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Biomedical subjects

F Martin

Publications and source records attributed to F Martin.

At least 73 records · Page 4Linked to original sources
PubMed2001

Population-based study of hospital trauma care in a rural state without a formal trauma system.

OBJECTIVE: Formalized systems of trauma care are believed to improve outcomes in an urban setting, but little is known of the applicability in a rural setting. METHODS: We conducted a population-based analysis of hospital survival after trauma comparing an American College of Surgeons-verified Level I trauma center (TC) with the pooled results of 13 small community hospitals (CH) in a rural state with no formal trauma system. All patients admitted to any hospital within the state of Vermont over a 5-year period (1995-1999) with a trauma discharge diagnosis were included. Elderly patients with isolated femur fractures were excluded from the database. International Classification of Diseases Injury Severity Scores (ICISSs) were calculated for each patient and used to control for injury severity in an omnibus logistic regression model that included age, ICISS, and hospital type (TC vs. CH) as predictors of survival. Patients who died were characterized on the basis of ICISS into "expected" (ICISS < 0.25), "indeterminate" (ICISS = 0.26-0.50), and "unexpected" (ICISS > 0.5). RESULTS: In 16,354 trauma admissions over the 5-year period in the rural state of Vermont, 370 (2.2%) died. There were 5,964 (36%) admitted to TC. Patients admitted to TC were more injured (ICISS 0.94 vs. 0.96) and had a higher mortality (3.1% vs. 1.8). Overall, care at the CH provided an improved survival (odds ratio = 1.75, 95% confidence internal = 1.31-2.18, p = 0.000). However, in the more severely injured cohort of trauma patients (expected and indeterminate; n = 133), overall survival was higher in the TC (16% CH vs. 38% TC, p = 0.02, chi2). Because the TC was known to provide care equivalent to Major Trauma Outcome Study norms during this time period (Z = -0.03, M = 0.894), we believe this study confirms that trauma care throughout the state is in accordance with national norms. CONCLUSION: In a rural state, without a statewide formal trauma system, survival after trauma is no worse at CH than TC when corrected for injury severity and age. Future expenditures of resources might better be concentrated in other areas such as discovery or prehospital care to further improve outcomes.

Adult↗
PubMed2001

A population-based study of geriatric trauma in a rural state.

BACKGROUND: Urban geriatric trauma patients are known to die more often than their younger counterparts. Little is known of the fate of geriatric trauma patients in a rural environment where delays to definitive treatment are frequent. We hypothesized that rural trauma patients would do worse than their urban counterparts because of prolonged delays to definitive care. METHODS: Five-year retrospective analysis of all trauma deaths occurring within a rural state and retrospective outcome analysis of trauma patients admitted to a tertiary care facility who were less than 55 years old (defined as young) and 55 or more years old (defined as old). Outcome analysis was performed comparing old and young rural hospitalized patients to the Major Trauma Outcome Study data set collected in major urban trauma centers. RESULTS: Of the total trauma deaths in the state, 32.5% were old. Old patients were less likely to die at the scene of the injury than were their younger counterparts (R2 = 0.84, p < 0.001). Hospitalized old patients had a significantly higher mean Revised Trauma Score and a significantly lower Injury Severity Score, a higher complication rate, and a higher mortality rate than did hospitalized young patients. The young group had a significantly better survival (W = 0.59, Z = -3.49, p = 0.0001) than the MTOS data set, but the old group had a significantly worse survival (W = -1.8, Z = -3.49, p = 0.001). CONCLUSION: In a rural environment, old trauma patients die more commonly in the hospital than their younger counterparts, who die more commonly at the scene. Old trauma patients who die in the hospital were less severely injured than their younger counterparts who died in the hospital. Old patients admitted to this rural trauma center have a significantly worse survival than their urban counterparts despite the fact that young rural trauma patients do significantly better than their urban counterparts. Understanding the demographics of rural geriatric trauma may be useful in allocating resources in rural trauma system design. It must be understood that despite relatively low injury severity and physiologic stability, there is a significant potential for rural geriatric trauma patients to do poorly.

Adolescent↗
PubMed2001

Salivary scintigraphy for assessing the protective effect of pilocarpine in head and neck irradiated tumours.

Patients with head and neck cancers can develop salivary hypofunction after radiotherapy. The use of pilocarpine during radiotherapy treatment has been shown to be an effective treatment, although its usefulness is being discussed. The aim of this study was: (1) to determine the value of a semiquantitative scintigraphy method for measuring the uptake and excretory salivary function of patients with head and neck irradiated tumours; and (2) to study the usefulness of pilocarpine as a salivary gland protector during radiotherapy. We prospectively studied 49 patients (mean age 61 years, range 29-87 years) with head and neck cancer in need of radiotherapy. Patients were divided into two groups consecutively: group P (26 patients) received 5 mg of pilocarpine three times per day starting the day before radiation therapy, and group NP (23 patients) received radiotherapy without pilocarpine and were used as the control group. Salivary gland scintigraphy and a visual analogue scale (VAS) of mouth dryness were obtained from each patient before radiotherapy and during the first year after treatment. The most frequent finding after radiotherapy was a quick impairment in parotid and submaxillary excretion (P < 0.001). There were no statistical differences comparing the pilocarpine group against the non-pilocarpine group. Parotid and submaxillary uptake significantly decreased after radiotherapy in both groups (P < 0.001). However, a tendency to recover within the pilocarpine group was observed in both the parotids and the submaxillary glands at 12 months. No differences were found comparing the VAS results in both groups. Strikingly, VAS data did not correlate with salivary gland dysfunction observed by means of scintigraphy. In conclusion, salivary scintigraphy is a useful technique to evaluate objectively the salivary gland function of patients with head and neck irradiated tumours as well as to test the response to pilocarpine. However, despite better results on the salivary uptake at 12 months, pilocarpine did not significantly improve salivary gland function.

Adult↗
PubMed2001

Postentry restriction to human immunodeficiency virus-based vector transduction in human monocytes.

Cells of the monocyte lineage can be infected with human immunodeficiency virus type 1 (HIV-1) both during clinical infection and in vitro. The ability of HIV-1-based vectors to transduce human monocytes, monocyte-derived macrophages, and dendritic cells (DCs) was therefore examined, in order to develop an efficient protocol for antigen gene delivery to human antigen-presenting cells. Freshly isolated monocytes were refractory to HIV-1-based vector transduction but became transducible after in vitro differentiation to mature macrophages. This maturation-dependent transduction was independent of the HIV-1 accessory proteins Vif, Vpr, Vpu, and Nef in the packaging cells and of the central polypurine tract in the vector, and it was also observed with a vesicular stomatitis virus-pseudotyped HIV-1 provirus, defective only in envelope and Nef. The level and extent of reverse transcription of the HIV-1-based vector was similar after infection of immature monocytes and of mature macrophages. However, 2LTR vector circles could not be detected in monocytes, suggesting a block to vector nuclear entry in these cells. Transduction of freshly isolated monocytes exposed to HIV-1-based vector could be rescued by subsequent differentiation into DCs. This rescue was induced by fetal calf serum in the DC culture medium, which promoted vector nuclear entry.

Antigen-Presenting Cells↗
PubMed2001

Anal intraepithelial neoplasia in HIV positive people.

OBJECTIVE: To review the current literature on HIV associated anal intraepithelial neoplasia (AIN). METHODS: A comprehensive Medline/Pubmed search was performed for the years 1980-2001 (January) for articles pertaining to HIV associated anal intraepithelial neoplasia. From the MeSH terms "anal intraepithelial neoplasia" and "anal cancer" the following subheadings were used: HIV, homosexual men, HPV, Epidemiology, Etiology, Mortality, Diagnosis, Screening, Drug Therapy, Surgical Therapy, Radio Therapy, Risk factors, ASIL. The search was limited to "human" for all searches. In the absence of enough "randomised controlled trials" the search was extended to clinical trials, reviews, and case reports. One analysis on cost effectiveness and two abstracts presented at 12th World AIDS Conference and 6th Conference on Retrovirus and Opportunistic Infections were included. The 44 publications referred to originate from the United Kingdom (9), the United States (26), and Denmark (5), with one each from Switzerland, Germany, Australia, and France. The Cochrane Database of systematic reviews yielded 11 complete reviews for "anal cancer" and none for "anal intraepithelial neoplasia." The textbook of AIDS-related cancers and their treatment was consulted. We also included our personal experience from the treatment of patients at the Chelsea and Westminster Hospital, one of the largest centres for the management of HIV disease in Europe. CONCLUSION: Routine anal cytological screening followed by appropriate management of AIN is an important issue for HIV infected patients. The natural history of AIN has not been fully established and this prevents clinicians from defining clear management protocols. There is early evidence that the benefits of highly active antiretroviral therapy (HAART) in terms of restoring immune function and reducing opportunistic infections and some neoplasms may not extend to regression of AIN. Under these circumstances it might be predicted that AIN and subsequent progression to invasive anal cancer would rise as HAART prolongs the lives of seropositive people. However, routine anal cytological screening will surely have to await an effective proved intervention for AIN and this would seem to be a pressing clinical goal.

Antiretroviral Therapy, Highly Active↗
PubMed2001

Chronic epilepsy with complex partial seizures is not always medically intractable--a long-term observational study.

OBJECTIVE: To study the prognosis of patients with complex partial seizures (CPS) with or without simple partial (SPS) and secondarily generalized tonic-clonic seizures (GTCS) and to analyze the factors related to the degree of medical responsiveness. MATERIAL AND METHODS: A total of 266 adult patients with CPS were included in a hospital based observational survey with a follow-up of 2 to 25 years. Clinical characteristics, seizure frequency, electroencephalography (EEG), cerebral computed tomography (CCT) and magnetic resonance imaging (MRI) findings were analyzed. Patients were categorized according to their degree of medical responsiveness into one of three groups: seizure free, improved control (>50% seizure reduction) and poor control. RESULTS: Mean age at follow-up was 44.7 years (SD 14.7, range 19-93). Mean age at seizure onset was 18.1 years (SD 14.7, median 15, range 1-79). Complete seizure control was achieved in 40%, improved seizure control in 36% and poor seizure control in 24%. Patients entered remission after a mean period of 15.7 years (SD 12.6, median 13, range 1-54) of active epilepsy. A third of all seizure-free patients were still in remission 6.1 years (SD 5.3, median 3.5, range 1-18) after discontinuation of antiepileptic drugs (AED). Patients with poor seizure control had a significantly younger age at onset (P<0.01), a higher initial seizure frequency (more than 3 per month) (P<0.01), abnormal neurological examination (P<0.01), and were more often mentally handicapped (P<0.01). Multiple logistic regression analysis revealed a high initial seizure frequency, mental handicap and an abnormal neurological examination as independent risk factors for poor seizure control. A positive family history, a history of febrile convulsions and/or psychosis, an abnormal EEG or MRI was not predictive of poor outcome. CONCLUSIONS: Not all patients with CPS were medically intractable. Seizure remission can be achieved after a long time of active epilepsy. Poor seizure control was associated with a high initial seizure frequency, mental handicap and abnormal neurological examination.

Adult↗
PubMed2001

Childhood cataracts.

OBJECTIVE: The aim of this study was to determine the surgical outcome of childhood cataracts. METHOD: Between 1990 and 1997, 137 patients less than 10 years of age (181 eyes) underwent cataract surgery at the Royal Alexandra Hospital for Children/New Children's Hospital. Data were collected retrospectively from medical records and supplemented with data from referrng ophthalmologists. RESULTS: In the majority of patients (53.0%), the cause of cataract was unknown. Sixty-two patients (45.3%) had bilateral and 75 patents (54.7%) had unilateral cataracts. In 84 patients (107 eyes: 59.1%), the onset of visually significant cataract was estimated to be before the age of 2 years. Reliable postoperatve visual acuity (VA) was obtained in 150 eyes from 116 patients. The mean LogMAR VA was 0.71 +/- 0.55 in bilateral cataracts and 1.17 +/- 0.68 in unilateral cataracts. Visual acuity was poor (LogMARVA > 1.0) in 71.8% of unilateral cataracts (compared to 27.9% in bilateral cataracts, P < 0.0001), and in 64.9% when onset was ess than 2 years (compared to 22.4% in later onset cataract, P < 0.0001). Posterior capsular opacity requiring laser or surgical removal was noted in 40 patients (44 eyes; 24.4%). Other complications included raised intraocular pressure in six patients (seven eyes: 3.7%), displaced intraocular lens or iris capture in 11 patients (12 eyes; 6.6%), ectopic pupil in five patients (five eyes; 2.7%), and severe inflammation in eight patients (12 eyes; 6.6%). There was cosmetically significant esotropia or exotropia in 24 patients (23 eyes; 12.7%). CONCLUSION: The prognosis for vision was significantly poorer in unilateral and earlier onset cataract. The importance of early diagnosis, prompt treatment and vigilant postoperative follow up in this subgroup cannot be overemphasized.

Cataract↗
PubMed2001

Inactivation of plasminogen activator inhibitor-1 accelerates thrombolysis of a platelet-rich thrombus in rat mesenteric arterioles.

To investigate the role of active plasminogen activator inhibitor 1 (PAI-1) in the evolution of a microthrombus generated in the arteriolar microcirculation, the monoclonal antibody, 33H1F7, which transforms active PAI-1 to a tissue type plasminogen activator (t-PA) substrate, was evaluated in an arteriolar thrombosis model in the rat mesentery. Arterioles (200-300 um) were stimulated electrically to create an endothelial lesion; ADP was then perfused for 2 min to induce the formation of a platelet-rich thrombus which lysed spontaneously in 140 +/- 24 s. Two successive ADP superfusions produced comparable thrombi which lysed in comparable times. Different doses of 33H1F7 were infused to rats for 30 min and the dose which inactivates rapidly and totally active rat PAI-1 (300 microg/kg/min) was selected to be tested on the thrombosis model. Infusion of 33H I F7 beginning 10 min before the ADP application significantly reduced the lysis time in comparison to the control (123 +/- 30 s versus 169 +/- 33 s, P < 0.05, paired Student's t-test) and the cumulative thrombus area during the lysis period was decreased by 56 +/- 7%. These results demonstrate that inactivation of PAI-1 is able to accelerate lysis of a platelet-rich clot in a mesenteric arteriole of the rat. Thus active PAI-1 most likely participates to the resistance to thrombolysis in the arteriolar microcirculation and its inactivation may shorten ischemic periods after microvascular obstruction such as e.g. during cerebral stroke.

Animals↗
PubMed2000

Kinase pathways in dominant and subordinate ovarian follicles during the first wave of follicular development in sheep.

The mechanism by which one or more dominant ovarian follicles continue development while other subordinate follicles regress is not known. The mitogen activated protein kinases (MAPKs) are a group of kinases that are activated by hormonal factors and form a cascade of processes that regulate cell growth, division and differentiation. The aim of the present experiment was to characterise the presence of the MAPKs, Erk 1/Erk 2 and Akt in healthy dominant follicles and regressing subordinate follicles. Following in vivo monitoring of ovarian follicle development, three ewes were ovariectomised and the follicular fluid and follicle wall (theca and granulosa cells) saved from the dominant and largest subordinate follicle. The dissected diameter and follicular fluid oestradiol concentration of the dominant follicle was larger (P<0.01) than the largest subordinate follicle (6.5+/-0.0mm and 41.3+/-4.9ng/ml versus 4.7+/-0.3mm and 0.6+/-0.4ng/ml). Western blot analyses showed that there was more Akt (202.7+/-6.4 versus 59.6+/-32.7 units; P<0.05) and Erk 1/Erk 2 (104.5+/-10.6 versus 0.3+/-0.2 units; P<0.01) present in follicle wall samples from the dominant compared to the largest subordinate follicles. Phosphorylated forms of Akt and Erk 1/Erk 2 were detected in samples from dominant but not subordinate follicles. We suggest that signal transduction pathways involving Akt and Erk 1/Erk 2 may play an important role in determining the outcome of ovarian follicle growth and development in sheep.

Animals↗
PubMed2000

Characterization and optimization of a real-time, parallel, label-free, polypyrrole-based DNA sensor by surface plasmon resonance imaging.

We describe in this paper a methodology to quantify multispot parallel DNA hybridizations and denaturations on gold surfaces by using, on one hand, a polypyrrole-based surface functionalization based on an electrospotting process and, on the other hand, surface plasmon resonance imaging allowing real-time measurements on several DNA spots at a time. Two characterization steps were performed in order to optimize the immobilization of oligonucleotide probes and, thus, to increase the signal-to-noise ratio of monitored hybridization signals: the first step consisted of characterizing the signal dependence upon the density of immobilized 15-mer probes, and, the second step, in analyzing the hybridization response versus spot thickness. We further demonstrated that a surface density of polypyrrole/DNA probes of approximately 130 fmol/ mm2 (590 pg/mm2) optimizes the hybridization signal that can be detected directly. Optimal thickness of the spot was found to be close to 11 nm. Specificity and regeneration steps on each spot have also been demonstrated successfully, showing this method to be very competitive and convenient in use.

Base Sequence↗
PubMed2000

Parkinson's disease-associated alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs.

Parkinson's disease (PD) is a neurodegenerative disorder that is pathologically characterized by the presence of intracytoplasmic Lewy bodies. Recently, two point mutations in alpha-synuclein were found to be associated with familial PD, but as of yet no mutations have been described in the homologous genes beta- and gamma-synuclein. alpha-Synuclein forms the major fibrillar component of Lewy bodies, but these do not stain for beta- or gamma-synuclein. This result is very surprising, given the extent of sequence conservation and the high similarity in expression and subcellular localization, in particular between alpha- and beta-synuclein. Here we compare in vitro fibrillogenesis of all three purified synucleins. We show that fresh solutions of alpha-, beta-, and gamma- synuclein show the same natively unfolded structure. While over time alpha-synuclein forms the previously described fibrils, no fibrils could be detected for beta- and gamma-synuclein under the same conditions. Most importantly, beta- and gamma-synuclein could not be cross-seeded with alpha-synuclein fibrils. However, under conditions that drastically accelerate aggregation, gamma-synuclein can form fibrils with a lag phase roughly three times longer than alpha-synuclein. These results indicate that beta- and gamma-synuclein are intrinsically less fibrillogenic than alpha-synuclein and cannot form mixed fibrils with alpha-synuclein, which may explain why they do not appear in the pathological hallmarks of PD, although they are closely related to alpha-synuclein and are also abundant in brain.

Amino Acid Sequence↗
PubMed2000

Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice.

Pantetheinase (EC 3.5.1.-) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti-oxidant. We show that the Vanin-1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin-1 transfectants and is immunodepleted by specific antibodies; (2) Vanin-1 is a GPI-anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin-1 null mice are deficient in membrane-bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane-bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin-1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

Amidohydrolases↗
PubMed2000

Flt3 ligand lessens the growth of tumors obtained after colon cancer cell injection in rats but does not restore tumor-suppressed dendritic cell function.

A defective function of the antigen-presenting cells may represent one of the ways used by cancer cells to escape the immune response. We have previously shown that human and rat colon carcinomas were infiltrated by dendritic cells that did not express the B7 co-stimulatory molecules required for inducing an efficient T-cell response. Flt3 ligand is a cloned hematopoietic growth factor that markedly augments the number of functional dendritic and NK cells in lymphoid and non-lymphoid tissues and exerts anti-tumor activity in various experimental models. We show here that repeated Flt3 ligand administration delays the s.c. growth of rat colon cancer cells in syngeneic animals without inducing tumor regression. In tumor-bearing animals, Flt3 ligand has a limited stimulatory effect on the antigen-presenting capacity of intra-tumoral and splenic dendritic cells, without restoring the high functional level of dendritic cells from tumor-free animals. Moreover, Flt3 ligand-mediated activation of NK cell cytotoxicity decreases when the tumor mass increases. Our results indicate that Flt3 ligand treatment of tumor-bearing animals does not sufficiently overcome tumor-induced immunosuppression to restore the inhibited functions of dendritic and NK cells and allow complete tumor regression.

Animals↗
PubMed2000

Cutting edge: the tumor counterattack hypothesis revisited: colon cancer cells do not induce T cell apoptosis via the Fas (CD95, APO-1) pathway.

The counterattack hypothesis, suggesting that cancer cells express Fas ligand (FasL) and are able to kill Fas-expressing tumor-infiltrating activated T cells, was supported by reports of the killing of Jurkat cells by FasL-expressing human colon cancer cell lines. Through the use of an improved cytotoxic assay in which soluble FasL and FasL-transfected KFL9 cells were used as positive controls, we show that none of seven human colon cancer cell lines induce apoptosis of two Fas-expressing target cell lines, Jurkat and L1210-Fas cells. Moreover, in coculture experiments, cancer cell monolayers do not inhibit the growth of Fas-expressing lymphoid cells. Although FasL mRNA and protein were detected in the extracts of the colon cancer cell lines, flow cytometry and confocal microscopy failed to detect the protein on the surface of tumor cells. These results suggest that the counterattack of tumor-infiltrating T lymphocytes by cancer cells may not account for immune tolerance toward tumor cells.

Animals↗
PubMed2000

IHG-2, a mesangial cell gene induced by high glucose, is human gremlin. Regulation by extracellular glucose concentration, cyclic mechanical strain, and transforming growth factor-beta1.

We used cloning in silico coupled with polymerase chain reaction to demonstrate that IHG-2 is part of the 3'-untranslated region of gremlin, a member of the DAN family of secreted proteins that antagonize the bioactivities of members of the transforming growth factor (TGF)-beta superfamily. Mesangial cell gremlin mRNA levels were induced by high glucose, cyclic mechanical strain, and TGF-beta1 in vitro, and gremlin mRNA levels were elevated in the renal cortex of rats with streptozotocin-induced diabetic nephropathy in vivo. gremlin expression was observed in parallel with induction of bone morphogenetic protein-2 (BMP-2), a target for gremlin in models of cell differentiation. Together these data indicate that (a) IHG-2 is gremlin, (b) gremlin is expressed in diabetic nephropathy in vivo, (c) both glycemic and mechanical strain stimulate mesangial cell gremlin expression in vitro, (d) high glucose induces gremlin, in part, through TGFbeta-mediated pathways, and (e) Gremlin is a potential endogenous antagonist of BMPs within a diabetic glomerular milieu.

3' Untranslated Regions↗
PubMed2000

Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system.

We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.

Amino Acid Sequence↗
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↗