Ectothermic organisms may expand their thermal tolerance by producing multiple protein isoforms with differing thermal sensitivities.While such isoforms commonly originate from allelic variation at a single locus(allo...Ectothermic organisms may expand their thermal tolerance by producing multiple protein isoforms with differing thermal sensitivities.While such isoforms commonly originate from allelic variation at a single locus(allozymes)or from gene duplication that gives rise to paralogs with distinct thermal responses,this study investigated mRNA editing as an alternative,post-transcriptional mechanism for generating mRNA variants.Cytosolic malate dehydrogenase(cMDH)was examined in foot tissue of two congeners of the marine mussel genus Mytilus,which occupy different thermal environments.Multiple editing events were detected within the mRNA coding region in both species.Editing sites were species-specific,with no shared positions identified.In M.coruscus,editing occurred at 117,123,135,190,195,204,279,and 444,while in M.galloprovincialis,editing was detected at 216 and 597.Each species exhibited multiple edited mRNA variants,and these isoforms were associated with differential protein expression.These findings suggest that mRNA editing may contribute an additional layer of molecular variation.The generation of diverse mRNA isoforms from a single DNA coding sequence may enhance enzymatic flexibility across temperature ranges,supporting eurythermal physiological performance and mitigating thermal stress.Moreover,the presence of multiple edited transcripts within individual organisms raises important caveats about the limitations of approaches that deduce amino acid sequences or estimate adaptive variation solely from genomic data.展开更多
AIM: to evaluate the expression of different insulinlike growth factor(IGF)-1 mRNA isoforms and IGF-1 receptor(IGF-1R) mRNA in hepatitis C virus(HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from c...AIM: to evaluate the expression of different insulinlike growth factor(IGF)-1 mRNA isoforms and IGF-1 receptor(IGF-1R) mRNA in hepatitis C virus(HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C(CH-C) patients were obtained before anti-viral therapy. Inflammatory activity(grading) and advancement of fibrosis(staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms(IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms(P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2(r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA(r = 0.891; r = 0.821, respectively), with IGF-1B mRNA(r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA(r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA(r = 0.956; r = 0.869, respectively), and B with C isoforms(r = 0.868)(P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading(G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data(e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.展开更多
AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and...AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.展开更多
Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exactbiological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavilydispute...Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exactbiological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavilydisputed, since data supporting both views have been reported in several independent studies. To clarify thisissue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the presentstudy, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis inthymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and afterinduction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering ofapoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptoticprocesses. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expressionwas restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolyticcleavage site and is therefore predicted to encode a monomeric protein. The biological function undernormal circumstances, however, will need further investigations for clarification. While apoptosis could notmodulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted inup-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control ofcell activation-mediated rather than apoptosis- induced signals.展开更多
基金supported by the National Key Research and Development Program of China(2022-24)National Natural Science Foundation of China(42025604,42376102)Fundamental Research Funds for the Central Universities。
文摘Ectothermic organisms may expand their thermal tolerance by producing multiple protein isoforms with differing thermal sensitivities.While such isoforms commonly originate from allelic variation at a single locus(allozymes)or from gene duplication that gives rise to paralogs with distinct thermal responses,this study investigated mRNA editing as an alternative,post-transcriptional mechanism for generating mRNA variants.Cytosolic malate dehydrogenase(cMDH)was examined in foot tissue of two congeners of the marine mussel genus Mytilus,which occupy different thermal environments.Multiple editing events were detected within the mRNA coding region in both species.Editing sites were species-specific,with no shared positions identified.In M.coruscus,editing occurred at 117,123,135,190,195,204,279,and 444,while in M.galloprovincialis,editing was detected at 216 and 597.Each species exhibited multiple edited mRNA variants,and these isoforms were associated with differential protein expression.These findings suggest that mRNA editing may contribute an additional layer of molecular variation.The generation of diverse mRNA isoforms from a single DNA coding sequence may enhance enzymatic flexibility across temperature ranges,supporting eurythermal physiological performance and mitigating thermal stress.Moreover,the presence of multiple edited transcripts within individual organisms raises important caveats about the limitations of approaches that deduce amino acid sequences or estimate adaptive variation solely from genomic data.
基金Minister of Education and Science,Warsaw,Poland,No.NN401009437
文摘AIM: to evaluate the expression of different insulinlike growth factor(IGF)-1 mRNA isoforms and IGF-1 receptor(IGF-1R) mRNA in hepatitis C virus(HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C(CH-C) patients were obtained before anti-viral therapy. Inflammatory activity(grading) and advancement of fibrosis(staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms(IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms(P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2(r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA(r = 0.891; r = 0.821, respectively), with IGF-1B mRNA(r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA(r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA(r = 0.956; r = 0.869, respectively), and B with C isoforms(r = 0.868)(P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading(G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data(e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.
文摘AIM:To investigate three isoforms of survivin in colorectal adenocarcinomas.METHODS:We used the LightCycler Technology(Roche),along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore(LC-Red 640).Real time quantitative polymerase chain reaction(PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues.In the patients group,carcinoembryonic antigen(CEA) and CA19-9 tumor markers were also measured immunochemically.RESULTS:Wild type survivin mRNA isoform was expressed in 48%of the 52 tumor samples,survivin-2b in 38%and survivin-ΔΕx3 in 29%,while no expression was found in normal tissues.The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b,survivin-ΔΕx3,survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin(P<0.001).The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion(P=0.006 and P<0.005,respectively).A significant difference was found between survivin-2b and morphologic cancer type.Also,the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis.No association was observed between the three isoforms and grade,metastasis,Dukes stage and gender.The three isoforms were not correlated with CEA and CA19-9.CONCLUSION:Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.
基金This work was supported by grant No. R05-2001-000-00464-0 from the Basic Research Program of the Korea Science and Engineering Foundation. The authors are very thankful to M.L. Cowan for corrections and suggestions to the text.
文摘Clusterin is a 75-80 kDa heterodimeric glycoprotein, that is produced in most tissues but which exactbiological role is still not clear. Particularly, its role in protection or promotion of apoptosis is heavilydisputed, since data supporting both views have been reported in several independent studies. To clarify thisissue, and also to determine whether clusterin expression itself might be affected by apoptosis, in the presentstudy, rat thymocytes were treated with dexamethasone, -a synthetic glucocorticoid that elicits apoptosis inthymocytes-, and clusterin mRNA expression was analyzed by semi-quantitative RT-PCR before and afterinduction of apoptosis. Interestingly, neither the treatment with dexamethasone in vitro nor triggering ofapoptosis in vivo up- regulated clusterin expression, opposing the view that clusterin is involved in apoptoticprocesses. On the other hand, a new clusterin mRNA isoform was detected and isolated, whose expressionwas restricted to freshly isolated thymocytes. This novel isoform lacks the post-translational proteolyticcleavage site and is therefore predicted to encode a monomeric protein. The biological function undernormal circumstances, however, will need further investigations for clarification. While apoptosis could notmodulate clusterin expression, activation of thymocytes with concanavalin A and interleukin-2 resulted inup-regulation of clusterin mRNA level, indicating that clusterin expression is rather under the control ofcell activation-mediated rather than apoptosis- induced signals.
