scholarly journals A Pitfall in Mouse Norovirus (MNV) Detection in Fecal Samples Using RT-PCR, and Construction of New MNV-Specific Primers

2013 ◽  
Vol 62 (2) ◽  
pp. 127-135 ◽  
Author(s):  
Masaru TAJIMA ◽  
Yuko KOTANI ◽  
Tsutomu KUROSAWA ◽  
Masayuki MIYASAKA
Keyword(s):  
Rt Pcr ◽  
Specific Primers ◽  
Fecal Samples

scholarly journals Detection of SARS-CoV-2 from patient fecal samples by whole genome sequencing

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Andreas Papoutsis ◽  
Thomas Borody ◽  
Siba Dolai ◽  
Jordan Daniels ◽  
Skylar Steinberg ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nasopharyngeal Swab ◽  
Whole Genome ◽  
Rt Pcr ◽  
Whole Genome Analysis ◽  
Fecal Samples ◽  
Oral Transmission ◽  
Study Participants ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background SARS-CoV-2 has been detected not only in respiratory secretions, but also in stool collections. Here were sought to identify SARS-CoV-2 by enrichment next-generation sequencing (NGS) from fecal samples, and to utilize whole genome analysis to characterize SARS-CoV-2 mutational variations in COVID-19 patients. Results Study participants underwent testing for SARS-CoV-2 from fecal samples by whole genome enrichment NGS (n = 14), and RT-PCR nasopharyngeal swab analysis (n = 12). The concordance of SARS-CoV-2 detection by enrichment NGS from stools with RT-PCR nasopharyngeal analysis was 100%. Unique variants were identified in four patients, with a total of 33 different mutations among those in which SARS-CoV-2 was detected by whole genome enrichment NGS. Conclusion These results highlight the potential viability of SARS-CoV-2 in feces, its ongoing mutational accumulation, and its possible role in fecal–oral transmission. This study also elucidates the advantages of SARS-CoV-2 enrichment NGS, which may be a key methodology to document complete viral eradication. Trial registration ClinicalTrials.gov, NCT04359836, Registered 24 April 2020, https://clinicaltrials.gov/ct2/show/NCT04359836?term=NCT04359836&draw=2&rank=1).

scholarly journals First Report of Mosaic Disease Caused by Colombian datura virus on Solanum lycopersicum Plants Commercially Cultivated in Japan

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 698-698 ◽  
Author(s):  
Y. Tomitaka ◽  
T. Usugi ◽  
R. Kozuka ◽  
S. Tsuda
Keyword(s):  
Nucleotide Sequence ◽  
Solanum Lycopersicum ◽  
Mosaic Disease ◽  
Causal Agent ◽  
Cultivated Plants ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Specific Primers ◽  
Tomato Plants ◽  
First Report

In 2009, some commercially grown tomato (Solanum lycopersicum) plants in Chiba Prefecture, Japan, exhibited mosaic symptoms. Ten plants from a total of about 72,000 cultivated plants in the greenhouses showed such symptoms. To identify the causal agent, sap from leaves of the diseased plants was inoculated into Chenopodium quinoa and Nicotiana benthamiana plants. Local necrotic lesions appeared on inoculated leaves of C. quinoa, but no systemic infection was observed. Systemic mosaic symptoms were observed on the N. benthamiana plants inoculated. Single local lesion isolation was performed three times using C. quinoa to obtain a reference isolate for further characterization. N. benthamiana was used for propagation of the isolate. Sap from infected leaves of N. benthamiana was mechanically inoculated into three individual S. lycopersicum cv. Momotaro. Symptoms appearing on inoculated tomatoes were indistinguishable from those of diseased tomato plants found initially in the greenhouse. Flexuous, filamentous particles, ~750 nm long, were observed by electron microscopy in the sap of the tomato plants inoculated with the isolate, indicating that the infecting virus may belong to the family Potyviridae. To determine genomic sequence of the virus, RT-PCR was performed. Total RNA was extracted from the tomato leaves experimentally infected with the isolate using an RNeasy Plant Mini kit (QIAGEN, Hilden, Germany). RT-PCR was performed by using a set of universal, degenerate primers for Potyviruses as previously reported (2). Amplicons (~1,500 bp) generated by RT-PCR were extracted from the gels using the QIAquick Gel Extraction kit (QIAGEN) and cloned into pCR-BluntII TOPO (Invitrogen, San Diego, CA). DNA sequences of three individual clones were determined using a combination of plasmid and virus-specific primers, showing that identity among three clones was 99.8%. A consensus nucleotide sequence of the isolate was deposited in GenBank (AB823816). BLASTn analysis of the nucleotide sequence determined showed 99% identity with a partial sequence in the NIb/coat protein (CP) region of Colombian datura virus (CDV) tobacco isolate (JQ801448). Comparison of the amino acid sequence predicted for the CP with previously reported sequences for CDV (AY621656, AJ237923, EU571230, AM113759, AM113754, and AM113761) showed 97 to 100% identity range. Subsequently, CDV infection in both the original and experimentally inoculated plants was confirmed by RT-PCR using CDV-specific primers (CDVv and CDVvc; [1]), and, hence, the causal agent of the tomato disease observed in greenhouse tomatoes was proved to be CDV. The first case of CDV on tomato was reported in Netherlands (3), indicating that CDV was transmitted by aphids from CDV-infected Brugmansia plants cultivated in the same greenhouse. We carefully investigated whether Brugmansia plants naturally grew around the greenhouses, but we could not find them inside or in proximity to the greenhouses. Therefore, sources of CDV inoculum in Japan are still unclear. This is the first report of a mosaic disease caused by CDV on commercially cultivated S. lycopersicum in Japan. References: (1) D. O. Chellemi et al. Plant Dis. 95:755, 2011. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) J. Th. J. Verhoeven et al. Eur. J. Plant. Pathol. 102:895, 1996.

scholarly journals Prevalence and molecular detection of shiga toxin producing Escherichia coli from diarrheic cattle

2016 ◽  
Vol 14 (1) ◽  
pp. 63-68 ◽  
Author(s):  
MM Akter ◽  
S Majumder ◽  
KH MNH Nazir ◽  
M Rahman
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Molecular Detection ◽  
Chain Reaction ◽  
Specific Primers ◽  
Fecal Samples ◽  
E Coli ◽  
Uremic Syndrome ◽  
Polymerase Chain ◽  
Positive Isolate

Shiga toxin-producing Escherichia coli (STEC) are zoonotically important pathogen which causes hemorrhagic colitis, diarrhea, and hemolytic uremic syndrome in animals and humans. The present study was designed to isolate and identify the STEC from fecal samples of diarrheic cattle. A total of 35 diarrheic fecal samples were collected from Bangladesh Agricultural University (BAU) Veterinary Teaching Hospital. The samples were primarily examined for the detection of E. coli by cultural, morphological and biochemical characteristics, followed by confirmation of the isolates by Polymerase Chain Reaction (PCR) using gene specific primers. Later, the STEC were identified among the isolated E. coli through detection of Stx-1 and Stx-2 genes using duplex PCR. Out of 35 samples, 25 (71.43%) isolates were confirmed to be associated with E. coli, of which only 7 (28%) isolates were shiga toxin producers, and all of them were positive for Stx-1. However, no Stx-2 positive isolate could be detected. From this study, it may be concluded that cattle can act as a reservoir of STEC which may transmit to human or other animals.J. Bangladesh Agril. Univ. 14(1): 63-68, June 2016

scholarly journals Double Infection of Onion yellow dwarf virus and Shallot latent virus in Garlic from Several Regions in Indonesia

2021 ◽  
Vol 25 (1) ◽  
pp. 40
Author(s):  
Nurenik Nurenik ◽  
Sedyo Hartono ◽  
Sri Sulandari ◽  
Susamto Somowiyarjo ◽  
Argawi Kandito
Keyword(s):  
Early Detection ◽  
Latent Virus ◽  
Yellow Dwarf ◽  
Dwarf Virus ◽  
Onion Yellow Dwarf Virus ◽  
Rt Pcr ◽  
Double Infection ◽  
Specific Primers ◽  
Shallot Latent Virus ◽  
Yellow Dwarf Virus

Viruses have been a problem on garlic cultivations in various countries. There are several viruses reported infecting garlic. Genera Potyvirus and Carlavirus are the most common viruses found infecting garlic. Mixed infection on garlic is often designated as a “garlic viral complex”. These viruses can be transmitted through imported garlic seeds. Therefore, it is necessary to conduct early detection of garlic seeds to prevent the epidemic of these viruses. This study aimed to detect Onion yellow dwarf virus (OYDV) and Shallot latent virus (SLV) on garlic. Garlic samples were obtained from Enrekang, Magelang, Temanggung, Tawangmangu, and Yogyakarta. Total RNA was extracted from the samples and subsequently used for RT-PCR using two pairs of specific primers SLV-F/SLV-R and OYDV-F/OYDV-R. Primary pair SLV-F/SLV-R in amplicons sized 276 bp, while OYDV-F/OYDV-R in amplicons sized 112 bp. RT-PCR results showed that OYDV was found in all samples tested in this study. Meanwhile, double infections (OYDV and SLV) were found in eight out of ten samples tested. These results indicated that double infections on garlic were common in Indonesia.

scholarly journals Development of cDNA normalization system and preliminary transcription analysis of KCS genes in apple tissues

2011 ◽  
Vol 59 (3) ◽  
pp. 9-12 ◽  
Author(s):  
Zsolt Albert ◽  
Cs. Deák ◽  
A. Miskó ◽  
M. Tóth ◽  
I. Papp
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Apple Fruit ◽  
Rt Pcr ◽  
Specific Primers ◽  
Specific Expression ◽  
Transcription Analysis ◽  
Tissue Specific

Wax production is an important aspect of apple (Malus domestica Borkh.) fruit development from both theoretical and practical point of views. The complex molecular mechanism that controls wax biosynthesis is still widely unknown but many studies focused on this topic. We aimed to develop further the experimental framework of these efforts with a description of an improved reference genes expression system. Results in the literature show that similarities exist among the expression of some housekeeping genes of different plant species. Based on these considerations and on gene expression data from Arabidopsis thaliana, some genes in apple were assigned for analysis. EST sequences of apple were used to design specific primers for RT-PCR experiments. Isolation of intact RNA from different apple tissues and performing RT-PCR reaction were also key point in obtaining expression patterns. To monitor DNA contamination of the RNA samples, specific primers were used that amplify intron-containing sequences from the cDNA. We found that actin primers can be used for the detection of intron containing genomic DNA, and tubulin primers are good internal controls in RT-PCR experiments. We were able to make a difference between tissue-specific and tissue-independent gene-expression, furthermore we found tissue specific differences between the expression patterns of candidate genes, that are potentially involved in wax-biosynthesis. Our results show that KCS1 and KCS4 are overexpressed in the skin tissue, this could mean that these genes have skin-specific expression in apple fruit.

scholarly journals Gastroenteric virus detection in fecal samples from women in Goiânia, State of Goiás, Brazil

2010 ◽  
Vol 43 (3) ◽  
pp. 240-243 ◽  
Author(s):  
Rui Gilberto Ferreira ◽  
Ana Maria Tavares Borges ◽  
Fabiola Souza Fiaccadori ◽  
Menira Borges de Lima Dias e Souza ◽  
Rodrigo Alessandro Togo Santos ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Virus Detection ◽  
Hiv Positive ◽  
Risk Of Infection ◽  
Rt Pcr ◽  
Fecal Samples ◽  
Hiv Positive Women ◽  
A Prospective Study ◽  
Immunoenzymatic Assays ◽  
Hiv Seropositive

INTRODUCTION: This was a prospective study that included women seen in the obstetrics and gynecology sector of Hospital das Clínicas, Federal University of Goiás, in Goiânia, State of Goiás, with the aim of detecting rotaviruses, adenoviruses, caliciviruses and astroviruses. Eighty-four women participated in the study and from these, 314 fecal samples were collected. Out of all of the women, 29 were seropositive for HIV and 55 were seronegative, and 45 and 39 were pregnant and non-pregnant, respectively. METHODS: Fecal samples were collected from each woman once every two months over the period from July 2006 to June 2007, and they were screened for rotaviruses by means of polyacrylamide gel electrophoresis and immunoenzymatic assays, for caliciviruses and astroviruses by means of RT-PCR and for adenovirus by means of immunoenzymatic assays. The astroviruses were genotyped using nested PCR. RESULTS: Among the 84 patients, 19 (22.6%) were positive for either calicivirus (14/19) or astrovirus (6/19), while one women was positive for both viruses in fecal samples collected on different occasions. Most of the positive samples were collected during the months of July and August (astrovirus) and September and October (calicivirus). None of the samples analyzed was positive for rotavirus or adenovirus. Gastroenteric viruses were detected in 13/19 (68.4%) of the pregnant women, whether HIV-seropositive or not. CONCLUSIONS: The results from the present study showed that neither pregnancy nor HIV-seropositive status among the women increased the risk of infection by any of the gastroenteric viruses studied. This study presents data on gastroenteric virus detection among pregnant and/or HIV-positive women.

scholarly journals Characteristic Differences in Reverse Transcription-Polymerase Chain Reaction Products of Ovine, Bovine, and Human Respiratory Syncytial Viruses

1993 ◽  
Vol 5 (3) ◽  
pp. 322-328 ◽  
Author(s):  
Richard D. Oberst ◽  
Michael P. Hays ◽  
Jim F. Evermann ◽  
Clayton L. Kelling
Keyword(s):  
Reverse Transcription ◽  
Reaction Products ◽  
Rt Pcr ◽  
Specific Primers ◽  
F Protein ◽  
Chain Reactions ◽  
Polymerase Chain Reactions ◽  
Respiratory Syncytial Viruses ◽  
Polymerase Chain ◽  
Syncytial Virus

In reverse transcription-polymerase chain reactions (RT-PCR) and DNA hybridizations using primers and an oligonucleotide probe to the fusion (F) protein mRNA of bovine respiratory syncytial virus (BRSV), all the BRSV isolates and a goat isolate could be distinguished from prototype isolates of human respiratory syncytial viruses (HRSV) and ovine (sheep and bighorn sheep) respiratory syncytial viruses (RSV). However, RT-PCR amplifications with primers to sequences of the HRSV F protein mRNA resulted in amplified products of ≍ 243 bp if mRNA templates of subgroup A HRSV strains were present and slightly larger amplified products with subgroup B HRSV strains. No amplified products were observed in HRSV-primed RT-PCR with BRSV or goat or ovine RSV mRNA templates. Although the ovine RSV isolates were antigenically cross-reactive with the goat RSV, HRSV and BRSV isolates, they were not amplified with either HRSV- or BRSV-specific primers in RT-PCR. These results confirm previous immunological comparisons suggesting that some ovine RSV isolates should be considered as distinct respiratory syncytial viruses.

Novel Fusion of the MALT1/MLT and MAP4 Genes Detected in a Diffuse Large B-Cell Lymphoma.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2838-2838
Author(s):  
Eva M. Murga Penas ◽  
Kristina Hinz ◽  
Petra Behrmann ◽  
Martin L. Hansmann ◽  
Claudia Becher ◽  
...  
Keyword(s):  
B Cell ◽  
B Cell Lymphoma ◽  
Low Grade ◽  
Rt Pcr ◽  
B Cell Lymphomas ◽  
Specific Primers ◽  
Interphase Fish ◽  
Exon 9 ◽  
Race Pcr ◽  
Large B Cell

Abstract The MALT1/MLT gene codes for a human paracaspase and plays a crucial role in NF-κB activation in response to TCR induction. Rearrangements of MALT1/MLT by the translocations t(11;18)(q21;q21) and t(14;18)(q32;q21) act by activating the NF-κB pathway and are the most frequent structural chromosomal abnormalities in extranodal marginal zone B-cell lymphomas of the MALT-type. They occur in 20 to 50% of low grade MALT lymphomas, but with only rare exception are absent in diffuse large B-cell lymphomas (DLBCL). By screening 118 diffuse large B-cell lymphomas and 28 Burkitt’s lymphomas by interphase FISH with probes flanking MALT1/MLT (Abbott Vysis), we found two cases with a break within MALT1/MLT. Further experiments with plasmid subclones from the BAMH1 fragments of PAC 152M5, which spans the entire coding region of MALT1/MLT, confirmed the break within MALT1/MLT in one case. This patient presented with a relapse of a DLBCL, centroblastic subtype, without any concomitant low-grade component or lymphoepithelial lesions. During relapse only nodal manifestations were seen (stage IIIA), however, at the time of primary diagnosis a lymphoma infiltration of one adrenal gland was present. FISH experiments with MALT1/MLT, API2 and IGH specific probes did not identify a t(11;18) or a t(14;18). 3′ RACE-PCR revealed a novel in frame fusion of exon 9 of the MALT1/MLT gene and exon 9 of the microtubule-associated protein 4 (MAP4) gene. RT-PCR with specific primers for MALT1/MLT and MAP4 confirmed the presence of the fusion transcript MALT1/MLT-MAP4. In addition, interphase FISH showed that the translocation was accompanied by a deletion of MALT1/MLT sequences distal to the breakpoint including the caspase-like domain, which is essential for activation of the NF-κB pathway in MALT lymphomas. Corresponding to this result, 5′ RACE-PCR with specific primers for MALT1/MLT and RT-PCR with nested primers for MAP4 and MALT1/MLT did not detect the reciprocal 5′MAP4-3′MALT1/MLT transcript. The MAP4 gene on chromosome 3p21 is the major microtubule-associated protein in non-neuronal tissues and promotes microtubule polymerization. Disruption of the microtubule-dynamics is induced by microtubule-interfering drugs, such as taxanes and Vinca alkaloids. We conclude that the 5′MALT1/MLT-3′MAP4 fusion is the pathogenetically relevant transcript in this non-reciprocal der(18)t(3;18)(p21;q21) translocation. The absence of the caspase like domain distinguishes this novel gene fusion, MALT1/MLT-MAP4, from the API2-MALT1/MLT in the t(11;18) and the IGH-MALT1/MLT in the t(14;18), in which the caspase like domain is invariably present and points to MAP4 as a new target gene with possible therapeutic implications in DLBCL.

MLLT10 Gene Promiscuity Unravels Involvement of RNA Processing Genes in Pediatric T-Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1431-1431
Author(s):  
Lucia Brandimarte ◽  
Valentina Pierini ◽  
Danika Di Giacomo ◽  
Paolo Gorello ◽  
Caterina Matteucci ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Rna Processing ◽  
Lymphoblastic Leukemia ◽  
Fusion Partner ◽  
Whole Genome ◽  
Rt Pcr ◽  
Specific Primers ◽  
Fusion Transcripts ◽  
Partner Gene

Abstract Abstract 1431 Background T-cell Acute Lymphoblastic Leukemia (T-ALL) affects about 15% of children with ALL. MLLT10 at 10p12 encodes for a transcription factor and is involved in leukemogenic fusions with PICALM or MLL in about 10% of childhood T-ALL with overexpression of HOXA cluster genes. In case of gene fusion MLLT10 retains the “octapeptide motif-leucine zipper” (OM-LZ) domain that appeared to be essential for leukemic transformation in mouse models (Deshpande AJ et al. Leukemia 2011) as it interacted with critical components of the chromatin modifying machinery, such as H3K79 methyltransferase hDOT1L (Okada Y et al. Cell 2005). Recently, in a case of early T-cell precursor ALL, NAP1L1 at 12q21 was identified by whole-genome sequencing as a new MLLT10 fusion partner gene (Zhang J et al. Nature 2012). We identified two new fusion transcripts involving MLLT10 in 2 cases of pediatric T-ALL suggesting MLLT10 is a promiscuous fusion partner gene in T-ALL. Aim Characterization of new MLLT10 fusion transcripts in 2 pediatric cases of T-ALL. Methods Total RNAs were extracted from cryopreserved bone marrow cells and retrotranscribed with MLLT10 specific reverse primer using 5'-RACE kit (Invitrogen). cDNAs were amplified in nested PCR using AAP/AUAP (Abridged Anchor Primer, Abridged Universal Amplification Primer, Invitrogen) as forward and MLLT10 specific primers as reverse. To confirm fusion transcripts we performed RT-PCR experiments using Thermoscript RT-PCR System (Invitrogen). cDNAs were amplified in nested PCR using HNRNPH1 and DDX3X specific primers as forward in patient 1 and 2 respectively and MLLT10 specific primers as reverse in both samples. PCR products were subcloned into pGEM-T easy vector (Promega) and sequenced. Whole genome analysis was performed applying Combined-Interphase Fluorescence In Situ Hybridization (CI-FISH) for 32 candidate genes as previously described (Gorello P et al. Haematologica 2010); Single Nucleotide Polymorphisms (SNPs) were performed following manifacturer's instructions (Affymetrix); Gene Expression Profiling (GEP) was applied to investigate the hypothesis that both new MLLT10 fusion genes shared leukemogenic properties with other MLLT10 fusions, particularly PICALM-MLLT10. Results In patient 1 the karyotype was 46,XX,inv(10)(p12q?)/46,XX. CI-FISH on bone marrow nuclei showed MLLT10 rearrangements (55%) and IKZF1 deletions (10%). The 10q disruption was located at 10q25.3 in a region of about 12 kb flanked by fosmid G248P87999G12, retained on inv(10), and RP11–411P18 translocated to chromosome 5. SNPs analysis was normal for copy number and LOH profile. In patient 2 cytogenetics failed. CI-FISH revealed a 9p deletion, with loss of PAX5 (9p13), CDKN2A/B (9p21) and JAK2 (9p24) in about 75% of interphase nuclei. MLLT10 break apart was abnormal in about 60% of nuclei, with break within RP11–418C1 spanning MLLT10 exons 1–3. SNPs analysis showed a 47,5Mb loss at 9p24.3-p11.2 and a 33,51Mb gain at 17q21.32-q25.3. 5'-RACE-PCR showed HNRNPH1 in patient 1 and DDX3X in patient 2 as two new MLLT10 partner genes in T-ALL. RT-PCR, cloning and sequencing confirmed these results. We found 2 in frame splicing variants in both cases: HNRNPH1 exon 11 (nt.1324)-MLLT10 exon 15 (nt. 2097) and HNRNPH1 intron 10 (nt. 6701)-MLLT10 exon 15 (nt. 2097) in patient 1; DDX3X exon 2 (nt. 958)-MLLT10 exon 3 (nt. 510) and DDX3X exon 1 (nt. 900)-MLLT10 exon 4 (nt. 590) in patient 2 (nucleotide numbers refer to GenBank accessions NM_005520.2 and NC_000005.9 for HNRNPH1, NM_001356.3 for DDX3X and NM_004641.3 for MLLT10). GEP showed that the new MLLT10 fusions were similar to 4 cases with PICALM-MLLT10 fusion but different from 5 other T-ALL, i.e. 2 with MLL translocations, 2 with inv(7)(p15;q34)/TCRB-HOXA and 1 with SET-NUP214 fusion, with a HOXA signature. Conclusions These two cases add new insights in multiple genomic recombinations affecting MLLT10 in pediatric T-ALL. Both new partner genes, i.e. HNRNPH1 and DDX3X, are involved in RNA processing and have not been reported to be involved in any genomic specific translocations, so far. The presence of the MLLT10 OM-LZ domain in both new fusions as well as the GEP of leukemic cells suggest that these MLLT10 recombinations activate the same leukemogenic pathways as identified for PICALM-MLLT10 and suggest promiscuity of MLLT10 in recombinations with leukemogenic genes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Insulin and SGK1 reduce the function of Na+/monocarboxylate transporter 1 (SMCT1/SLC5A8)

2016 ◽  
Vol 311 (5) ◽  
pp. C720-C734 ◽  
Author(s):  
Adriana López-Barradas ◽  
Tania González-Cid ◽  
Norma Vázquez ◽  
Marisol Gavi-Maza ◽  
Adriana Reyes-Camacho ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Insulin Treatment ◽  
Kinase Activity ◽  
Acid Metabolism ◽  
Monocarboxylate Transporter ◽  
Human Pancreatic Cancer ◽  
Rt Pcr ◽  
Specific Primers ◽  
Monocarboxylate Transporter 1

SMCTs move several important fuel molecules that are involved in lipid, carbohydrate, and amino acid metabolism, but their regulation has been poorly studied. Insulin controls the translocation of several solutes that are involved in energetic cellular metabolism, including glucose. We studied the effect of insulin on the function of human SMCT1 expressed in Xenopus oocytes. The addition of insulin reduced α-keto-isocaproate (KIC)-dependent 22Na+ uptake by 29%. Consistent with this result, the coinjection of SMCT1 with SGK1 cRNA decreased the KIC-dependent 22Na+ uptake by 34%. The reduction of SMCT1 activity by SGK1 depends on its kinase activity, and it was observed that the coinjection of SMCT1 with S442D-SGK1 (a constitutively active mutant) decreased the KIC-dependent 22Na+ uptake by 50%. In contrast, an SMCT1 coinjection with K127M-SGK1 (an inactive mutant) had no effect on the KIC-dependent Na+ uptake. The decreasing SMCT1 function by insulin or SGK1 was corroborated by measuring [1-14C]acetate uptake and the electric currents of SMCT1-injected oocytes. Previously, we found that SMCT2/Slc5a12-mRNA, but not SMCT1/Slc5a8-mRNA, is present in zebrafish pancreas (by in situ hybridization); however, SLC5a8 gene silencing was associated with the development of human pancreatic cancer. We confirmed that the mRNA and protein of both transporters were present in rat pancreas using RT-PCR with specific primers, Western blot analysis, and immunohistochemistry. Additionally, significant propionate-dependent 22Na+ uptake occurred in pancreatic islets and was reduced by insulin treatment. Our data indicate that human SMCT1 is regulated by insulin and SGK1 and that both SMCTs are present in the mammalian pancreas.

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