Expression Of Triosephosphate Isomerase Research Articles

Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Expression of vimentin, TPI and MAT2A in human dermal microvascular endothelial cells during angiogenesis in vitro.

IntroductionIn vitro assays of angiogenesis face immense problems considering their reproducibility based on the inhomogeneous characters of endothelial cells (ECs). It is necessary to detect influencing factors, which affect the angiogenic potency of ECs.ObjectiveThis study aimed to analyse expression profiles of vimentin (VIM), triosephosphate isomerase (TPI) and adenosylmethionine synthetase isoform type–2 (MAT2A) during the whole angiogenic cascade in vitro. Furthermore, the impact of knocking down vimentin (VIM) on angiogenesis in vitro was evaluated, while monitoring TPI and MAT2A expression.MethodsA long–term cultivation and angiogenic stimulation of human dermal microvascular ECs was performed. Cells were characterized via VEGFR–1 and VEGFR–2 expression and a shRNA–mediated knockdown of VIM was performed. The process of angiogenesis in vitro was quantified via morphological staging and mRNA–and protein–levels of all proteins were analysed.ResultsWhile native cells ran through the angiogenic cascade chronologically, knockdown cells only entered beginning stages of angiogenesis and died eventually. Cell cultures showing a higher VEGFR–1 expression survived exclusively and displayed an upregulation of MAT2A and TPI expression. Native cells highly expressed VIM in early stages, MAT2A mainly in the beginning and TPI during the course of angiogenesis in vitro.ConclusionVIM knockdown led to a deceleration of angiogenesis in vitro and knockdown cells displayed expressional changes in TPI and MAT2A. Cell populations with a higher number of stalk cells emerged as being more stable against manipulations and native expression profiles provided an indication of VIM and MAT2A being relevant predominantly in beginning stages and TPI during the whole angiogenic cascade in vitro.

Differential regulation of genes co-involved in aroma production and stress amelioration during salt acclimation in indica rice cultivars

The present study investigated the effects of salt acclimation on the expression of genes, viz., proline dehydrogenase (PDH), Δ1-pyrroline-5-carboxylate-synthetase (P5CS), P5C dehydrogenase (P5CDH), ornithine aminotransferase (OAT), betaine aldehyde dehydrogenase 1 (BADH1), BADH2, triose phosphate isomerase (TPI), ornithine decarboxylase (ODC), arginine decarboxylase (ADC), S-adenosyl-methionine decarboxylase (SAMDC), spermidine synthase (SPDS), spermine synthase (SPMS), diamine oxidase (DAO) and polyamine oxidase (PAO) in four indigenous aromatic varieties, viz., Tulaipanji (TP), Radhunipagal (RP), Kalonunia (KN) and Gobindobhog (GB) along with a non-aromatic cultivar, IR-64. The PDH, P5CS, P5CDH and OAT expression showed the regulation of the P5C pathway of aroma synthesis, with OAT appearing to regulate the crosstalk between the P5C and the polyamine (PA) pathway for the production of aroma. The role of PA pathway was further validated by monitoring the expression of ODC, ADC, SAMDC, SPDS, SPMS, DAO and PAO. The BADH2 expression reflected the fact that the enzyme encoded by the gene was directly and negatively associated with aroma content in rice. The TPI expression established that the enzyme encoded by this gene leading to the synthesis of methylglyoxal (MG) is also a key regulator for aroma synthesis. The current study established the varietal differences in the expression of the aforementioned genes in the selected rice cultivars. The salt-stressed aromatic varieties followed the P5C pathway to synthesize aroma. TP and RP exhibited the capability to generate aroma via PA-dependent pathway. Salt-stressed KN, on the contrary, accumulated higher BADH2 and lower TPI transcripts, which correlated with our previous report of KN accumulating low aroma during salinization due to efficient metabolite channelization towards stress amelioration.

Changes of B cell subsets in central pathological process of autoimmune encephalomyelitis in mice

BackgroundMultiple sclerosis is a demyelinating and autoimmune disease and its immune response is not fully elucidated. This study was conducted to examine the pathological changes and B cell subsets in experimental autoimmune encephalomyelitis (EAE) mice, and analyze the expression of triosephosphate isomerase (TPI) and GADPH to define the role of B cell subsets in the disease.ResultsFemale C57BL/6 mice were randomly divided into EAE group (n = 18) and control (n = 18). During the experiments, the weight and nerve function scores were determined. The proportions of B cell subsets in the peripheral blood were measured by flow cytometry. Seven, 18 and 30 days after immunization, the brain and spinal cord tissues were examined for the infiltration of inflammatory cells using hematoxylin-eosin (HE) HE staining and the demyelination using Luxol fast blue staining. The expression of B cell-related proteins was detected immunohistochemistrially and the expression of antigenic TPI and GADPH was analyzed using enzyme-linked immunosorbent assay (ELISA). HE staining showed that mice had more severe EAE 18 d than 7 d after modelling, while the symptoms were significantly relieved at 30 d. The results were consistent with the weight measurements and neural function scores. Immunohistochemistry studies showed that B cells aggregated in the spinal cord, but not much in the brain. Flow cytometry studies showed that there were more B cells in control than in EAE models from day 7 and the difference was narrowed at day 30. The level of plasma cells increased continuously, reached the top at day 21 and obviously declined at day 30. On other hand, the numbers of memory B cells increased gradually over the experimental period. The numbers of plasma and memory B cells were similar between the control and EAE mice. ELISA data revealed that the brain contents of TPI and GAPDH were higher in EAE mice than in control at day 7, while at day 18, the levels were reversed.ConclusionsIn the central pathological process of EAE mice, B cells exert role through the mechanism other than producing antibodies and the levels of brain TPI and GADPH are related to the severity of autoimmune induced-damage.

Increased expression of triosephosphate isomerase 1 in ovine pulmonary adenocarcinoma revealed by two-dimensional electrophoresis and immunohistochemistry

Increased expression of triosephosphate isomerase 1 in ovine pulmonary adenocarcinoma revealed by two-dimensional electrophoresis and immunohistochemistry

Triose-phosphate isomerase is a novel target of miR-22 and miR-28, with implications in tumorigenesis.

Aerobic glycolysis is the hallmark of many cancer cells that results in a high rate of adenosine triphosphate (ATP) production and, more importantly, biosynthetic intermediates, which are required by the fast-growing tumor cells. The molecular mechanism responsible for the increased glycolytic influx of tumor cells is still not fully understood. In the present study, we have attempted to address the above question by exploring the role of the glycolytic enzyme, triose-phosphate isomerase (TPI), in the cancer cells. The western blot analysis of the 30 human colorectal cancer samples depicted higher post-transcriptional expression of TPI in the tumor tissue relative to the normal tissue. In addition, we identified two novel microRNAs, miR-22 and miR-28, that target the TPI messenger RNA (mRNA) and regulate its expression. miR-22 and the miR-28 showed significant inverse expression status viz-a-viz the expression of the TPI. The specificity of the miR-22/28 regulation of the TPI mRNA was confirmed by various biochemical and mutagenic assays. Moreover, the hypoxia conditions resulted in an increased expression of the TPI protein, with a concomitant decrease in miR-22/28. The physiological significance of the TPI and miR-22/28 interaction for the glycolytic influx was confirmed by the l-lactate production in the HCT-116+/+ cells. Overall, our data demonstrate the novel microRNA mediated post-transcriptional regulation of the TPI glycolytic enzyme, which may be one of the possible reasons for the increased glycolytic capacity of the tumor cells.

Identification of salinity-related genes in ENO2 mutant (eno2−) of Arabidopsis thaliana

Abiotic stress poses a great threat to plant growth and can lead to huge losses in yield. Gene enolase2 (ENO2) is important in resistance to abiotic stress in various organisms. ENO2 T-DNA insertion mutant (eno2−) plants of Arabidopsis thaliana showed complete susceptibility to sodium chloride treatment when were analyzed either as whole plants or by measuring root growth during NaCl treatment. Quantitative real-time RT-PCR (RT-qPCR) was performed to investigate the expression profile of ENO2 in response to NaCl stress in Arabidopsis. The transcript level of ENO2 was rapidly elevated in 300 mmol L−1 NaCl treatment. ENO2 also responded to 300 mmol L−1 NaCl treatment at the protein level. To illuminate the mechanism underlying ENO2 resistance to salt at the transcriptional level, we studied the wild-type and eno2− Arabidopsis lines that were treated with 300 mmol L−1 NaCl for 18 h using 454 GS FLX, which resulted in an expressed sequence tag (EST) dataset. A total of 961 up-regulated and 746 down-regulated differentially expressed genes (DEGs) were identified in the pairwise comparison WT-18 h:eno2−−18 h. The DEGs were identified and functionally annotated using the databases of Gene Ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG). The identified unigenes were subjected to GO analysis to determine biological, molecular, and cellular functions. The biological process was enriched in a total of 20 GO terms, the cellular component was enriched in 13 GO terms, and the molecular function was enriched in 11 GO terms. Using KEGG mapping, DEGs with pathway annotations contributed to 115 pathways. The top 3 pathways based on a statistical analysis were biosynthesis of the secondary metabolites (KO01110), plant-pathogen interactions (KO04626), and plant hormone signal transduction (KO04075). Based on these results, ENO2 contributes to increased resistance to abiotic stress. In particular, ENO2 is involved in some of the metabolic stress response pathways in Arabidopsis. Our work also demonstrates that this EST dataset will be a powerful resource for further studies of ENO2, such as functional analyses, investigations of biological roles, and molecular breeding. Additionally, 3-phosphoglycerate kinase (PGK), 3-phosphoglycerate kinase 1 (PGK1), triosephosphate isomerase (TPI), and pyruvate kinase (PK) in glycolysis interactions with ENO2 were verified using the yeast two-hybrid experiment, and ENO2 may regulate the expression of PGK, PGK1, TPI, and PK. Taken together, the results from this study reflects that ENO2 gene has an important role in the response to the high salt stress.

Clinical significance and prognostic value of Triosephosphate isomerase expression in gastric cancer.

Triosephosphate isomerase (TPI) is highly expressed in many human cancers and is involved in migration and invasion of cancer cells. However, TPI clinicopathological significance and prognostic value in gastric cancer (GC) are not yet well defined. The aim of the present work was to evaluate TPI expression in GC tissue and its prognostic value in GC patients.TPI expression was analyzed in 92 primary GC tissues and 80 adjacent normal mucosa tissues from GC patients undergoing gastrectomy by immunohistochemical analysis of tissue microarrays (TMAs). Univariate and multivariate analyses were performed to investigate TPI prognostic significance in GC patients.Immunohistochemical staining score showed that TPI expression in cancer tissues was significantly higher than in adjacent normal mucosa (P < .001). Univariate analysis revealed that TPI expression, depth of invasion, lympho node metastasis, tumor node metastasis (TNM) stage, and tumor diameter were associated with negative prognostic predictors for overall survival in GC patients (P < .05). High TPI expression represented a significant predictor of shorter survival in GC patients with positive lymphatic metastasis (P = .022) and tumor diameter >5 cm (P = .018). Cox multivariate analysis identified TPI expression, TNM stage, and tumor diameter as independent prognostic factors in GC patients.TPI expression might be considered as a novel prognostic factor to evaluate GC patients’ survival.

Role of triosephosphate isomerase and downstream functional genes on gastric cancer.

Triosephosphate isomerase (TPI) is highly expressed in many types of human tumors and is involved in migration and invasion of cancer cells. However, TPI clinicopathological significance and malignant function in gastric cancer(GC) have not been well defined. The present study aimed to examine TPI expression in GC tissue and its biological functions. Furthermore, we investigated its downstream genes by gene chip technology. Our results showed that TPI expression was higher in gastric cancer tissues than adjacent tissues, although no statistical differences were found between TPI expression and clinicopathological factors. TPI overexpression in human gastric carcinoma cell line BGC-823 enhanced cell proliferation, invasion and migration, but did not change cell cycle distribution, while TPI knockdown suppressed proliferation, invasion and migration, induced apoptosis and increased G2/M arrest of human gastric carcinoma cell line MGC-803. Since the cell division cycle associated 5 (CDCA5) was identified as the one with the most decreased expression after TPI knockdown, we investigated its role in MGC-803 cells. The results showed that CDCA5 knockdown also inhibited proliferation, migration, induced apoptosis and increased G2/M arrest similarly to TPI knockdown. CDCA5 overexpression promoted MGC-803 cell proliferation, clone formation and migration abilities. These results indicated that TPI expression level might affect GC cell behavior, suggesting that both TPI and CDCA5 might be considered as potential tumor markers related with GC development and might be potential new targets in GC treatment.

Pharmacological Blockade of Cannabinoid CB1 Receptors in Diet-Induced Obesity Regulates Mitochondrial Dihydrolipoamide Dehydrogenase in Muscle.

Cannabinoid CB1 receptors peripherally modulate energy metabolism. Here, we investigated the role of CB1 receptors in the expression of glucose/pyruvate/tricarboxylic acid (TCA) metabolism in rat abdominal muscle. Dihydrolipoamide dehydrogenase (DLD), a flavoprotein component (E3) of α-ketoacid dehydrogenase complexes with diaphorase activity in mitochondria, was specifically analyzed. After assessing the effectiveness of the CB1 receptor antagonist AM251 (3 mg kg-1, 14 days) on food intake and body weight, we could identified seven key enzymes from either glycolytic pathway or TCA cycle—regulated by both diet and CB1 receptor activity—through comprehensive proteomic approaches involving two-dimensional electrophoresis and MALDI-TOF/LC-ESI trap mass spectrometry. These enzymes were glucose 6-phosphate isomerase (GPI), triosephosphate isomerase (TPI), enolase (Eno3), lactate dehydrogenase (LDHa), glyoxalase-1 (Glo1) and the mitochondrial DLD, whose expressions were modified by AM251 in hypercaloric diet-induced obesity. Specifically, AM251 blocked high-carbohydrate diet (HCD)-induced expression of GPI, TPI, Eno3 and LDHa, suggesting a down-regulation of glucose/pyruvate/lactate pathways under glucose availability. AM251 reversed the HCD-inhibited expression of Glo1 and DLD in the muscle, and the DLD and CB1 receptor expression in the mitochondrial fraction. Interestingly, we identified the presence of CB1 receptors at the membrane of striate muscle mitochondria. DLD over-expression was confirmed in muscle of CB 1 -/- mice. AM251 increased the pyruvate dehydrogenase and glutathione reductase activity in C2C12 myotubes, and the diaphorase/oxidative activity in the mitochondria fraction. These results indicated an up-regulation of methylglyoxal and TCA cycle activity. Findings suggest that CB1 receptors in muscle modulate glucose/pyruvate/lactate pathways and mitochondrial oxidative activity by targeting DLD.

Characterization and Proteome Analysis of Inosine 5-Monophosphate Dehydrogenase in Epidemic Streptococcus suis Serotype 2

Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe disease symptoms in pigs and humans. In the present study, we found one isogenic mutant lacking inosine 5-monophosphate dehydrogenase (IMPDH) ΔZY05719 was attenuated in pigs compared with the wild-type SS2 strain ZY05719. Comparative proteome analysis of the secreted proteins expression profiles between ZY05719 and ΔZY05719 allowed us to identify Triosephosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH), which were down expressed in the absence of the IMPDH. Both of them are glycolytic enzymes participating in the glycolytic pathway. Compared with ZY05719, ΔZY05719 lost the ability of utilize mannose, which might relate to down expression of TPI and GAPDH. In addition, GAPDH is a well-known factor that involved in adhesion to host cells, and we demonstrated ability of adhesion to HEp-2 and PK15 by ΔZY05719 was significantly weakened, in contrast to ZY05719. The adhesion to host cells is the crucial step to cause infection for pathogen, and the reduction adhesion of ΔZY05719, to some extent illustrates the attenuated virulence of ΔZY05719.

Aging-related changes of triose phosphate isomerase in hippocampus of senescence accelerated mouse and the intervention of acupuncture

Glycometabolism disorder induced by triose phosphate isomerase (TPI) is closely related to Alzheimer's disease (AD). Senescence accelerated mouse (SAM) is often employed as an AD model characteristic of early cognitive impairment. In order to investigate the variation of TPI with aging, SAM prone 8 (SAMP8) and SAM resistant 1 (SAMR1) were divided into 2-month, 6-month, 8-month and 12-month group. For the analysis of acupuncture intervention, SAMP8 were divided into SAMP8 control group (Pc), SAMP8 acupoint group (Pa), SAMP8 non-acupoint group (Pn) and SAMR1 control group (Rc). Grading score of senescence and Morris water maze results showed that SAMP8 presented aging-related deterioration of learning and memory, and that acupuncture could improve the learning and memory ability of SAMP8. TPI activity and expression were detected by colorimetric method and Western blot analysis, respectively. When compared to SAMR1, TPI activity in 6-, 8- and 12-month SAMP8 decreased significantly. However, acupuncture intervention markedly up-regulated TPI activity in hippocampus of Pa. These findings suggested that the learning and memory deterioration of SAMP8 with aging might be associated with the lower TPI activity and that acupuncture could improve the cognitive impairment by increasing TPI activity, thus correcting the abnormal glycolysis metabolism and maintaining the brain homeostasis and internal environment.

Effects of platelets on the protein expression in aortic segments: A proteomic approach

It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor-α (TNF-α)-preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton-related proteins expression was modified by PRP. However, when PRP was coincubated with TNF-α pre-stimulated aortic segments lesser number of cytoskeleton-related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic-related proteins. However, in TNF-α-preincubated segments the presence of PRP upexpressed glyceraldehyde-3-phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF-α-preincubated segments the expression of fructose 1,6-bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α-chain. In addition, the activity of fructose 1,6-bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall.

Identification of triosephosphate isomerase as an anti-drug resistance agent in human gastric cancer cells using functional proteomic analysis

Proteomic study was used to explore new multidrug resistance (MDR)-related proteins and clarify novel mechanism of MDR in gastric cancer. Two-dimensional gel electrophoresis and the PDQuest software analysis were applied to compare the differential expression of MDR-related proteins in gastric cancer SGC7901 cells and drug-resistant SGC7901 cells (SGC7901/VCR) induced by vincristine sulfate (VCR). The differential protein dots were excised and further analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (MALDI-TOF-MS). Nine differential expression proteins between the two cell lines were successfully identified by MALDI-TOF-MS. Triosephosphate isomerase (TPI), a glycolytic pathway enzyme, was identified as a downregulated protein in SGC7901/VCR cells. Further, Western blot analysis and semiquantitative RT-PCR confirmed its decreased expression in SGC7901/VCR cells. Sense vector pcDNA3.1-TPI was constructed and transfected into SGC7901/VCR. The sensitivity of TPI-SGC7901/VCR cells to adriamycin (ADR), VCR, 5-fluorouracil and cis-dichlorodiamine platinum, as well as the accumulation and retention to ADR, were significantly increased when compared to their control cell lines. These results provide new MDR-related protein candidates, which are differentially expressed in the MDR cell line and its parental cell line including TPI, which may participate in the VCR-mediated MDR in human gastric cancer. Upregulation of TPI expression could partially reverse multidrug-resistant phenotype of SGC7901/VCR, which suggests that TPI may be an anti-drug resistance agent in gastric cancer and the candidate target to develop novel therapeutics for better treatment of gastric cancer.

Hypoxia up-regulates triosephosphate isomerase expression via a HIF-dependent pathway.

The glycolytic enzyme triosephosphate isomerase (TPI) catalyses the reversible conversion of dihydroxyacetone phosphate into glyceraldehyde-3-phosphate. We report here that the expression of TPI at both the mRNA and protein levels is increased by hypoxia in vivo and in vitro. The temporal pattern of hypoxic TPI induction is very similar to that of genes triggered by the hypoxia-inducible transcription factor (HIF) and is mimicked characteristically by cobalt and by deferoxamine, but is absent in cells with a defective aryl hydrocarbon receptor nuclear translocator (ARNT, here HIF-1beta) and in cells lacking HIF-1alpha protein. We conclude from these findings that the expression of TPI is regulated via the HIF pathway and thus belongs to the family of classic oxygen-regulated genes. The physiological meaning of an increased expression of TPI in hypoxygenated tissues is probably to increase the flow of triosephosphates through the glycolytic cascade thus leading to an increase of anaerobic energy generation.

Quantitative proteomics analysis of specific protein expression and oxidative modification in aged senescence-accelerated-prone 8 mice brain

The senescence-accelerated mouse (SAM) is a murine model of accelerated senescence that was established using phenotypic selection. The SAMP series includes nine substrains, each of which exhibits characteristic disorders. SAMP8 is known to exhibit age-dependent learning and memory deficits. In our previous study, we reported that brains from 12-month-old SAMP8 have greater protein oxidation, as well as lipid peroxidation, compared with brains from 4-month-old SAMP8 mice. In order to investigate the relation between age-associated oxidative stress on specific protein oxidation and age-related learning and memory deficits in SAMP8, we used proteomics to identify proteins that are expressed differently and/or modified oxidatively in aged SAMP8 brains. We report here that in 12 month SAMP8 mice brains the expressions of neurofilament triplet L protein, lactate dehydrogenase 2 (LDH-2), heat shock protein 86, and α-spectrin are significantly decreased, while the expression of triosephosphate isomerase (TPI) is increased compared with 4-month-old SAMP8 brains. We also report that the specific protein carbonyl levels of LDH-2, dihydropyrimidinase-like protein 2, α-spectrin and creatine kinase, are significantly increased in the brain of 12-month-old SAMP8 mice when compared with the 4-month-old SAMP8 brain. These findings are discussed in reference to the effect of specific protein oxidation and changes of expression on potential mechanisms of abnormal alterations in metabolism and neurochemicals, as well as to the learning and memory deficits in aged SAMP8 mice.

Identification and characterization of a novel rat triosephosphate isomerase gene in remnant ileum after massive small bowel resection.

This paper describes the identification and characterization of a novel cDNA encoding a putative protein of 254 amino acids that is highly homologous to triosephosphate isomerase. The cDNA was isolated by subtractive hybridization and was differentially expressed in the remnant rat ileum after massive small bowel resection. The novel triosephosphate isomerase was named rsTPI (resection-induced TPI) and the putative protein encoded RSTPI. The nucleotide and amino acid sequences of rsTPI and RSTPI were about 60% and 62% homologous to Giardia lamblia TPI and TPI, respectively. Active catalytic sites (Lys 13, His 95, and Glu 167) and the peptide motifs, AYEPVWSIGT and GGASLKPEF found in other triosephosphate isomerases were conserved in RSTPI. rsTPI expression was detected in normal ileum and pancreas by reverse transcription-polymerase chain reaction. Expression of rsTPI in remnant rat ileum was detectable by northern blot analysis one week after massive small bowel resection. Expression increased significantly by 2.8-fold between one and two weeks after surgery. High levels were maintained for at least one month after surgery. The up-regulation of triosephosphate isomerase expression in the remnant small intestine after massive resection indicates that it may play an important role in the adaptive process.

Expression of Triosephosphate Isomerase Transcripts in Rat Testis: Evidence for Retinol Regulation and a Novel Germ Cell Transcript1

Vitamin A is essential for mammalian spermatogenesis. To isolate retinol-induced cDNAs from rat testis, vitamin A-deficient (VAD) rats were treated with retinol for 4 h and mRNA isolated from their testes was used to construct a subtractive cDNA library. One cDNA isolated from this library contained a sequence for triosephosphate isomerase (TPI). Northern blot analysis showed that the TPI cDNA hybridized with two transcripts in testis. A 1.4-kb transcript, which is the sole transcript found in most tissues, was expressed in the somatic cells of testis, whereas a novel 1.5-kb transcript was detected only in haploid spermatids. However, only the level of the shorter transcript increased with retinol treatment of the testis or of cultured Sertoli cells. Furthermore, screening of an adult rat testis cDNA library with the TPI cDNA yielded a cDNA putatively corresponding to the 1.5-kb transcript. Sequence analyses of the two TPI cDNAs revealed a 100-bp deletion in one cDNA that may be due to use of an alternative polyadenylation signal. These results suggest independent processing mechanisms for TPI expression in the somatic cells and the haploid germ cells of testis.