Wen-Chuan Lee

Animal technology institute Taiwan, T’ai-pei, Taipei, Taiwan

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Publications (18)31.94 Total impact

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    ABSTRACT: Background Immune complex (IC) vasculitis can be experimentally induced in animal models by intravenous injection of horse serum (HS), and the findings of HS-induced IC vasculitis in swine were very similar to that of Kawasaki disease (KD). The IC mechanism may be involved in the pathogenesis of vasculitis in KD. Here, we studied the two-dimensional (2D) echocardiographic and histopathological findings of acute, subacute, and healing phases of vasculitis induced by two different types of HS, and the reproducibility of IC vasculitis in swine. Methods and results Our study group consisted of 24 pure-bred landrace male piglets of 1.5–3 months of age. They were divided into three HS groups (n = 17), namely, Group A (n = 8) receiving gamma globulin-free HS, and Group B (n = 6) receiving donor herd HS, three doses at 5-day intervals, and Group C (n = 3) that received only one dose of donor herd HS on Day 1, and the saline group (n = 7) that received three doses of intravenous normal saline (NS) at 5-day intervals. The 2D echocardiography was performed every 3–4 days, and all piglets were killed for histopathological studies at different dates from Days 2 to Day 60. All the HS groups developed rashes and demonstrated significant dilation (54–150%) of coronary arteries in Groups A and B; when compared (p < 0.02) with 9–53% dilation in Group C and the saline group. Histopathological changes of test groups were asymmetric coronary vasculitis in various stages, whereas none of the piglets in the control group developed vasculitis. No significant difference in the echocardiographic and histopathological findings was observed among the piglets that received two types of HS. Conclusion HS can induce IC vasculitis in swine. The rashes and 2D echocardiographic and histopathological studies of the acute to healing phases showed close similarities with KD, and it is concluded that swine may serve as a unique experimental model for IC vasculitis and for various therapeutic trials.
    Pediatrics & Neonatology 01/2014; · 0.93 Impact Factor
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    ABSTRACT: A dual-functional Escherichia coli expression vector capable of producing soluble recombinant proteins with high immunogenicity in animals is introduced. This vector expresses polypeptides fused to a PTD-J-domain peptide. The J-domain peptide is derived from murine Hsp40 by using optimized codons for E. coli. The association of the J-domain to the nucleotide binding domain of the DnaK chaperone increases the probability that the fused polypeptide will be folded by the DnaK and hence increases the solubility of the recombinant protein. The PTD-J-domain can also enhance the immunogenicity of the fused chicken IGF-I polypeptide as well as an oligo-peptide derived from haptoglobin in rodents, possibly via the association with either the extracellular or intracellular Hsp70 proteins.
    Protein Expression and Purification 01/2009; 65(1):51-6. · 1.43 Impact Factor
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    ABSTRACT: Sperm capacitation and the acrosome reaction are fundamentally important to fertilization. Nitric oxide (NO) has been shown to have various functions in male reproduction. This work investigates whether boar sperm can generate NO, as well as the effects of NO and geldanamycin (GA), a heat-shock protein 90 (HSP90)-specific inhibitor, on the capacitation of boar spermatozoa. Observations showed that porcine sperm produced low levels of NO under non-capacitating conditions. However, the NO concentration almost doubled under capacitating conditions (P<0.001). Treatment with NG-nitro-L-arginine methyl ester (L-NAME) reduced the production of NO by 30-40% in capacitating sperm (P<0.05). GA treatment increased it by 23-75% in a dose-dependent manner (P<0.05). L-NAME treatment reduced the percentage of sperm undergoing the acrosome reaction, whereas sodium nitroprusside, an NO-releasing compound, and GA treatment increased the percentage of sperm undergoing the acrosome reaction (P<0.05). GA treatment promoted the production of NO and the acrosome reaction. The increase in NO production by GA treatment was similar to that caused by the calcium ionophore, A23187, suggesting that the GA-induced acrosome reaction may be triggered by an increase of the intracellular calcium concentration. The signaling pathway involved in GA-mediated NO production and its biological function in fertilizing boar spermatozoa will be elucidated in further studies.
    Animal Reproduction Science 02/2008; 104(1):56-68. · 1.90 Impact Factor
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    ABSTRACT: We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5'-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.
    Animal Reproduction Science 10/2007; 101(1-2):113-24. · 1.90 Impact Factor
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    ABSTRACT: Serum is believed to harbor thousands of distinct proteins that are either actively secreted or leak from various blood cells or tissues. Exploring protein composition in serum may accelerate the discovery of novel protein biomarkers for specific economic traits in livestock species. This study analyzed serum protein composition to establish a 2-DE reference map, and monitored protein dynamics of single-comb White Leghorn hens at 8, 19 and 23 weeks after hatching. A total of 119 CBB-stained and 315 silver-stained serum protein spots were analyzed by MALDI-TOF MS. Of these, 98 CBB-stained and 94 silver-stained protein spots were significantly matched to existing chicken proteins. The identified spots represented 30 distinctive proteins in the serum of laying hens. To compare protein expression during development, expression levels of 47 protein spots were quantified by relative spot volume with Melanie 3 software. Ten protein spots increased and 3 protein spots decreased as hen age increased. Previous research has suggested that some of these proteins play critical roles in egg production. The differentially expressed proteins with unknown identities will be valuable candidates for further explorations of their roles in egg production of laying hens.
    PROTEOMICS 05/2006; 6(7):2217-24. · 4.13 Impact Factor
  • Journal of Molecular and Cellular Cardiology - J MOL CELL CARDIOL. 01/2006; 40(6):1009-1009.
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    ABSTRACT: The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.
    PROTEOMICS 12/2005; 5(16):4205-12. · 4.13 Impact Factor
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    ABSTRACT: Heat-shock proteins (HSPs) are important in spermatogenesis. This study investigated developmental changes in the expression of major HSPs in porcine testis. The testis from five immature (mean age 2.9+/-0.1 months) and five mature boars (35.7+/-14.0 months) were examined. Two-dimensional polyacrylamide gel electrophoresis was conducted and proteins were identified by Western blotting and/or matrix-assisted laser desorption/ionization mass spectrometry. Moreover, the 90, 70, and 60 kDa HSPs, 70 kDa heat-shock cognate protein (HSC 70), tubulin, and actin were quantified on two-dimensional gels. Protein spots were quantified by densitometry, combined with a computer-assisted image analysis system. Immunohistochemistry was performed to analyze the expression pattern of major HSPs and beta-tubulin in testis. One isoform of HSP 90 (HSP 90 alpha), two isoforms of HSC 70 (HSC 70a and HSC 70c), one isoform of HSP70 (HSP 70e), and tubulin increased after sexual maturation (P<0.05). A testis-specific HSP70 (P70t) was markedly increased in the testes of sexually mature boars. Meanwhile, levels of actin and some isoforms of HSPs including 60 kDa HSP remained similar in both groups. These observations were further confirmed by immunohistochemistry; therefore, the upregulation of protein expression in the adult testis could be attributed to a higher level of protein expression and the number of cells that were HSPs-positive already resided in the immature testis. The differential expression of major HSPs suggested that they may be important in porcine spermatogenesis.
    Theriogenology 12/2005; 64(9):1940-55. · 2.08 Impact Factor
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    ABSTRACT: A high environmental temperature affects the economic performance of pigs. Heat shock protein 70 (HSP70) has been reported to participate importantly in thermotolerance. This study aims to produce transgenic pigs overexpressing porcine HSP70.2, the highly inducible one of HSP70 members, and to prove the cellular thermotolerance in the primary fibroblasts from the transgenics. A recombinant plasmid in which the sequence that encodes the porcine HSP70.2 gene is fused to green fluorescence protein (GFP) was constructed under the control of cytomegalovirus (CMV) enhancer and promoter. Two transgenic pigs were produced by microinjecting pCMV-HSP70-GFP DNA into the pronucleus of fertilized eggs. Immunoblot assay revealed the varied overexpression level (6.4% and 1.4%) of HSP70-GFP in transgenic pigs. After heating at 45C for 3 h, the survival rate (78.1%) of the primary fibroblast cells from the highly expressing transgenic pig exceeded that from the non-transgenic pig (62.9%). This result showed that primary fibroblasts overexpressing HSP70-GFP confer cell thermotolerance. We suggest that transgenic pigs overexpressing HSP70 might improve their thermotolerance in summer and therefore reduce the economic loss in animal production.
    Asian Australasian Journal of Animal Sciences 01/2005; 18(1). · 0.64 Impact Factor
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    ABSTRACT: An attempt was made to induce immune complex vasculitis by horse serum (HS) infusions in piglets, hoping to produce experimental coronary artery lesions that mimic Kawasaki disease. A total of 21 purebred male piglets of 1.5, 2.5, and 3 mo were divided into HS (n = 14) and control, normal saline (NS; n = 7) groups. In seven piglets, 5 mL/kg of HS was infused, then repeated with 10 mL/kg 10 d later. In another seven piglets, 10 mL/kg of HS was infused three times at 5-d intervals. In three piglets in the control group, 5 and 10 mL/kg of NS was infused at 10-d intervals. In another four piglets of the control group, 10 mL/kg of NS was infused three times at 5-d intervals. Two-dimensional echocardiographic examinations for visualization and measurement of the coronary arteries were done before and after infusions at 4- to 5-d interval. Hematology examination showed that white blood cells and platelets decreased, then increased. The animals were killed at 14-60 d after the first infusion of HS or NS, for histopathologic and immunohistochemical studies. All HS groups developed skin rashes and echocardiographic evidence of coronary artery dilation and histopathologic changes of vasculitis. None in the NS group developed vasculitis. The main changes of the coronary vasculitis were intimal proliferation, smooth muscle cell necrosis, and vacuolization changes. Those that received three HS infusions developed more skin rashes than those that received two infusions. It is concluded that piglets may serve as an experimental model for immune complex vasculitis involving the coronary arteries with skin rashes mimicking Kawasaki disease.
    Pediatric Research 03/2004; 55(2):211-9. · 2.67 Impact Factor
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    ABSTRACT: The purpose of this study was to investigate the relationship between swine health status and the concentration of the serum acute phase proteins, haptoglobin (HP), and C-reactive protein (CRP). A total of 378 clinically healthy pigs from farms A and B, plus 20 pigs culled from farm A due to poor growth, were used in this experiment. Each pig was examined and blood samples were collected during slaughter. The HP concentration was measured by using an HP-hemoglobin binding assay. The CRP concentration was measured by using a CRP enzyme immunoassay. Gross and histopathological lesions were examined and recorded at slaughter. Representative samples were then collected in order to isolate pathogens. Swine enzootic pneumonia, found in 47.7% of the pigs, was the most common lesion. Other lesions included pleuropneumonia (32.7%), suppurative pneumonia (10.3%), fibrinous pericardititis (4.3%), Ascaris migration in the liver (33.9%), and intestinal serositis (3.0%). On farm A, the percentage of pigs with 1 or more lesions was 88.2%. For culled pigs from farm A, the mean serum concentrations of HP and CRP were 2.23 +/- 0.14 mg/mL and 252.93 +/- 11.62 microg/mL, which were significantly higher than concentrations in clinically normal pigs (1.42 +/- 0.02 mg/mL and 84.88 +/- 2.61 microg/mL, respectively, P < 0.01). Moreover, among clinically normal farm A pigs, the mean HP concentration in pigs with lesions (1.43 +/- 0.02 mg/mL) was significantly higher than in pigs without lesions (1.32 +/- 0.07 mg/mL) (P < 0.05). However, the mean serum CRP concentrations in these animals were not significantly different. On farm B, the percentage of pigs with one or more lesions was 50.0%. Interestingly, the mean serum HP concentration in clinically normal pigs with lesions was significantly lower in farm B pigs (1.23 +/- 0.07 mg/mL) than in the farm A pigs (1.43 +/- 0.02 mg/mL; P < 0.01). However, serum CRP concentrations in farm A and B pigs were not significantly different. Serum HP concentration, which is a better indicator of inflammatory reactions in pig herds than serum CRP concentration, provides an important marker for swine health status.
    Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire 11/2003; 67(4):283-90. · 1.19 Impact Factor
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    ABSTRACT: The purpose of this study was to determine the relationship between the serum level of C-reactive protein (CRP) and lactation and health status. Blood samples were collected every 2 wk for 12 mo from 29 randomly selected dairy cattle on 3 farms. At the time the blood samples were collected, the stage of pregnancy, lactation status, breeding records, general health condition, reproductive status, and body condition score were recorded for each cow. Serum CRP was detected with sodium dodecyl sulfate polyacrylamide gel electrophoresis and western immunoblotting. C-reactive protein levels were measured with a densitometer and expressed as an optimal dose value. C-reactive protein levels were correlated with the body condition score, lactation status, and animal health (P < 0.05), but not with ambient temperature, animal age, or parity. C-reactive protein levels increased with milk production, peaking during high lactation (2 to 4 mo of pregnancy), and decreased when lactation ceased. In addition, the CRP level was highest during naturally occurring infections, such as mastitis and other tissue inflammation. Thus, the CRP level can confirm the presence of inflammation. The stress effect of taking blood samples as measured by the CRP level, was also examined. The CRP level became rapidly elevated 12 h after the blood samples were taken but returned to normal 36 h later. In conclusion, the stresses resulting from overall poor health, heavy lactation, and blood sampling caused the elevation of serum CRP. C-reactive protein is a marker or tool for evaluating the health status of a herd. C-reactive protein should also be considered as a useful criteria to assess the stress levels and may be useful in early surveillance of disease conditions in a dairy herd.
    Canadian journal of veterinary research = Revue canadienne de recherche vétérinaire 06/2003; 67(2):102-7. · 1.19 Impact Factor
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    ABSTRACT: Higher environmental temperature affects the economic performance of pigs. Heat shock protein 70 has been shown to play an important role in thermoresistance. The purpose of this study was to assess the effect of a single nucleotide polymorphism in the 5'-flanking region of porcine HSP70.2 on growth performance in Taiwanese Duroc. The genotype of this nt 393 polymorphic site could be verified by digestion with Bsa WI restriction enzyme of a PCR product. Pigs with TT and TC genotypes have thinner backfats than those with CC type (p
    Asian Australasian Journal of Animal Sciences 01/2003; 16(1). · 0.64 Impact Factor
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    ABSTRACT: Ultrasonography is one of the most common, noninvasive techniques used for cardiovascular diagnosis because it provides reliable information and enhances patient safety. Two-dimensional (2-D) and M-mode echocardiography is conducted to assess the severity and distribution of myocardial hypertrophy. Hypertrophic cardiomyopathy (HCM) is a primary myocardial disease that has variable manifestations because interactions between the many facets of systolic and diastolic dysfunction of the heart are complex. The objective of the study reported here was to characterized clinical HCM in pigs. A commercial Vingmed (CFM-800) 3.25 MHz transducer was used to perform 2-D and M-mode echocardiography. Experimental pigs (about 100 kg in body weight) were anesthetized and positioned in left lateral recumbency. Echocardiographic images (2-D) were acquired in parasternal short-axis and long-axis views. The 2-D images provided M-mode under direct anatomic visualization. The pigs were sacrificed for pathologic study after echocardiographic examination. In typical HCM cases (n = 8), the interventricular septum thickness increased, the left ventricular (LV) end-systolic and end-diastolic dimensions decreased, and the left atrial dimensions and the indexes of systolic function, such as ejection fraction and velocity of fiber shortening, increased. The LV outflow tract narrowed, particularly when gross upper septal hypertrophy was evident. Moreover, systolic cranial motion (SCM) of the septal leaflet of the mitral valve was observed. Doppler evidence of mitral regurgitation often was associated with SCM. The echocardiographic findings from pigs with HCM resembled those from humans. Thus, porcine HCM may serve as a spontaneous animal model for the study of HCM in humans.
    Comparative medicine 07/2002; 52(3):238-42. · 1.12 Impact Factor
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    ABSTRACT: As widely believed treating cells with trichostatin A (TSA), an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. This compound is often compared with other potential anticancer drugs in cell cycle, proliferation and differentiation research. Furthermore, geldanamycin (GA), a 90-kDa heat shock protein (HSP90) specific inhibitor, is a well-known potential anticancer agent. This study examines whether GA can affect the cellular functions induced by TSA. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 microg/ml GA for 30 min and an addition of 50 ng/ml TSA (GA + TSA) apparently averted cell death. Our results indicated that the cell survival rate was only approximately 20% when prolonged treatment was undertaken with 50 ng/ml TSA (TSA) alone for 24 h. In contrast, the cell survival rate was enhanced by two folds when treating with GA + TSA. Furthermore, DNA fragmentation assay revealed that fragmented DNA was produced 8 h after prolonged treatment with TSA alone. Within 16 h, the apoptotic percentages of TSA-treated cells were between 15-25%. In contrast, the other treatments did not exceed 6%. Furthermore, GA inhibited TSA-induced histone H4 hyperacetylation. Western blotting analysis further demonstrated that the HSP70 levels did not significantly increase in TSA-treated cells. However, the accumulated 70-kDa heat shock protein (HSP70) markedly increased up to 2 to 3 folds at 8 h in GA- and GA + TSA-treated cells, and the maximum amount up to 5 to 7 folds at 20 h. Conversely, HSP90 did not markedly increase in all treatments. Based on the results in this study, we suggest that apoptosis induced by TSA can be prevented by GA-induced increment of heat shock proteins, particularly HSP70.
    Life Sciences 04/2002; 70(15):1763-75. · 2.56 Impact Factor
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    ABSTRACT: We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90alpha. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [gamma-32P]ATP/Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [gamma-32P]ATP x Mg2+ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 alpha, and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK > kinase FA/GSK-3 alpha.
    Journal of Protein Chemistry 02/2002; 21(2):111-21.
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    ABSTRACT: Molecular mechanisms of whole-body thermotolerance (WBT) in mammals have not been investigated thoroughly. The purpose of this study was to assess the induction of the 70 kDa heat shock protein (HSP70) and antioxidant enzyme activity in animal WBT, which was induced by whole-body hyperthermia (WBH) in mice. As a preconditioning treatment, WBH was applied to mice to induce WBT. Synthesis of inducible HSP70 (HSP70i) and quantification of its increased level in liver were investigated by one- and two-dimensional polyacrylamide gel electrophoresis and immunoblotting. HSP70i synthesis in mice liver was induced by non-lethal WBH (41 degrees C, 30 min). When compared to control animals, the level of liver HSP70i increased substantially (by 3.6-fold; P<0.0001). When exposed to 30 min of hyperthermia preconditioning, and after recovery for 48 h, the survival rate was 88.2 %, which was significantly higher than that of the control group (37.5 %; P<0.01). Moreover, the survival rate of animals subjected to preconditioning for 15 min was 72.2 %, which was also significantly higher than that of the control group (P<0.05). In contrast, the survival rate of animals subjected to preconditioning for 45 min was 63.5 %, which was not different from the control group. Nonetheless, the protection index of the group subjected to 15 min and 30 min of preconditioning was 1.93 and 2.37, respectively. Furthermore, to assess their contributions to WBT, the activities of antioxidant enzymes were also measured. After 48 h of recovery in preconditioned animals, hepatic antioxidant enzyme activities, including superoxide dismutase, catalase and glutathione peroxidase, had not changed significantly. To study the molecular mechanism of WBT, we successfully developed a mouse model and suggest that, rather than the activities of antioxidant enzymes, it is HSP70i that has a role to help animals survive during severe heat stress.
    Journal of Experimental Biology 01/2002; 205(Pt 2):273-8. · 3.24 Impact Factor
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    ABSTRACT: We have isolated and sequenced cDNA clones encoding a 90-kDa heat shock protein (HSP90) from a porcine brain cDNA library. The sequence of the 2202-nucleotide coding region showed 88.6% homology with that of the human homologue. Moreover, the deduced amino acid sequence of the porcine hsp90 cDNA was 99.7% identical to that of the human counterpart, with a difference of only three amino acids in a total of 733 residues. Expression of the gene was greatly increased in cultured cells during recovery from heat shock treatment at 45°C for 60 min. Three major transcripts 2.2, 3.0, and 4.1 kb in size were detected by Northern blot hybridization. These transcripts were further identified in a whole-pig hyperthermia experiment. These three hsp90 transcripts were constitutively expressed in porcine tissues including kidney, liver, brain, and heart, and their levels were markedly enhanced during recovery from 30-min hyperthermia treatment at 43°C. Furthermore, we found that HSP90 was preferentially expressed in pituitary gland, brain, adrenal gland, and testis, in comparison to the other tissues.
    Gene 02/1999; · 2.20 Impact Factor