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J H Lim

Publications and source records attributed to J H Lim.

At least 37 records · Page 2Linked to original sources
PubMed2001

The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation.

Unfolding is an essential process during translocation of preproteins into mitochondria; however, controversy exists as to whether mitochondria play an active role in unfolding. We have established an in vitro system with a kinetic saturation of the mitochondrial import machinery, yielding translocation rates comparable to in vivo import rates. Preproteins with short N-terminal segments in front of a folded domain show a characteristic delay of the onset of translocation (lag phase) although the maximal import rate is similar to that of longer preproteins. The lag phase is shortened by extending the N-terminal segment to improve the accessibility to matrix heat shock protein 70 and abolished by unfolding of the preprotein. A mutant mtHsp70 defective in binding to the inner membrane prolongs the lag phase and reduces the translocation activity. A direct comparison of the rate of spontaneous unfolding in solution with that during translocation demonstrates that unfolding by mitochondria is significantly faster, proving an active unfolding process. We conclude that access of mtHsp70 to N-terminal preprotein segments is critical for active unfolding and initiation of translocation.

Animals↗
PubMed2001

Group IIA secretory phospholipase A(2) stimulates inducible nitric oxide synthase expression via ERK and NF-kappaB in macrophages.

The mammalian group IIA secretory phospholipase A(2) (sPLA(2)) is believed to play an important role in inflammation and cell injury. The present study underlines the importance of group IIA sPLA(2) in the regulation of iNOS. Treatment of cells with sPLA(2) induced protein expression and mRNA accumulation of iNOS in a dose-dependent manner. The pretreatment of cells with rho-BPB or SCA, selective sPLA(2) inhibitors, inhibited sPLA(2)-induced iNOS expression. sPLA(2) stimulated the simultaneous activation of two classes of mitogen-activated protein kinases ERK and JNK, but did not stimulate p38 MAPK. PD98059, a selective MEK inhibitor, inhibited sPLA(2)-induced nitrite production and iNOS expression as well as ERK phosphorylation. In addition, pretreatment of rho-BPB or SCA also resulted in inhibition of sPLA(2)-induced ERK phosphorylation. The sPLA(2) signaling mechanisms involving the activation of transcription factor NF-kappaB were studied in the same cells. That stimulation of cells with sPLA(2) caused NF-kappaB activation in a time-dependent manner was shown by the detection of NF-kappaB-specific DNA-protein binding and by IkappaBalpha degradation. sPLA(2)-induced NF-kappaB activation was prevented in the presence of rho-BPB. Furthermore, the NF-kappaB inhibitor PDTC suppressed sPLA(2)-induced nitrite production and iNOS expression as well as IkappaBalpha degradation. The results strongly suggest that group IIA sPLA(2) induces iNOS in macrophages and that this induction occurs through ERK and NF-kappaB.

Animals↗
PubMed2001

Molecular cloning and characterization of thermostable DNA ligase from Aquifex pyrophilus, a hyperthermophilic bacterium.

A DNA ligase gene from the hyperthermophilic bacterium Aquifex pyrophilus (Ap) was cloned and sequenced. An open reading frame of 2,157 bp that codes for a 82-kDa protein showed 40%-60% homology with a series of NAD+-dependent DNA ligases from different organisms. The recombinant enzyme Ap DNA ligase expressed in Escherichia coli was purified to homogeneity and characterized. The activity of Ap DNA ligase gradually increased in proportion to the concentration of monovalent salt up to 200 mM NaCl, 150 mM KCl, 200 mM NH4Cl, and 350 mM potassium glutamate. The optimum temperature and pH of Ap DNA ligase were greater than 65 degrees C and 8.0-8.6, respectively, for nick-closing activity. More than 75% of the ligation activity was retained after incubation at 95 degrees C for 60 min, whereas the half-lives of Thermus aquaticus and Escherichia coli DNA ligases at 95 degrees C were < or =15 min and 5 min, respectively. Thermostable Ap DNA ligase was applied to repeat expansion detection (RED) and could be a useful enzyme in DNA diagnostics.

Amino Acid Sequence↗
PubMed2001

Pregnancies resulting from in vitro matured oocytes collected from women with regular menstrual cycle.

PURPOSE: To demonstrate that human immature oocytes retrieved from women with regular menstrual cycles can undergo maturation and fertilization, and that the resulting embryos can establish pregnancies. METHODS: Immature oocytes (n = 568) were retrieved from women with regular menstrual cycle. The intact immature oocytes (n = 506) were allowed to mature in YS medium supplemented with 70% human follicular fluid (hFF); the matured oocytes were fertilized with husband sperm. Two pronuclei oocytes were cocultured with cumulus cells in YS medium supplemented with 10% hFF until 2 or 3 days after insemination. The cleaved embryos were transferred in uteri. RESULTS: Follicles were aspirated on Day 9.2 +/- 5.3 of 63 natural cycles from 51 patients (mean age = 34.8 +/- 4.0 years). The average number of retrieved immature oocytes was 9.0. The maturation rate was 74.3% (376/506). The two PN and cleavage rates were 72.6% (273/376) and 89.0% (243/273), respectively. Embryo transfer was achieved in 51 cycles and clinical pregnancy rate was 17.6% (9/51). CONCLUSIONS: The results suggest that in vitro matured oocytes can undergo fertilization and the resulting embryos may successfully lead to pregnancies. However, further research is needed to improve IVM technique to achieve success rate comparable to gonadotrophin stimulated cycles.

Adult↗
PubMed2001

Alternative embryo transfer on day 3 or day 5 for reducing the risk of multiple gestations.

PURPOSE: This study was carried out to reduce the possibility of high-order multiple gestations and the failure of embryo transfer by determining their replacement date based on the number and quality of 2-day embryos. METHODS: All zygotes were cocultured with cumulus cells in 10 microliters of YS medium containing 10% human follicular fluid (hFF) for 48 or 96 hr. In period I, all embryos were transferred on day 3 (1032 cycles). In period II, the embryos were transferred on either day 3 or day 5 by determining their replacement date based on the number and quality of 2-day embryos: there were 2701 patients in whom embryos were replaced on day 3 (in the case that the number of zygotes was less than eight and the number of good-quality embryos was less than three) and 1952 patients less than 40 years old in whom embryos were replaced on day 5 (in the case that the number of zygotes was eight or more and/or the number of good-quality embryos was three or more). On the other hand, patients who were 40 years old or more were alloted to day 3 transfer cycles, regardless of the number and quality of the 2-day embryos, due to the possibility of their not producing blastocyst-stage embryos in vitro. RESULTS: The number of embryos transferred in period II was 2.9 +/- 0.6, while that in period I was 3.7 +/- 0.5. The multiple pregnancy rate was significantly decreased in period II (30.7%) compared to that (49.6%) in period I, while the pregnancy and implantation rates in period II (36.1 and 16.4%, respectively) were not lower than those (34.9 and 16.1%, respectively) in period I. The rate of triplet or more gestations was significantly minimized in period II (2.3%) compared to that in period I (26.5%). CONCLUSIONS: We propose that determination of the date on which embryos should be transferred based on the number and quality of embryos on day 2 may help to maintain an acceptable pregnancy rate, while minimizing embryo transfer failure and high-order multiple gestations.

Adult↗
PubMed2001

Constitutive over-expression of AtGSK1 induces NaCl stress responses in the absence of NaCl stress and results in enhanced NaCl tolerance in Arabidopsis.

GSK3/shaggy-like protein kinases have been shown to play diverse roles in development and signal transduction pathways in various organisms. An Arabidopsis homologue of GSK3/shaggy-like kinase, AtGSK1, has been shown to be involved in NaCl stress responses. In order to further clarify the role of AtGSK1 in NaCl stress responses in plants, we generated transgenic Arabidopsis plants that over-expressed AtGSK1 mRNA. These plants showed enhanced resistance to NaCl stress when assayed either as whole plants or by measurement of root growth on NaCl plates. In addition, AtGSK1 transgenic plants in the absence of NaCl stress showed phenotypic changes, such as accumulation of anthocyanin, that were similar to those observed in wild-type plants under NaCl stress. Transgenic plants accumulated 30-50% more Na+ than did wild-type plants when subjected to NaCl stress, and Ca2+ content was increased by 15-30% in the transgenic plants regardless of the NaCl stress level. Northern blotting revealed that AtGSK1 over-expression induced expression of the NaCl stress-responsive genes AtCP1, RD29A and CHS1 in the absence of NaCl stress. In addition, AtCBL1 and AtCP1 were super-induced in the NaCl-stressed transgenic plants. Taken together, these results suggest that AtGSK1 is involved in the signal transduction pathway(s) of NaCl stress responses in Arabidopsis.

Adaptation, Physiological↗
PubMed2001

The clinical significance of common bile-duct dilatation in patients without biliary symptoms or causative lesions on ultrasonography.

BACKGROUND AND STUDY AIMS: Although abdominal ultrasonography (US) is a good initial screening method for detection of biliary tract disease, we sometimes encounter patients who only have findings of dilatation of the common bile duct (CBD) on US, without specific biliary symptoms or jaundice. This study aimed to evaluate the causes and clinical significance of dilatation of the CBD in patients without biliary symptoms, jaundice, or causative lesions at US. PATIENTS AND METHODS: A total of 77 patients who had no biliary symptoms and whose internal CBD diameter was more than 7 mm, without definite causative lesions on US, were enrolled. Of these, 49 underwent endoscopic retrograde cholangiopancreatography (ERCP) and 28 underwent follow-up US or computed tomography (CT) instead of ERCP. We excluded patients whose bilirubin level had increased beyond the upper normal level or who had previous history of upper abdominal surgery including cholecystectomy. RESULTS: The ERCP findings were as follows: no lesion in 20 patients (40.8%), juxtapapillary duodenal diverticulum (JDD) in 11 (22.5%), benign stricture in ten (20.4%), distal CBD mass in two (4.1%), choledochal cyst in two (4.1%), anomalous union of the pancreaticobiliary duct (AUPBD) in two (4.1%), and choledochal cyst with AUPBD in two (4.1%). There were no differences in age or in alkaline phosphatase or gamma-glutamyl transpeptidase (GGT) levels between the patients who had causative lesions revealed at ERCP and those who did not. Among the 28 patients who did not undergo ERCP, 12 had returned to normal and eight had no change in CBD diameter on follow-up US. Among eight patients who underwent CT, there were four with normal findings, one with JDD, and three with suspected choledochal cysts. CONCLUSIONS: We detected a significant number of causative biliary tract lesions in asymptomatic adults with dilatation of the CBD on routine abdominal US; no laboratory or demographic parameters were useful for discrimination. Further diagnostic study will be helpful for the early diagnosis of biliary tract disease in such patients.

Adult↗
PubMed2001

The analysis of resectability and survival in pancreatic cancer patients with vascular invasion.

One of the major limitations of curative resection in patients with pancreatic cancer is local tumor extension to the mesenteric vessels. Thus, the purposes of our study were to assess the clinical value of contrast-enhanced spiral computed tomography (CT) in predicting the resectability and survival of patients with pancreatic cancer with suspicious vascular invasion and to assess the influence of curative resection on the survival of these patients. We enrolled 40 patients with pancreatic cancer who were suspected of having an involvement of the adjacent large vessels and who subsequently underwent operation with curative intent in the study. Resectability and survival were correlated with CT findings such as segment length, degree of encasement, and type and number of vessels involved. The survival rate was compared between the curative and palliative resection groups, and survival rate was compared between the resected and unresected groups. Of the 40 patients with adenocarcinoma of the pancreas, 14 had curative resections and 26 had palliative resections. The probability of curative resection was higher in patients with segment lengths less than 2 cm, as compared with segment lengths more than 2 cm. However, there was no difference in survival between the two groups. There were no differences in resectability and survival according to the degree of encasement and type and number of vessels involved. There was no difference in survival between the curative and palliative resection groups. There was no difference in survival between the resected and unresected groups. A survival benefit was not achieved by curative resection in patients with pancreatic cancer with vascular invasion. Therefore, it would be better to avoid aggressive surgery in patients with pancreatic cancer with vascular invasion.

Humans↗
PubMed2001

Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors.

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

Fungal Proteins↗
PubMed2001

Radiologic spectrum of cholangiocarcinoma: emphasis on unusual manifestations and differential diagnoses.

Most cholangiocarcinomas are ductal adenocarcinomas that arise from both intra- and extrahepatic bile duct epithelium, and their typical growth pattern can be classified as exophytic, infiltrative, polypoid, or a combination of these. Those of unusual histologic type (eg, mucin-hypersecreting cholangiocarcinoma, squamous adenocarcinoma, biliary cystadenocarcinoma, and mucinous carcinoma) show a growth pattern different from that of the typical ones (ie, ductal). Cholangiocarcinomas frequently develop in patients with any of a variety of preexisting bile duct diseases, some of which are considered precursors of cholangiocarcinoma (eg, biliary lithiasis, clonorchiasis, recurrent pyogenic cholangitis, and primary sclerosing cholangitis). Some bulky hepatic tumors of either primary or secondary origin mimic exophytic peripheral cholangiocarcinoma. Some variants of hepatocellular carcinoma, such as sclerosing, fibrolamellar, and cholangiohepatocellular carcinoma, resemble exophytic peripheral cholangiocarcinoma, while that with intraductal growth resembles polypoid cholangiocarcinoma. Among benign bile duct diseases, tumorous conditions (eg, benign biliary tumors) may mimic polypoid cholangiocarcinoma, whereas benign stricture of various causes (eg, cholangitides, traumatic and postsurgical sequelae, chronic pancreatitis, papillary stenosis) usually mimics infiltrative cholangiocarcinoma.

Bile Duct Neoplasms↗
PubMed2001

Conspicuity of hepatocellular nodular lesions in cirrhotic livers at ferumoxides-enhanced MR imaging: importance of Kupffer cell number.

PURPOSE: To correlate the conspicuity of hepatocellular carcinomas and dysplastic nodules on ferumoxides-enhanced magnetic resonance (MR) images with the number of Kupffer cells in the hepatic lesions, as compared with that in background liver in histopathologic findings. MATERIALS AND METHODS: Sixty-nine histopathologically proved moderately or poorly differentiated hepatocellular carcinomas, 10 well-differentiated hepatocellular carcinomas, and 19 dysplastic nodules were retrospectively studied in 68 patients with cirrhosis who underwent ferumoxides-enhanced MR imaging. The contrast-to-noise ratio between the nodules and surrounding parenchyma was calculated at T2-weighted fast spin-echo imaging, and the difference in the number of Kupffer cells between the nodules and surrounding hepatic tissue was calculated histopathologically. The results of MR imaging and histopathologic examination were correlated. RESULTS: All 69 moderately or poorly differentiated hepatocellular carcinomas had high contrast-to-noise ratios at MR imaging and large differences in the number of Kupffer cells. Six of the 10 well-differentiated hepatocellular carcinomas had contrast-to-noise ratios of zero or nearly zero, and five of these had little difference in the number of Kupffer cells. All 19 dysplastic nodules had contrast-to-noise ratios of zero or nearly zero, and there were virtually no differences in the number of Kupffer cells. CONCLUSION: Hepatocellular nodule conspicuity at ferumoxides-enhanced MR imaging depends on differences in the number of Kupffer cells within a nodule and the surrounding cirrhotic liver; moderately or poorly differentiated hepatocellular carcinomas can be distinguished from well-differentiated hepatocellular carcinomas and dysplastic nodules.

Adult↗
PubMed2001

Hepatocellular carcinoma treated with percutaneous radio-frequency ablation: evaluation with follow-up multiphase helical CT.

PURPOSE: To determine serial changes in hepatocellular carcinomas (HCCs) treated with percutaneous radio-frequency (RF) ablation at long-term follow-up multiphase helical computed tomography (CT). MATERIALS AND METHODS: There were 43 nodular HCCs in 40 patients at follow-up CT performed not less than 12 months after RF ablation. All patients underwent follow-up multiphase helical CT immediately, 1 month, and then every 3 months after percutaneous RF ablation. The serial changes in attenuation, enhancement pattern, shape, other findings, and volume of the ablated lesions were analyzed at follow-up CT. RESULTS: Thirty-eight (88%) of 43 ablated lesions were of low attenuation, with absence of contrast material enhancement at immediate and 1-month follow-up CT, which is suggestive of successful treatment. The remaining five lesions (12%) showed peripheral nodular enhancement, suggesting residual viable tumor. Compared with volume changes at immediate follow-up CT, the mean percentages of volume change at 1, 4, 10, 16, and 19 months were 79%, 50%, 27%, 11%, and 6%, respectively. Of 43 ablated lesions, 24 (56%) were mostly round at immediate CT and remained unchanged at subsequent follow-up CT. Peripheral rim enhancement was seen in 34 (79%) of 43 lesions at immediate CT but resolved in all 34 lesions at 1-month follow-up CT. Other associated findings included iatrogenic arteriovenous shunt in 10 patients, perihepatic hemorrhage in three, and pneumothorax in one. CONCLUSION: Follow-up multiphase helical CT of HCCs treated with percutaneous RF ablation showed variable findings in the treated lesions and surrounding liver parenchyma.

Adult↗
PubMed2001

Detection of hepatocellular carcinomas and dysplastic nodules in cirrhotic liver: accuracy of ultrasonography in transplant patients.

The purpose of this study was to assess the usefulness of ultrasonography in the detection of hepatocellular carcinomas and dysplastic nodules in patients with liver cirrhosis. Pretransplantation sonograms in 52 patients with liver cirrhosis who underwent orthotopic liver transplantation were evaluated retrospectively. The numbers of hepatocellular carcinomas and dysplastic nodules were assessed in the explanted liver specimens and compared with pretransplantation ultrasonographic results. Eighteen hepatocellular carcinomas in 16 patients and 20 dysplastic nodules in 11 patients were present in the explanted livers. The size of hepatocellular carcinomas ranged from 0.6 to 5.0 cm (mean, 2.1 cm) in diameter, and that of dysplastic nodules ranged from 0.5 to 1.7 cm (mean, 1.0 cm) in diameter. Pretransplantation ultrasonography enabled detection of 6 of 18 hepatocellular carcinoma and 0 of 20 dysplastic nodule lesions; lesion detection sensitivity for hepatocellular carcinomas and dysplastic nodules was 33% and 0%, respectively. Patient sensitivity and specificity for hepatocellular carcinomas were 38% (6 of 16) and 92% (33 of 36), and those for dysplastic nodules were 0% and 95% (39 of 41), respectively. On the basis of our results, ultrasonography is insensitive for detection of hepatocellular carcinomas and dysplastic nodules in patients with advanced liver cirrhosis.

Adult↗
PubMed2001

Differential regulation of Dlx gene expression by a BMP morphogenetic gradient.

Three members of the vertebrate Distal-less gene family, Dlx3, 5 and 6, are transcribed in early gastrula embryos of Xenopus laevis. This expression is confined to ectoderm and is excluded from the presumptive neural plate region. Expression of all three genes is dependent upon BMP signaling, with significant differences in how the three genes respond to the BMP antagonist chordin. This correlates with the different expression domain boundaries in vivo for Dlx3 compared to Dlx5 and 6, suggesting that BMP signal attenuation could be the primary factor in determining these different patterns in the gastrula ectoderm.

Animals↗
PubMed2000

Acetaminophen inhibits iNOS gene expression in RAW 264.7 macrophages: differential regulation of NF-kappaB by acetaminophen and salicylates.

Acetaminophen is a widely used analgesic and anti-inflammatory drug that is considered a good alternative to salicylates for individuals who cannot tolerate salicylates. Nitric oxide (NO) synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation. Recent evidence suggests that anti-inflammatory effect of salicylates lies in the inhibition of iNOS, but nothing has been reported about the direct effect of iNOS expression by acetaminophen. The present study was designed to elucidate sequentially the action mechanisms of acetaminophen and salicylates (aspirin and sodium salicylate) on lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)-induced iNOS expression in RAW 264.7 macrophages. Both acetaminophen and salicylates inhibited NO production and iNOS protein expression in a dose dependent manner. Acetaminophen inhibited iNOS mRNA expression, promoter activity of iNOS gene and nuclear factor-kappa B (NF-kappaB) binding activity induced by LPS plus IFN-gamma, whereas salicylates did not show any effect on them. In addition, salicylates did not affect on iNOS mRNA stability induced by LPS plus IFN-gamma. Furthermore, the inhibition of iNOS protein expression and NO production by salicylates was disappeared when salicylates were added for only 5 h to inhibit the early event of iNOS expression. Aspirin also dose dependently inhibited iNOS enzyme activity in cell-free extracts, whereas no significant differences were observed in extracts treated with sodium salicylate or acetaminophen. These findings suggest that acetaminophen may exert analgesic or anti-inflammatory effect by inhibiting iNOS expression induced by LPS plus IFN-gamma at transcriptional level by suppression of NF-kappaB binding activity, whereas salicylates exert its effect by inhibiting iNOS expression at the translational or posttranslational level.

Acetaminophen↗
PubMed2000

Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation.

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.

Animals↗
PubMed2000

Cyclophilin-promoted folding of mouse dihydrofolate reductase does not include the slow conversion of the late-folding intermediate to the active enzyme.

Cyclophilins accelerate slow protein folding reactions in vitro by catalyzing the cis/trans isomerization of peptidyl-prolyl bonds. Cyclophilins were reported to be involved in a variety of cellular functions, including the promotion of protein folding by use of the substrate mouse dihydrofolate reductase (DHFR). The interaction of cyclophilin with DHFR has only been studied under limited conditions so far, not taking into account that native DHFR exists in equilibrium with a non-native late-folding intermediate. Here we report a systematic analysis of catalysis of DHFR folding by cyclophilins. The specific ligand methotrexate traps DHFR in its native state, permitting a specific analysis of the action of cyclophilin on both denatured DHFR with non-native prolyl bonds and denatured DHFR with all-native prolyl bonds. Cyclophilins from yeast and Neurospora crassa as well as the related prolyl isomerase b from Escherichia coli promote the folding of different forms of DHFR to the enzymatically active form, demonstrating the generality of cyclophilin-catalyzed folding of DHFR. The slow equilibrium between the late-folding intermediate and native DHFR suggests that prolyl isomerization may be required for this final phase of conversion to native DHFR. However, by reversible trapping of the intermediate, we analyze the slow interconversion between native and late-folding conformations in the backward and forward reactions and show a complete independence of cyclophilin. We conclude that cyclophilin catalyzes folding of DHFR, but surprisingly not in the last slow folding step.

Animals↗