator of this activity may be related to TLR4, a receptor recognizing bacterial LPS. In the present study, these factors were produced not only in the conditions of LPS stimulation but also in the absence of LPS, which indicates that cecropin A- and puromycin-modified biomaterials may upregulate pathways leading to humoral antibacterial immune response.
Assuntos
Anti-Infecciosos , Materiais Biocompatíveis , Materiais Biocompatíveis/farmacologia , Lipopolissacarídeos , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Polímeros/farmacologia , Staphylococcus epidermidis , PuromicinaRESUMO
Experimental glomerulonephritis results in hypertension that is sensitive to salt. Nevertheless, salt retention alone cannot explain the increase in blood pressure. Angiotensin antagonistic therapy reduces hypertension caused by puromycin amino nucleosides (PAN). We investigated the hypothesis that PAN modifies renal vascular reactivity through processes dependent on angiotensin. Long-Evans rats were given an intraperitoneal injection of either puromycin (150 mg/kg) or saline (controls). Group 1 was fed a normal sodium diet (NSD, n = 9). Group 2 was given 30 mg/L of quinapril (Q) in addition to NSD (NSD + Q; n = 6). Group 3 received a high sodium diet (HSD, n = 7), and Group 4 received HSD + Q (n = 7). Systolic blood pressure (SBP), plasma creatinine, proteinuria, and sodium balance were monitored for 12 days. On day 15, renal vascular reactivity was assessed by administering increasing doses of angiotensin II, acetylcholine (ACh), and sodium nitroprusside (SNP) directly into the renal artery. SBP progressively increased in all PAN groups. This increase in SBP was greater in the HSD groups and was not significantly altered by Q treatment. SBP increased by 22 ± 4% (NSD), 51 ± 5% (NSD + Q), 81 ± 10% (HSD), and 65 ± 8% (HSD + Q). The renal blood flow of PAN rats did not return to baseline despite their normal renal vasoconstrictor responses to angiotensin II. Additionally, they showed reduced renal vasodilator responses to SNP and Ach. The vasodilator responses to both vasodilators were surprisingly unaffected by the inhibition of the angiotensin-converting enzyme (ACE). Renal vasodilator responses to both endothelium-dependent and independent variables were reduced in early PAN-induced hypertension. We found that the angiotensin-mediated mechanism is not responsible for this altered renal vasoreactivity.
Assuntos
Angiotensina II , Rim , Animais , Angiotensina II/farmacologia , Ratos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Masculino , Ratos Long-Evans , Pressão Sanguínea/efeitos dos fármacos , Puromicina/farmacologia , Nitroprussiato/farmacologia , Puromicina Aminonucleosídeo , Acetilcolina/farmacologia , Nefropatias/induzido quimicamenteRESUMO
Computational pharmacogenomics can potentially identify new indications for already approved drugs and pinpoint compounds with similar mechanism-of-action. Here, we used an integrated drug repositioning approach based on transcriptomics data and structure-based virtual screening to identify compounds with gene signatures similar to three known proteasome inhibitors (PIs; bortezomib, MG-132, and MLN-2238). In vitro validation of candidate compounds was then performed to assess proteasomal proteolytic activity, accumulation of ubiquitinated proteins, cell viability, and drug-induced expression in A375 melanoma and MCF7 breast cancer cells. Using this approach, we identified six compounds with PI properties ((-)-kinetin-riboside, manumycin-A, puromycin dihydrochloride, resistomycin, tegaserod maleate, and thapsigargin). Although the docking scores pinpointed their ability to bind to the ß5 subunit, our in vitro study revealed that these compounds inhibited the ß1, ß2, and ß5 catalytic sites to some extent. As shown with bortezomib, only manumycin-A, puromycin dihydrochloride, and tegaserod maleate resulted in excessive accumulation of ubiquitinated proteins and elevated HMOX1 expression. Taken together, our integrated drug repositioning approach and subsequent in vitro validation studies identified six compounds demonstrating properties similar to proteasome inhibitors.
Assuntos
Bortezomib , Reposicionamento de Medicamentos , Inibidores de Proteassoma , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/química , Reposicionamento de Medicamentos/métodos , Bortezomib/farmacologia , Transcriptoma , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular Tumoral , Células MCF-7 , Simulação de Acoplamento Molecular , Antineoplásicos/farmacologia , Antineoplásicos/química , Puromicina/farmacologia , Perfilação da Expressão Gênica , Sobrevivência Celular/efeitos dos fármacosRESUMO
Staphylococcus aureus is highly prevalent among patients with atopic dermatitis (AD), and this pathogen may trigger and aggravate AD lesions. The aim of this study was to determine the prevalence of S. aureus in the nares of pediatric subjects and verify the phenotypic and molecular characteristics of the isolates in pediatric patients with AD. Isolates were tested for antimicrobial susceptibility, SCCmec typing, and Panton-Valentine Leukocidin (PVL) genes. Lineages were determined by pulsed-field gel electrophoresis and multilocus sequence typing (MLST). AD severity was assessed with the Scoring Atopic Dermatitis (SCORAD) index. Among 106 patients, 90 (85%) presented S. aureus isolates in their nares, and 8 also presented the pathogen in their skin infections. Two patients had two positive lesions, making a total of 10 S. aureus isolates from skin infections. Methicillin-resistant S. aureus (MRSA) was detected in 24 (26.6%) patients, and PVL genes were identified in 21 (23.3%), including 6 (75%) of the 8 patients with skin lesions but mainly in patients with severe and moderate SCORAD values (P=0.0095). All 24 MRSA isolates were susceptible to trimethoprim/sulfamethoxazole, while 8 isolates had a minimum inhibitory concentration (MIC) to mupirocin >1024 μg/mL. High lineage diversity was found among the isolates including USA1100/ST30, USA400/ST1, USA800/ST5, ST83, ST188, ST718, ST1635, and ST2791. There was a high prevalence of MRSA and PVL genes among the isolates recovered in this study. PVL genes were found mostly among patients with severe and moderate SCORAD values. These findings can help clinicians improve the therapies and strategies for the management of pediatric patients with AD.
Assuntos
Animais , Masculino , Camundongos , Ratos , Nefropatias/metabolismo , Rim/metabolismo , Podócitos/metabolismo , Transdução de Sinais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica , Redes Reguladoras de Genes , Immunoblotting , Nefropatias/induzido quimicamente , Nefropatias/genética , Rim/patologia , Rim/fisiopatologia , Microscopia Eletrônica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Puromicina , Podócitos/patologia , Podócitos/ultraestrutura , Proteômica/métodos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
There have been numerous reports that infusion of either natural bile or bile salts into the duodenum evokes a rapid increase in pancreatic secretion through the release of the hormone secretin from the duodenal mucosa. We have extended this observation by the demonstration of an additional late increase in secretion which persisted for many hours and have sought to identify the processes underlying this increase. In anaesthetised rats, infusion of 20 mM taurocholate into the duodenum caused a staircase-like increase in the weight of pancreatic secretion which extended over many hours during which, the HCO −3 and protein output of the secretion showed only minimal changes. This effect was also reproduced with intra-duodenal infusion of natural bile which was inferred to act though its taurocholate content. Since the stimulatory action was also obtained with superfusion of taurocholate or natural bile onto the small intestine and by intravenous injection of taurocholate, it was concluded that taurocholate acted by being absorbed into the bloodstream and then by exerting a stimulatory action on the exocrine pancreas. This action was inhibited by puromycin (a protein synthesis inhibitor), by (..) (AU)
Assuntos
Animais , Ratos , Suco Pancreático , Pâncreas/fisiologia , Ácido Taurocólico/farmacocinética , Bile , Puromicina/farmacocinética , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Células Acinares/fisiologiaRESUMO
This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.
Assuntos
Animais , Vetores Genéticos , Transfecção , Trypanosoma cruzi , Dados de Sequência Molecular , PuromicinaRESUMO
Proteolysis of endogenous proteins may play a key role in the adaptation of T. cruzi to the different host environments to which it is exposed during its complex life cycle. For this reason, we have attempted to study the intracellular pathways of protein degradation in the non infective epimastigotes form (EP strain) of T. cruzi. Following intracellular proteolysis by pulse chase experiments with 35 S methionine, we observed a significant inhibition (50 percent) of the degradation of endogenous proteins in log phase parasites in the presence of inhibitors of lysosomal functions, such as chloroquine and E 64. A significant increase in proteolysis was observed in stationary phase parasites which was reverted to log phase values by supplementing the chase medium with 0.5 percent glucose or 10 percent serum, or in the presence of chloroquine. Under this condition of nutritional stress, we could observe an increase in the activity of acid proteases. A significant increase in the degradation rates was observed when abnormal proteins were induced in the parasite by amino acid analogs and puromycin. This increase was not affected by E 64, suggesting the participation of non lysosomal mechanisms in the degradation of rapidly degradable abnormal proteins. Under these conditions, we could observe an increase in high molecular weight conjugates of ubiquitin with respect to endogenous proteins. These results suggest the importance of lysosomal mechanisms in the degradation of cellular proteins in nutritional optimal conditions and during nutritional deprivation, and the possible involvement of the ubiquitin system in the degradation of high turnover proteins