RESUMEN
Work-based learning (WBL) provides relevant contemporary experience of working environments. Potential benefits for students include developing invaluable skills (clinical, personal, cultural, and professional) and gaining greater awareness of the profession and future career opportunities. However, there are also challenges related to running and sustaining a successful WBL program. In the context of this study, WBL refers to external placements undertaken by final-year students. The aims of the study were to identify ways to optimize the benefits while managing the challenges in delivering WBL in a veterinary curriculum. An in-depth study was undertaken at Chattogram Veterinary and Animal Sciences University (CVASU), Bangladesh, where a WBL program has been in place for 20 years. Final-year veterinary students at CVASU were surveyed to ascertain WBL experiences; survey findings were further explored in focus groups with students, recent graduates, faculty, and placement providers. Most agreed that they had sufficient opportunities to observe, assist, and directly handle pet and farm animals with top skills learned, including clinical diagnosis and communication, and recognized the value of learning in professional workplaces. Based on suggested areas of improvement, the following recommendations can be made: carefully selecting placements, adjusting time allocation, improving communication and building strong collaborations with placement providers, allowing students to customize more placements to align with their career preferences, and staffing adequately to arrange placements and manage a WBL program. Overall, results suggest the current WBL arrangements at CVASU are reasonably good, but there are some specific areas for improvement.
RESUMEN
The hepatotoxic bile acid glycochenodeoxycholate (GCDC) modulates hepatocyte cell death through activation of JNK, Akt, and Erk. The nonhepatotoxic bile acid taurocholate activates Akt and Erk through the sphingosine-1-phosphate receptor 2 (S1PR2). The role of the S1PR2 in GCDC-mediated apoptosis and kinase activation is unknown. Studies were done in rat hepatocytes, HUH7 cells, and HUH7 cells stably transfected with rat Ntcp (HUH7-Ntcp). Cells were treated with GCDC and apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for the active cleaved fragment of caspase 3. Kinase activation was determined by immunoblotting with phospho-specific antibodies. JTE-013, an inhibitor of S1PR2, significantly attenuated morphological evidence of GCDC-induced apoptosis and prevented caspase 3 cleavage in rat hepatocytes and HUH7-Ntcp cells. In hepatocytes, JTE-013 mildly suppressed, augmented, and had no effect on GCDC-induced JNK, Akt, and Erk phosphorylation, respectively. Similar results were seen in HUH7-Ntcp cells except for mild suppression of JNK and Erk phosphorylation. Knockdown of S1PR2 in HUH7-Ntcp augmented Akt, inhibited JNK, and had no effect on Erk phosphorylation. GCDC failed to induce apoptosis or kinase activation in HUH7 cells. In conclusion, SIPR2 inhibition attenuates GCDC-induced apoptosis and inhibits and augments GCDC-induced JNK and Akt phosphorylation, respectively. In addition, GCDC must enter hepatocytes to mediate cell death or activate kinases. These results suggest that SIPR2 activation is proapoptotic in GCDC-induced cell death but that this effect is not due to direct ligation of the S1PR2 by the bile acid.
Asunto(s)
Apoptosis , Carcinoma Hepatocelular/metabolismo , Ácido Glicoquenodesoxicólico/metabolismo , Hepatocitos/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Animales , Línea Celular Tumoral , Células Cultivadas , Ácido Glicoquenodesoxicólico/toxicidad , Humanos , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Ratas , Ratas Wistar , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Esfingosina-1-FosfatoRESUMEN
In hepatocytes, cAMP both activates p38 mitogen-activated protein kinase (MAPK) and increases the amount of multidrug resistance-associated protein-2 (MRP2) in the plasma membrane (PM-MRP2). Paradoxically, taurolithocholate (TLC) activates p38 MAPK but decreases PM-MRP2 in hepatocytes. These opposing effects of cAMP and TLC could be mediated via different p38 MAPK isoforms (α and ß) that are activated differentially by upstream kinases (MKK3, MKK4, and MKK6). Thus we tested the hypothesis that p38α MAPK and p38ß MAPK mediate increases and decreases in PM-MRP2 by cAMP and TLC, respectively. Studies were conducted in hepatocytes isolated from C57BL/6 wild-type (WT) and MKK3-knockout (MKK3(-/-)) mice and in a hepatoma cell line (HuH7) that overexpresses sodium-taurocholate cotransporting polypeptide (NTCP) (HuH-NTCP). Cyclic AMP activated MKK3, p38 MAPK, and p38α MAPK and increased PM-MRP2 in WT hepatocytes, but failed to activate p38α MAPK or increase PM-MRP2 in MKK3(-/-) hepatocytes. In contrast to cAMP, TLC activated total p38 MAPK but decreased PM-MRP2, and did not activate MKK3 or p38α MAPK in WT hepatocytes. In MKK3(-/-) hepatocytes, TLC still decreased PM-MRP2 and activated p38 MAPK, indicating that these effects are not MKK3-dependent. Additionally, TLC activated MKK6 in MKK3(-/-) hepatocytes, and small interfering RNA knockdown of p38ß MAPK abrogated TLC-mediated decreases in PM-MRP2 in HuH-NTCP cells. Taken together, these results suggest that p38α MAPK facilitates plasma membrane insertion of MRP2 by cAMP, whereas p38ß MAPK mediates retrieval of PM-MRP2 by TLC.
Asunto(s)
Membrana Celular/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Colagogos y Coleréticos/farmacología , AMP Cíclico/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Transporte de Proteínas , Ácido Taurolitocólico/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Transhepatic solute transport provides the osmotic driving force for canalicular bile formation. Choleretic and cholestatic agents affect bile formation, in part, by altering plasma membrane localizations of transporters involved in bile formation. These short-term dynamic changes in transporter location are highly regulated posttranslational events requiring various cellular signaling pathways. Interestingly, both choleretic and cholestatic agents activate the same intracellular signaling kinases, such as phosphoinositide-3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein kinase (MAPK). An emerging theme is that choleretic and cholestatic effects may be mediated by different isoforms of these kinases. This is most evident for PKC-mediated regulation of plasma membrane localization of Na+-taurocholate cotransporting polypeptide (NTCP) and multidrug resistance-associated protein 2 (MRP2) by conventional PKCα (cPKCα), novel PKCδ (nPKCδ), nPKCε, and atypical PKCζ (aPKCζ). aPKCζ may mediate choleretic effects by inserting NTCP into the plasma membrane, and nPKCε may mediate cholestatic effects by retrieving MRP2 from the plasma membrane. On the other hand, cPKCα and nPKCδ may be involved in choleretic, cholestatic, and anticholestatic effects by inserting, retrieving, and inhibiting retrieval of transporters, respectively. The effects of PKC isoforms may be mediated by phosphorylation of the transporters, actin binding proteins (radixin and myristoylated alanine-rich C kinase substrate), and Rab proteins. Human NTCP plays an important role in the entry of hepatitis B and D viruses into hepatocytes and consequent infection. Thus, PKCs, by regulating NTCP trafficking, may also play an important role in hepatic viral infections.
Asunto(s)
Bilis/metabolismo , Colestasis/enzimología , Proteína Quinasa C/metabolismo , Animales , Humanos , Isoformas de Proteínas/metabolismoRESUMEN
The SLC10A transporter gene family consists of seven members and substrates transported by three members (SLC10A1, SLC10A2 and SLC10A6) are Na(+)-dependent. SLC10A1 (sodium taurocholate cotransporting polypeptide [NTCP]) and SLC10A2 (apical sodium-dependent bile salt transporter [ASBT]) transport bile salts and play an important role in maintaining enterohepatic circulation of bile salts. Solutes other than bile salts are also transported by NTCP. However, ASBT has not been shown to be a transporter for non-bile salt substrates. While the transport function of NTCP can potentially be used as liver function test, interpretation of such a test may be complicated by altered expression of NTCP in diseases and presence of drugs that may inhibit NTCP function. Transport of bile salts by NTCP and ASBT is inhibited by a number of drugs and it appears that ASBT is more permissive to drug inhibition than NTCP. The clinical significance of this inhibition in drug disposition and drug-drug interaction remains to be determined. Both NCTP and ASBT undergo post-translational regulations that involve phosphorylation/dephosphorylation, translocation to and retrieval from the plasma membrane and degradation by the ubiquitin-proteasome system. These posttranslational regulations are mediated via signaling pathways involving cAMP, calcium, nitric oxide, phosphoinositide-3-kinase (PI3K), protein kinase C (PKC) and protein phosphatases. There appears to be species difference in the substrate specificity and the regulation of plasma membrane localization of human and rodent NTCP. These differences should be taken into account when extrapolating rodent data for human clinical relevance and developing novel therapies. NTCP has recently been shown to play an important role in HBV and HDV infection by serving as a receptor for entry of these viruses into hepatocytes.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Animales , Humanos , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Transporte de Proteínas , Sodio/metabolismo , Simportadores/antagonistas & inhibidoresRESUMEN
Retained bile acids, which are capable of inducing cell death, activate protein kinase Cδ (PKC-δ) in hepatocytes. In nonhepatic cells, both pro- and antiapoptotic effects of PKC-δ are described. The aim of this study was to determine the role of PKC-δ in glycochenodeoxycholate (GCDC)-induced apoptosis in rat hepatocytes and human HUH7-Na-taurocholate-cotransporting polypeptide (Ntcp) cells. Apoptosis was monitored morphologically by Hoechst staining and biochemically by immunoblotting for caspase 3 cleavage. The role of PKC-δ was evaluated with a PKC activator (phorbol myristate acetate, PMA) and PKC inhibitors (chelerythrine, H-7, or calphostin), PKC-δ knockdown, and wild-type (WT) or constitutively active (CA) PKC-δ. PKC-δ activation was monitored by immunoblotting for PKC-δ Thr505 and Tyr311 phosphorylation or by membrane translocation. JNK and Akt phosphorylation and the amount of total bisindolylmaleimide (BIM) were determined by immunoblotting. GCDC induced the translocation of PKC-δ to the mitochondria and/or plasma membrane in rat hepatocytes and HUH7-Ntcp cells and increased PKC-δ phosphorylation on Thr505, but not on Tyr311, in HUH7-Ntcp cells. GCDC-induced apoptosis was attenuated by PMA and augmented by PKC inhibition in rat hepatocytes. In HUH-Ntcp cells, transfection with CA or WT PKC-δ attenuated GCDC-induced apoptosis, whereas knockdown of PKC-δ increased GCDC-induced apoptosis. PKC-δ silencing increased GCDC-induced JNK phosphorylation, decreased GCDC-induced Akt phosphorylation, and increased expression of BIM. GCDC translocated BIM to the mitochondria in rat hepatocytes, and knockdown of BIM in HUH7-Ntcp cells decreased GCDC-induced apoptosis. Collectively, these results suggest that PKC-δ does not mediate GCDC-induced apoptosis in hepatocytes. Instead PKC-δ activation by GCDC stimulates a cytoprotective pathway that involves JNK inhibition, Akt activation, and downregulation of BIM.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Ácido Glicoquenodesoxicólico/farmacología , Hepatocitos/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Apoptosis/fisiología , Proteína 11 Similar a Bcl2 , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Hepatocitos/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Transducción de Señal/fisiologíaRESUMEN
Rab proteins (Ras homologous for brain) play an important role in vesicle trafficking. Rab4 and Rab11 are involved in vesicular trafficking to the plasma membrane from early endosomes and recycling endosomes, respectively. Tauroursodeoxycholate (TUDC) and cAMP increase bile formation, in part, by increasing plasma membrane localization of multidrug resistance-associated protein 2 (MRP2). The goal of the present study was to determine the role of these Rab proteins in the trafficking of MRP2 by testing the hypothesis that Rab11 and/or Rab4 facilitate cAMP- and TUDC-induced MRP2 translocation to the plasma membrane. Studies were conducted in HuH-NTCP cells (HuH7 cells stably transfected with human NTCP), which constitutively express MRP2. HuH-NTCP cells were transfected with Rab11-WT and GDP-locked dominant inactive Rab11-GDP or with Rab4-GDP to study the role of Rab11 and Rab4. A biotinylation method and a GTP overlay assay were used to determine plasma membrane MRP2 and activation of Rab proteins (Rab11 and Rab4), respectively. Cyclic AMP and TUDC increased plasma membrane MRP2 and stimulated Rab11 activity. Plasma membrane translocation of MRP2 by cAMP and TUDC was increased and inhibited in cells transfected with Rab11-WT and Rab11-GDP, respectively. Cyclic AMP (previous study) and TUDC increased Rab4 activity. However, cAMP- and TUDC-induced increases in MRP2 were not inhibited by Rab4-GDP. Taken together, these results suggest that Rab11 is involved in cAMP- and TUDC-induced MRP2 translocation to the plasma membrane.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , AMP Cíclico/farmacología , Ácido Tauroquenodesoxicólico/farmacología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Línea Celular , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Transporte de Proteínas , Simportadores/genética , Simportadores/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab4/genética , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
UNLABELLED: Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. CONCLUSION: Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Quinasa C-delta/fisiología , Ácido Taurolitocólico/metabolismo , Animales , Línea Celular Tumoral , Humanos , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , RatasRESUMEN
BACKGROUND: Factors influencing parasitosis in cattle in Bangladesh remain inadequately explored, necessitating a comprehensive investigation for interventions and sustainable livestock farming. OBJECTIVES: We conducted this study to estimate the prevalence and distribution of gastrointestinal parasites, exploring their intricate relationship with farm management practices across a spectrum of small-, medium-, and large-scale commercial farms. METHODS: We conducted this study in the Chattogram district of Bangladesh. We collected a total of 189 freshly voided faecal samples from different farms. We recorded the age, breed, milking status, sex, body condition score, and anthelmintic use history of the sampled animals. We processed the samples using the direct smear method, with the identification of one egg per sample being considered positive. RESULTS: We estimated the prevalence of gastrointestinal parasite infection in large-scale (52.1%), medium-scale (54.5%), and small-scale farms (70.0%), with statistically significant differences (p ≤ 0.05). Both pregnant and lactating cows, as well as indigenous cattle, were more likely to have gastrointestinal parasites (p ≤ 0.05). The predominant parasites across farms of all sizes were trematodes (Paramphistomum spp. and Schistosomas spp.) and protozoa (Balantidium coli and Coccidia spp.). CONCLUSION: Poor farm management practices, such as no pasture management and inadequate deworming regimens, may contribute to the elevated prevalence and infection load observed on small-scale farms. The increased parasitosis in previously dewormed animals can be attributed to the development of anthelmintic resistance against gastrointestinal parasites. Implementing proper and effective deworming strategies is crucial to preventing gastrointestinal parasitosis and mitigating the risk of anthelmintic resistance.
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Antihelmínticos , Enfermedades de los Bovinos , Enfermedades Gastrointestinales , Parasitosis Intestinales , Femenino , Animales , Bovinos , Lactancia , Bangladesh/epidemiología , Antihelmínticos/uso terapéutico , Parasitosis Intestinales/epidemiología , Parasitosis Intestinales/veterinaria , Parasitosis Intestinales/parasitología , Enfermedades Gastrointestinales/epidemiología , Enfermedades Gastrointestinales/veterinaria , Enfermedades de los Bovinos/parasitologíaRESUMEN
The Na(+) taurocholate (TC) cotransporting polypeptide Ntcp/NTCP mediates TC uptake across the sinusoidal membrane of hepatocytes. Previously, we demonstrated that nitric oxide (NO) inhibits TC uptake through S-nitrosylation of a cysteine residue. Our current aim was to determine which of the eight cysteine residues of Ntcp is responsible for NO-mediated S-nitrosylation and inhibition of TC uptake. Thus, we tested the effect of NO on TC uptake in HuH-7 cells transiently transfected with cysteine-to-alanine mutant Ntcp constructs. Of the eight mutants tested, only C44A Ntcp displayed decreased total and plasma membrane (PM) levels that were also reflected in decreased TC uptake. C266A Ntcp showed a decrease in TC uptake that was not explained by a decrease in total expression or PM localization, indicating that C266 is required for optimal uptake. We speculated that NO would target C266 since a previous report had shown the thiol reactive compound [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) inhibits TC uptake by wild-type NTCP but not by C266A NTCP. We confirmed that MTSET targets C266 of Ntcp, but, surprisingly, we found that C266 was not responsible for NO-mediated inhibition of TC uptake. Instead, we found that C96 was targeted by NO since C96A Ntcp was insensitive to NO-mediated inhibition of TC uptake. We also found that wild-type but not C96A Ntcp is S-nitrosylated by NO, suggesting that C96 is important in regulating Ntcp function in response to elevated levels of NO.
Asunto(s)
Óxido Nítrico/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Cisteína , Humanos , Mutagénesis Sitio-Dirigida , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Simportadores/genéticaRESUMEN
Cyclic AMP stimulates translocation of Na(+)/taurocholate cotransporting polypeptide (NTCP) from the cytosol to the sinusoidal membrane and multidrug resistance-associated protein 2 (MRP2) to the canalicular membrane. A recent study suggested that protein kinase Cδ (PKCδ) may mediate cAMP-induced translocation of Ntcp and Mrp2. In addition, cAMP has been shown to stimulate NTCP translocation in part via Rab4. The aim of this study was to determine whether cAMP-induced translocation of NTCP and MRP2 require kinase activity of PKCδ and to test the hypothesis that cAMP-induced activation of Rab4 is mediated via PKCδ. Studies were conducted in HuH-NTCP cells (HuH-7 cells stably transfected with NTCP). Transfection of cells with wild-type PKCδ increased plasma membrane PKCδ and NTCP and increased Rab4 activity. Paradoxically, overexpression of kinase-dead dominant-negative PKCδ also increased plasma membrane PKCδ and NTCP as well as Rab4 activity. Similar results were obtained in PKCδ knockdown experiments, despite a decrease in total PKCδ. These results raised the possibility that plasma membrane localization rather than kinase activity of PKCδ is necessary for NTCP translocation and Rab4 activity. This hypothesis was supported by results showing that rottlerin, which has previously been shown to inhibit cAMP-induced membrane translocation of PKCδ and NTCP, inhibited cAMP-induced Rab4 activity. In addition, LY294002 (a phosphoinositide-3-kinase inhibitor), which has been shown to inhibit cAMP-induced NTCP translocation, also inhibited cAMP-induced PKCδ translocation. In contrast to the results with NTCP, cAMP-induced MRP2 translocation was inhibited in cells transfected with DN-PKCδ and small interfering RNA PKCδ. Taken together, these results suggest that the plasma membrane localization rather than kinase activity of PKCδ plays an important role in cAMP-induced NTCP translocation and Rab4 activity, whereas the kinase activity of PKCδ is necessary for cAMP-induced MRP2 translocation.
Asunto(s)
Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Proteína Quinasa C-delta/metabolismo , Simportadores/metabolismo , Proteínas de Unión al GTP rab4/metabolismo , Técnicas de Cultivo de Célula , Regulación de la Expresión Génica , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Transporte de ProteínasRESUMEN
The sodium-taurocholate (TC) cotransporting polypeptide (NTCP) facilitates bile formation by mediating sinusoidal Na(+)-TC cotransport. During sepsis-induced cholestasis, there is a decrease in NTCP-dependent uptake of bile acids and an increase in nitric oxide (NO) levels in hepatocytes. In rat hepatocytes NO inhibits Na(+)-dependent uptake of taurocholate. The aim of this study was to extend these findings to human NTCP and to further investigate the mechanism by which NO inhibits TC uptake. Using a human hepatoma cell line stably expressing NTCP (HuH-NTCP), we performed experiments with the NO donors sodium nitroprusside and S-nitrosocysteine and demonstrated that NO inhibits TC uptake in these cells. Kinetic analyses revealed that NO significantly decreased the V(max) but not the K(m) of TC uptake by NTCP, indicating noncompetitive inhibition. NO decreased the amount of NTCP in the plasma membrane, providing a molecular mechanism for the noncompetitive inhibition of TC uptake. One way that NO can modify protein function is through a posttranslational modification known as S-nitrosylation: the binding of NO to cysteine thiols. Using a biotin switch technique we observed that NTCP is S-nitrosylated under conditions in which NO inhibits TC uptake. Moreover, dithiothreitol reversed NO-mediated inhibition of TC uptake and S-nitrosylation of NTCP, indicating that NO inhibits TC uptake via modification of cysteine thiols. In addition, NO treatment led to a decrease in Ntcp phosphorylation. Taken together these results indicate that the inhibition of TC uptake by NO involves S-nitrosylation of NTCP.
Asunto(s)
Óxido Nítrico/farmacología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Ácido Taurocólico/antagonistas & inhibidores , Ácido Taurocólico/farmacocinética , Línea Celular Tumoral , Membrana Celular/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacología , Ditiotreitol/farmacología , Humanos , Transportadores de Anión Orgánico Sodio-Dependiente/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , S-Nitrosotioles/farmacología , Simportadores/antagonistas & inhibidoresRESUMEN
The World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic on March 11, 2020. COVID-19, the current global health emergency, is wreaking havoc on human health systems and, to a lesser degree, on animals globally. The outbreak has continued since the first report of COVID-19 in China in December 2019, and the second and third waves of the outbreak have already begun in several countries. COVID-19 is expected to have adverse effects on crop production, food security, integrated pest control, tourism, the car industry, and other sectors of the global economy. COVID-19 induces a range of effects in livestock that is reflected economically since human health and livelihood are intertwined with animal health. We summarize the potentially harmful effects of COVID-19 on livestock and possible mitigation steps in response to this global outbreak. Mitigation of the negative effects of COVID-19 and future pandemics on livestock requires the implementation of current guidelines.
RESUMEN
BACKGROUND & AIMS: Glycochenodeoxycholate (GCDC) and taurolithocholate (TLC) are hepatotoxic and cholestatic bile salts, whereas tauroursodeoxycholate (TUDC) is cytoprotective and anticholestatic. Yet they all act, in part, through phosphatidylinositol-3-kinase(PI3K)-dependent mechanisms ("PI3K-paradox"). Hepatocytes express three catalytic PI3K Class I isoforms (p110α/ß/γ), specific functions of which, in liver, are unclear. In other cell types, p110γ is associated with detrimental effects. To shed light on the PI3K enigma, we determined whether hydrophobic and hydrophilic bile salts differentially activate distinct p110 isoforms in hepatocytes, and whether p110γ mediates bile salt-induced hepatocyte cell death. METHODS: Isoform-specific PI3K activity assays were established to determine isoform activation by bile salts in rat hepatocytes. Activation of Akt and JNK was determined by immunoblotting. Following stimulation with hydrophobic bile salts, hepatocellular apoptosis was determined morphologically after Hoechst staining and by analysis of caspase-3/-7 activity or caspase-3 cleavage. Activity or expression of PI3K p110γ was inhibited pharmacologically (AS604850) or by knock-down using specific siRNA. RESULTS: All bile salts tested activated p110ß, while p110α was activated by TUDC and GCDC. Intriguingly, only hydrophobic bile salts activated p110γ. Inhibition of p110γ attenuated GCDC-induced Akt- and JNK-activation, but did not alter TUDC- or cAMP-induced Akt-signaling in rat hepatocytes. Inhibition or knock-down of p110γ markedly attenuated hydrophobic bile salt-induced apoptosis in rat hepatocytes and human hepatoma cell lines but did not alter Fas-, tumor necrosis factor α- and etoposide-induced apoptosis. Depletion of Ca(++) prevented GCDC-induced toxicity in rat hepatocytes but did not affect GCDC-induced Akt- and JNK-activation. CONCLUSIONS: PI3K p110γ is activated by hydrophobic, but not hydrophilic bile salts. Bile salt-induced hepatocyte apoptosis is partly mediated via a PI3K p110γ dependent signaling pathway, potentially involving JNK.
Asunto(s)
Apoptosis/efectos de los fármacos , Ácidos y Sales Biliares/farmacología , Carcinoma Hepatocelular/patología , Fosfatidilinositol 3-Quinasa Clase Ib/fisiología , Hepatocitos/efectos de los fármacos , Neoplasias Hepáticas/patología , Animales , Células Cultivadas , Dioxoles/farmacología , Activación Enzimática/efectos de los fármacos , Ácido Glicoquenodesoxicólico/toxicidad , Células Hep G2 , Hepatocitos/patología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Tiazolidinedionas/farmacologíaRESUMEN
Cyclic AMP (cAMP) induces translocation of multidrug resistant protein 2 (Mrp2) to the canalicular membrane and activates p38 MAPK in rat hepatocytes. In this study, we tested the hypothesis that cAMP-induced Mrp2 translocation may be mediated via p38 MAPK. Studies were conducted in rat hepatocytes and in a human hepatoma cell line, HuH-7. In rat hepatocytes, cAMP increased Mrp2 translocation and p38 MAPK activity. These effects of cAMP were inhibited by SB203580, an inhibitor of p38 MAPK. Wortmannin, a specific inhibitor of phosphoinositide-3-kinase (PI3K), did not inhibit cAMP induced activation of p38 MAPK, indicating PI3K-independent activation of p38 MAPK by cAMP. To further define the role of p38 MAPK, molecular approaches were used to up- or downregulate p38 MAPK activity in HuH-7 cells using constitutively active (CA) and dominant-negative (DN) MAPK kinase 3 and 6 (MKK3/6). MKK3/6 are upstream kinases responsible for the activation of p38 MAPK. Cells transfected with CAMKK6 showed increased p38 MAPK activity and MRP2 translocation compared with empty vector. cAMP-induced activation of p38 MAPK was inhibited in cells transfected with DNMKK3/6 and DNMKK3, but not with DNMKK6. DNMKK3/6 and DNMKK3 also inhibited cAMP-induced MRP2 translocation. cAMP selectively activated p38alpha MAPK in HuH-7 cells. Knockdown of p38alpha MAPK by short heterodimer RNA resulted in decreased level of p38 MAPK and failure of cAMP to stimulate MRP2 translocation. Taken together, these results suggest that cAMP-induced MRP2 translocation in hepatic cells is mediated via PI3K-independent and MKK3-mediated activation of p38alpha MAPK.
Asunto(s)
AMP Cíclico/fisiología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Androstadienos/farmacología , Animales , Línea Celular Tumoral , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Imidazoles/farmacología , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Piridinas/farmacología , Ratas , Transfección , WortmaninaRESUMEN
Antibiotic-free broiler meat production is becoming increasingly popular worldwide due to consumer perception that it is superior to conventional broiler meat. Globally, broiler farming impacts the income generation of low-income households, helping to alleviate poverty and secure food in the countryside and in semi-municipal societies. For decades, antibiotics have been utilized in the poultry industry to prevent and treat diseases and promote growth. This practice contributes to the development of drug-resistant bacteria in livestock, including poultry, and humans through the food chain, posing a global public health threat. Additionally, consumer demand for antibiotic-free broiler meat is increasing. However, there are many challenges that need to be overcome by adopting suitable strategies to produce antibiotic-free broiler meat with regards to food safety and chicken welfare issues. Herein, we focus on the importance and current scenario of antibiotic use, prospects, and challenges in the production of sustainable antibiotic-free broiler meat, emphasizing broiler farming in the context of Bangladesh. Moreover, we also discuss the need for and challenges of antibiotic alternatives and provide a future outlook for antibiotic-free broiler meat production.
RESUMEN
The cholestatic bile acid taurolithocholate (TLC) inhibits biliary secretion of organic anions and hepatic uptake of taurocholate (TC). TLC has been suggested to induce retrieval of Mrp2 from the canalicular membrane via the phosphoinositide-3-kinase (PI3K)/PKB-dependent activation of novel protein kinase Cepsilon (nPKCepsilon) in rat hepatocytes. The aim of the present study was to determine whether TLC-induced inhibition of TC uptake may also involve PI3K-dependent activation of PKCepsilon in HuH7 cells stably transfected with human Na(+)-dependent TC-cotransporting polypeptide (NTCP) (HuH-NTCP cells). To avoid direct competition for uptake, cells were pretreated with TLC, washed, and then incubated with (3)H-TC to determine TC uptake. TLC produced time- and dose-dependent inhibition of TC uptake. TLC inhibited TC uptake competitively without affecting NTCP membrane translocation. A PI3K inhibitor failed to reverse TLC-induced TC uptake inhibition and TLC-inhibited PKB phosphorylation. TLC did activate nPKCepsilon as evidenced by increased membrane translocation and nPKCepsilon-Ser(729) phosphorylation. Overexpression of dominant negative-nPKCepsilon reversed TLC-induced inhibition of PKB phosphorylation but not of TC uptake. Finally, cAMP prevented TLC-induced inhibition of TC uptake via the PI3K pathway, and the prevention is due to the sum of cAMP-induced stimulation and TLC-induced inhibition of TC uptake. Taken together, these results suggest that TLC-induced inhibition of PKB, but not of TC uptake, is mediated via nPKCepsilon. Activation of nPKCepsilon and inhibition of TC uptake by TLC are not mediated via the PI3K/PKB pathway.
Asunto(s)
Hepatocitos/enzimología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Simportadores/metabolismo , Ácido Taurocólico/metabolismo , Ácido Taurolitocólico/metabolismo , Animales , Unión Competitiva , Transporte Biológico , Línea Celular Tumoral , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Activación Enzimática , Humanos , Cinética , Masculino , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Fosforilación , Transporte de Proteínas , Ratas , Ratas Wistar , Simportadores/genética , TransfecciónRESUMEN
UNLABELLED: Cyclic adenosine monophosphate (cAMP) stimulates translocation of Na(+)-taurocholate (TC) cotransporting polypeptide (Ntcp) and multidrug resistant associated protein 2 (Mrp2) to the plasma membrane. Because cAMP activates phosphoinositide-3-kinase (PI3K) and protein kinase C (PKC) activation is PI3K-dependent, the aim of the current study was to determine whether cAMP activates conventional and novel PKCs in hepatocytes and whether such activation plays a role in cAMP-stimulated Ntcp and Mrp2 translocation. The effect of cAMP on PKCs, TC uptake, and Ntcp and Mrp2 translocation was studied in isolated rat hepatocytes using a cell-permeable cAMP analog, CPT-cAMP. The activity of PKCs was assessed from membrane translocation of individual PKCs, and phospho-specific antibodies were used to determine PKCdelta phosphorylation. TC uptake was determined from time-dependent uptake of (14)C-TC, and a cell surface biotinylation method was used to determine Ntcp and Mrp2 translocation. CPT-cAMP stimulated nPKCdelta but not cPKCalpha or nPKCepsilon, and induced PI3K-dependent phosphorylation of nPKCdelta at Thr(505). Rottlerin, an inhibitor of nPKCdelta, inhibited cAMP-induced nPKCdelta translocation, TC uptake, and Ntcp and Mrp2 translocation. Bistratene A, an activator of nPKCdelta, stimulated nPKCdelta translocation, TC uptake, and Ntcp and Mrp2 translocation. The effects of cAMP and bistratene A on TC uptake and Ntcp and Mrp2 translocation were not additive. CONCLUSION: These results suggest that cAMP stimulates Ntcp and Mrp2 translocation, at least in part, by activating nPKCdelta via PI3K-dependent phosphorylation at Thr(505).
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , AMP Cíclico/metabolismo , Hepatocitos/enzimología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Proteína Quinasa C-delta/metabolismo , Simportadores/metabolismo , Acetamidas/farmacología , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Hepatocitos/metabolismo , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Quinasa C-delta/antagonistas & inhibidores , Piranos/farmacología , Ratas , Ratas Wistar , Compuestos de Espiro/farmacología , Ácido Taurocólico/metabolismoRESUMEN
UNLABELLED: Cyclic adenosine monophosphate (cAMP) stimulates hepatic bile acid uptake by translocating sodium-taurocholate (TC) cotransporting polypeptide (Ntcp) from an endosomal compartment to the plasma membrane. Rab4 is associated with early endosomes and involved in vesicular trafficking. This study was designed to determine the role of Rab4 in cAMP-induced TC uptake and Ntcp translocation. HuH-Ntcp cells transiently transfected with empty vector, guanosine triphosphate (GTP) locked dominant active Rab4 (Rab4(GTP)), or guanosine diphosphate (GDP) locked dominant inactive Rab4 (Rab4(GDP)) were used to study the role of Rab4. Neither Rab4(GTP) nor Rab4(GDP) affected either basal TC uptake or plasma membrane Ntcp level. However, cAMP-induced increases in TC uptake and Ntcp translocation were enhanced by Rab4(GTP) and inhibited by Rab4(GDP). In addition, cAMP increased GTP binding to endogenous Rab4 in a time-dependent, but phosphoinositide-3-kinase-independent manner. CONCLUSION: Taken together, these results suggest that cAMP-mediated phosphoinositide-3-kinase-independent activation of Rab4 facilitates Ntcp translocation in HuH-Ntcp cells.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , AMP Cíclico/farmacología , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Proteínas de Unión al GTP rab4/fisiología , Transporte Biológico/efectos de los fármacos , Carcinoma Hepatocelular , Línea Celular Tumoral , Membrana Celular/metabolismo , Endosomas/metabolismo , Humanos , Neoplasias Hepáticas , Transporte de Proteínas , TransfecciónRESUMEN
A cross-sectional observation and an intervention study were conducted in Chittagong, Bangladesh in 2015 to assess the status of antimicrobial residues in chicken and fish. The samples were tested for selected antimicrobials (amoxicillin, ciprofloxacin, oxytetracycline and enrofloxacin) using thin-layer chromatography (TLC). The TLC-based overall prevalence of residues was 87.9% in chicken (N = 182) and 56.9% in fish (N = 153). The prevalences in chicken in June (N = 91) and in October (N = 91), respectively, were 91.2% and 83.5% (amoxicillin), 1.1% and 1.1% (enrofloxacin), 1.1% and 0% (ciprofloxacin), and 0% and 6.6% (oxytetracycline). In fish, the prevalence in September (N = 74) and in October (N = 79) was 52.7% and 44.3% (amoxicillin) and 1.4% and 27.8% (oxytetracycline), respectively. The mean concentration of amoxicillin residue was evaluated by ultra-high-performance liquid chromatography to be 508.4 mg/kg (chicken) and 515.4 mg/kg (fish). The random effect model identified market (Chawkbazar vs. Boalkhali: OR 5.7; Steelmill bazar vs. Boalkhali: OR 5.6) as significant factors for amoxicillin residue in chicken. Amoxicillin concentration was significantly reduced in chicken of Kazirhat (ß= - 1.3) and Chawkbazar (ß= - 1.1) and increased in October (ß= 0.77) based on a generalized linear model (GLM). Climbing perch fish had significantly more risk of having amoxicillin residue than that of Bombay duck (OR = 0.05). All samples were treated by washing, boiling and cooking with spices, and then, TLC-based screening of amoxicillin residues was done. A subset of each treated group was evaluated by UHPLC. Treatment reduced amoxicillin residue levels significantly.