Association of the ADIPOQ Rs2241766 and Rs266729 Polymorphisms with Metabolic Syndrome in the Chinese Population: A Meta-analysis.

OBJECTIVE: This meta-analysis was performed to summarize the association of the ADIPOQ rs2241766 and rs266729 polymorphisms with metabolic syndrome (MS) in the Chinese population. METHODS: We searched for articles in MEDLINE via PubMed, EMBASE, HuGE Navigator, CNKI, and Wanfang databases and calculated odds ratios (ORs) with 95% confidence intervals (CIs) to determine the strength of associations in fixed- or random-effects models. RESULTS: We included 21 articles in the meta-analysis: 17 reports of ADIPOQ rs2241766 with 3628 cases and 3000 controls and 8 of rs266729 with 2021 cases and 2226 controls. We found an increased risk of MS with the ADIPOQ rs2241766 polymorphism in some genetic models (allele model: OR=1.12, 95% CI: 1.03-1.21; dominant model: OR=1.15, 95% CI: 1.04-1.28; homozygote model: OR=1.22, 95% CI: 1.00-1.49) but no association with the ADIPOQ rs266729 polymorphism (allele model: OR=0.98, 95% CI: 0.82-1.17; dominant model: OR=0.90, 95% CI: 0.79-1.02; recessive model: OR=1.09, 95% CI: 0.85-1.39; homozygote model: OR=1.03, 95% CI: 0.80-1.33). CONCLUSION: The results of this meta-analysis suggest an association between the ADIPOQ rs2241766 polymorphism and MS in the Chinese population. G allele of ADIPOQ rs2241766 increases the risk of MS. Better designed studies with different ethnic populations and larger sample sizes are needed for assessing the relationship between ADIPOQ rs2241766 and rs266729 polymorphisms and MS in the future.

Association of TCF7L2 and GCG Gene Variants with Insulin Secretion, Insulin Resistance, and Obesity in New-onset Diabetes.

This cohort study was designed to evaluate the association of transcription factor 7-like 2 (TCF7L2) and proglucagon gene (GCG) variants with disordered glucose metabolism and the incidence of type 2 diabetes mellitus (T2DM) in a rural adult Chinese population. A total of 7,751 non-T2DM participants ⋝18 years old genotyped at baseline were recruited. The same questionnaire interview and physical and blood biochemical examinations were performed at both baseline and follow-up. During a median 6 years of follow-up, T2DM developed in 227 participants. After adjustment for potential contributory factors, nominally significant associations were seen between TT genotype and the recessive model of TCF7L2 rs7903146 and increased risk of T2DM [hazard ratio (HR)=4.068, 95% confidence interval (CI): 1.270-13.026; HR=4.051, 95% CI: 1.268-12.946, respectively]. The TT genotype of rs7903146 was also significantly associated with higher fasting plasma insulin level and the homeostasis model assessment of insulin resistance in case of new-onset diabetes. In addition, the TCF7L2 rs290487 TT genotype was associated with abdominal obesity and the GCG rs12104705 CC genotype was associated with both general obesity and abdominal obesity in case of new-onset diabetes.

The association between androgen receptor gene CAG polymorphism and polycystic ovary syndrome: a case-control study and meta-analysis.

PURPOSE: Many studies have been carried out to confirm the relationship between androgen receptor gene CAG repeat polymorphism and polycystic ovary syndrome (PCOS), without consistent results. Hence we conducted the current study to research this relationship. METHODS: 224 Chinese Han women with PCOS and 223 in vitro fertilization and embryo transplantation (IVF-ET) infertile women with tubal factor or male infertility served as the controls were recruited in our study. PCR-based assays were applied to genotype the (CAG)n repeat alleles. A meta-analysis including 1,536 PCOS patients and 1,807 controls was conducted to produce a pooled estimate. RESULTS: We observed that the CAG bi-allelic mean lengths were similar in PCOS patients and controls (22.65 ± 2.5 vs. 23.09 ± 2.1, P = 0.116). When CAG bi-allelic were divided into two categories (mean repeats ≤22, >22), the short AR-CAG bi-allelic showed more frequent in PCOS group than in controls (56.25% vs 29.14%, P < 0.001). Further analysis presented that, in PCOS, there was a lower mean CAG repeat lengths in mean bi-allelic lengths (22.3 ± 2.5 vs. 23.9 ± 2.2, P = 0.008) and long bi-allelic lengths (24.3 ± 1.4 vs. 25.9 ± 1.6, P = 0.05) among patients with testosterone less than 0.7 ng/ml compared with those whose testosterone was more than 0.7 ng/ml. Besides, the testosterone were positively correlated with the CAG polymorphism (r = 0.237, P = 0.008), which accorded with our meta-analysis results. CONCLUSIONS: The distribution of AR-CAG allele differed between PCOS patients and controls, and polymorphism of CAG repeat lengths may contribute to hyperandrogenism in PCOS.

Braided thin-walled biodegradable ureteral stent: preliminary evaluation in a canine model.

OBJECTIVES: To evaluate a novel designed degradable ureteral stent. METHODS: A total of 24 male Beagles, each with bilateral stents implanted (a biodegradable ureteral 4.5-Fr stent and a standard 4-Fr biostable stent) were divided into four groups. Intravenous pyelography, B-mode ultrasonography, and blood and urine tests were carried out before the procedure (0 weeks), and at 1-, 2-, 3- and 4-week intervals. Meanwhile, the mechanical characteristics of stents were tested, and scanning electron microscopy images of the biodegradable braided stents were obtained at different time-points postoperatively. In addition, histopathological changes were compared between the two different stents. RESULTS: All biodegradable braided stents began degrading at 1 week, and had completely degraded by 4 weeks. Hydronephrosis was equivalent during the first 2 weeks, but less with the biodegradable stents than with the control biostable stents at 3 and 4 weeks. Preoperative and postoperative blood and urine results were similar. The mechanical properties of the biodegradable stents were better than conventional biostable stents. Scanning electron microscopy images obtained at different weekly intervals showed that stents degraded in a predictable fashion. Histological testing of the urinary tract showed that the stent-related tissue reactivity of the two different stents were similar. CONCLUSIONS: Our novel braided thin-walled biodegradable stents provide temporary renal drainage as good as commercially available biostable stents. They also have good biocompatibility and physical characteristics. Therefore, they might have clinical application.

Identification of a novel infection-enhancing epitope on dengue prM using a dengue cross-reacting monoclonal antibody.

BACKGROUND: Dengue virus (DENV) infection is the most important arthropod- borne viral disease in human, but antiviral therapy and approved vaccines remain unavailable due to antibody-dependent enhancement (ADE) phenomenon. Many studies showed that pre-membrane (prM)-specific antibodies do not efficiently neutralize DENV infection but potently promote ADE infection. However, most of the binding epitopes of these antibodies remain unknown. RESULTS: In the present study, we characterized a DENV cross-reactive monoclonal antibody (mAb), 4D10, that neutralized poorly but potently enhanced infection of four standard DENV serotypes and immature DENV (imDENV) over a broad range of concentration. In addition, the epitope of 4D10 was successfully mapped to amino acid residues 14 to18 of DENV1-4 prM protein using a phage-displayed peptide library and comprehensive bioinformatics analysis. We found that the epitope was DENV serocomplex cross-reactive and showed to be highly immunogenic in Balb/c mice. Furthermore, antibody against epitope peptide PL10, like 4D10, showed broad cross-reactivity and weak neutralizing activtity with four standard DENV serotypes and imDENV but significantly promoted ADE infection. These results suggested 4D10 and anti-PL10 sera were infection-enhancing antibodies and PL10 was infection-enhancing epitope. CONCLUSIONS: We mapped the epitope of 4D10 to amino acid residues 14 to18 of DENV1-4 prM and found that this epitope was infection-enhancing. These findings may provide significant implications for future vaccine design and facilitate understanding the pathogenesis of DENV infection.

[Cardio-myogenic differentiation of human amniotic fluid colony derived stem cells in the form of embryonic body-like structure].

OBJECTIVE: To investigate cardio-myogenic differentiation potential of human amniotic fluid colony derived stem cells (HAFCDSC) in the form of embryonic body (EB)-like structure in vitro. METHODS: HAFCDSC were isolated from second trimester amniotic fluid which was backup of amniocentesis specimens. The forth passage of HAFCDSC were cultured by hanging-drop preparation in complete medium for 5 days to form EB-like structures followed by inducing medium in regular tissue culture dishes for 2 weeks (experiment group). The EB-like structures cultured in complete medium were served as control group. Cardiomyocyte phenotypes were detected by RT-PCR and immunofluorescence staining. RESULTS: HAFCDSC could form EB-like structures 24 h after hanging drop culture, and the diameter of EB-like structures gradually increased with culture duration and the mean diameter of EB-like structures reached 237.3 ± 26.7 µm on 5(th) day. The formation rate of EB-like structures was 93.5%. RT-PCR analysis showed EB-like structures expressed stem cell related mRNA. Immunofluorescence staining demonstrated that some cardiomyocyte phenotypes such as Desmin, α-Actinin and Tn I were expressed by HAFCDSCs in EB-like structures after cardio-myogenic induction. Cardiomyocytes specific mRNA including Desmin, α-Actin, Tn I, Tn T, T-box and NK2.5 were also transcripted as detected by RT-PCR. No positive results was found in control group. CONCLUSION: HAFCDSC can form uniform EB-like structures after hanging drop culture for 5 days. HAFCDSC differentiate into cardiomyocyte-like cells through the form of EB-like structures after induction by 5-Aza.

Structural rearrangements and the unfolding mechanism of a Trigger Factor mutant studied by multiple structural probes.

Trigger Factor (TF) is a three-domain chaperone which catalyzes nascent peptide folding and harbors peptidyl-prolyl cis-trans isomerase activity. The multi-domain structure of TF makes it an interesting and challenging candidate for studies of the structural properties and functional behavior of individual domains or combined domain constructs. Here we constructed a TF mutant, NC, combining the N- and C-domains that are responsible for TF's chaperone function, and compared structural changes and unfolding characteristics of NC with wild-type TF by monitoring fluorescence spectra, far-UV CD, chemical crosslinking, DSC and binding with hydrophobic probes (ANS or bis-ANS). The results showed that the NC construct, like intact TF, could bind to hydrophobic probes, form dimers in solution, and showed a similar 3-state guanidine-induced unfolding profile. However, the NC fragment showed reduced stability towards both guanidine unfolding and thermal denaturation, suggesting that the presence of the M-domain of TF contributes to the stability of the intact TF structure.

Alcohol oxidase (AOX1) from Pichia pastoris is a novel inhibitor of prion propagation and a potential ATPase.

Previous results suggest that methylotrophic yeasts may contain factors that modulate prion stability. Alcohol oxidase (AOX), a key enzyme in methanol metabolism, is an abundant protein that is specific to methylotrophic yeasts. We examined the effect of Pichia pastoris AOX1 on prion phenotypes in Saccharomyces cerevisiae. The S. cerevisiae prion states [PSI(+)] and [URE3] arise from aggregation of the proteins Sup35p and Ure2p respectively, and correlate with the ability of Sup35p and Ure2p to form amyloid-like fibrils in vitro. We found that expression of P. pastoris AOX1 in S. cerevisiae had no effect on propagation of the [PSI(+)] prion, but inhibited propagation of [URE3]. Addition of AOX1 early in the time-course of fibril formation inhibits Ure2p fibril formation in vitro. AOX1 has not previously been identified as an ATPase. However, we discovered that in addition to its flavin adenine dinucleotide-dependent AOX activity, AOX1 possesses ATPase activity. This study identifies AOX1 as a novel prion inhibitory factor and a potential ATPase.

Bladder reconstruction using autologous smooth muscle cell sheets grafted on a pre-vascularized capsule.

Rationale: Construction of functional vascularized three-dimensional tissues has been a longstanding objective in the field of tissue engineering. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate functional vascularized smooth muscle tissue flaps for bladder reconstruction in a rabbit model was tested. Methods: Skin tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Smooth muscle cells and endothelial progenitor cells were harvested and cocultured to form pre-vascularized smooth muscle cell sheet. Then repeated transplantation of triple-layer cell sheet grafts onto the vascularized capsular tissue was performed at 2-day intervals to prefabricate functional vascularized smooth muscle tissue flaps. Bladder muscular wall defects were created and repaired by six-layer cell sheet graft (sheet only), capsule flap (capsule only) and vascularized capsule prelaminated with smooth muscle cell sheet (sheet plus capsule). The animals were followed for 3 months after implantation and their bladders were explanted serially. Results: Bladder capacity and compliance were maintained in sheet plus capsule group throughout the 3 months. Tissue bath stimulation demonstrated that contractile responses to carbachol and KCl among the three groups revealed a significant difference (p < 0.05). Histologically, inflammation was evident in the capsule only group at 1 month and fibrosis was observed in sheet only group at 3 months. The vessel density in capsule only and sheet plus capsule group were significantly higher than in the sheet only group at each time point (p < 0.05). Comparison of the smooth muscle content among the three groups revealed a significant difference (p < 0.05). Conclusion: These results proved that the capsule may serve as an induced vascular bed for vascularized smooth muscle tissue flap prefabrication. The prefabricated functional vascularized smooth muscle tissue flap has the potential for reliable bladder reconstruction and may create new opportunities for vascularization in 3-D tissue engineering.

Tissue expander capsule as an induced vascular bed to prefabricate an axial vascularized buccal mucosa-lined flap for tubularized posterior urethral reconstruction: preliminary results in an animal model.

Surgical repair of complex posterior urethral disruptions remains one of the most challenging problems in urology. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate axial vascularized buccal mucosa-lined flaps for tubularized posterior urethral reconstruction in a rabbit model was tested. The experiments were performed in three stages. First, silicone tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Next, buccal mucosa grafts were transplanted into the newly formed capsular tissue supplied by axial vessels for buccal mucosa-lined flap prefabrication. Then, circumferential posterior urethral defects were created and repaired with the buccal mucosa graft (Group 1), the capsule flap (Group 2), and the prefabricated capsule buccal mucosa composite flap (Group 3). After surgery, notable contracture of the tubularized buccal mucosa graft was observed in the neourethra, and none of the rabbits in Group 1 maintained a wide urethral caliber. In Group 2, the retrieved neourethra showed little evidence of epithelial lining during the study period, and the lumen caliber was narrowed at the 3-month evaluation. In Group 3, the buccal mucosa formed the lining in the neourethra and maintained a wide urethral caliber for 3 months. The capsule may serve as an induced vascular bed for buccal mucosa-lined flap prefabrication. The prefabricated buccal mucosa-lined flap may serve as a neourethra flap for posterior urethral replacement.

Thermal unfolding of Escherichia coli trigger factor studied by ultra-sensitive differential scanning calorimetry.

Temperature-induced unfolding of Escherichia coli trigger factor (TF) and its domain truncation mutants, NM and MC, were studied by ultra-sensitive differential scanning calorimetry (UC-DSC). Detailed thermodynamic analysis showed that thermal induced unfolding of TF and MC involves population of dimeric intermediates. In contrast, the thermal unfolding of the NM mutant involves population of only monomeric states. Covalent cross-linking experiments confirmed the presence of dimeric intermediates during thermal unfolding of TF and MC. These data not only suggest that the dimeric form of TF is extremely resistant to thermal unfolding, but also provide further evidence that the C-terminal domain of TF plays a vital role in forming and stabilizing the dimeric structure of the TF molecule. Since TF is the first molecular chaperone that nascent polypeptides encounter in eubacteria, the stable dimeric intermediates of TF populated during thermal denaturation might be important in responding to stress damage to the cell, such as heat shock.

Infection and replication of avian influenza H5N1 virus in an infected human.

The highly pathogenic avian influenza H5N1 viruses usually cause severe diseases and high mortality in infected humans. However, the tissue tropism and underlying pathogenesis of H5N1 virus infection in humans have not been clearly elucidated yet. In this study, an autopsy was conducted to better understand H5N1 virus distributions in tissues of infected humans, and whether H5N1 virus can replicate in extrapulmonary tissues. We found that the lungs had the higher viral load than the spleen, whereas no detectable viruses in tissues of heart, liver, kidney, large intestine, small intestine, or brain. Specifically, the viral load was higher in the left lung (7.1 log10 copies per ml) in relation to the right lung (5.7 log10 copies per ml), resulting in more severe pathological damage in the left lung, and lung tissues contained both positive- and negative-stranded viral RNA. However, there existed a low level of H5N1 viruses in the spleen (3.8 log10 copies per ml), with the absence of positive-stranded viral RNA. Our results indicate that replication of H5N1 viruses mainly occurs in the lungs, and the degree of lung damage is highly correlated with the viral load in the lungs. The low-load viruses in the spleen might be introduced through blood circulation or other ways.

Tubularized urethral reconstruction using a prevascularized capsular tissue prelaminated with buccal mucosa graft in a rabbit model.

Tubularized graft urethroplasty fails largely because of inadequate graft take. Prefabrication of buccal mucosa lined flap has theoretical indications for constructing neourethra with an independent blood supply. The efficacy of using a tissue expander capsule as an induced vascular bed to prefabricate an axial vascularized buccal mucosa-lined flap for tubularized urethral reconstruction in a rabbit model was tested. The experiments were performed in three stages. First, silicone tissue expanders were inserted into the groin to induce vascularized capsule pouch formation. Next, buccal mucosa grafts were transplanted to the newly formed capsular tissue supplied by the axial vessel for buccal mucosa-lined flap prefabrication. Then, circumferential urethral defects were created and repaired by buccal mucosa graft (Group 1), capsule flap (Group 2) and prefabricated capsule buccal mucosa composite flap (Group 3). With retrograde urethrography, no rabbits in Group 1 maintained a wide urethral caliber. In Group 2, the discontinued epithelial layer regenerated at 1 month, and the constructed neourethra narrowed even though the lumen surface formed intact urothelial cells at 3 months. In Group 3, buccal mucosa formed the lining in the neourethra and kept a wide urethral caliber for 3 months. The capsule may serve as an induced vascular bed for buccal mucosa-lined flap prefabrication. The prefabricated buccal mucosa-lined flap may serve as a neourethra flap for circumferential urethral replacement.

Computational prediction and identification of dengue virus-specific CD4(+) T-cell epitopes.

In this study, we tried to identify dengue virus-specific CD4(+) T-cell epitopes, which can induce PBMC (peripheral blood mononuclear cells) isolated from DF convalescent patients (dengue virus type 1 infection) to secrete IFN-gamma. PBMC of DF convalescent patients were stimulated in vitro with dengue virus-derived peptides, which were prepared based on the prediction of dengue virus-specific CD4(+) T-cell epitopes by using RANKpep online software. Subsequently, the frequency of IFN-gamma producing T cells and percentage of IFN-gamma(+) CD4(+) T cells were measured by using ELISPOT assay and ICS assay (intracellular cytokine straining), respectively. The positive response of PBMC by ELISPOT showed that the numbers of SFC (spots forming cells) ranged from 50 to 310 SFC/1x10(6) PBMC. The positive response of PBMC by ICS assay showed that the percentage of IFN-gamma(+) CD4(+) T cells ranged from 0.03 to 0.27%. As a result, C(45-57) (KLVMAFIAFLRFL), E(396-408) (SSIGKMFEATARG), NS3(23-35) (YRILQRGLLGRSQ), and NS3(141-155) (NREGKIVGLYGNGVV) were identified as dengue virus-specific CD4(+) T-cell epitopes.

Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor.

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.

Substitution of the precursor peptide prevents anti-prM antibody-mediated antibody-dependent enhancement of dengue virus infection.

Antibody-dependent enhancement (ADE) is currently considered as the mechanism underlying the pathogenesis of severe dengue disease. Many studies have shown that precursor (pr) peptide-specific antibodies do not efficiently neutralize infection but potently promote ADE of dengue virus (DENV) infection. To explore the effect of pr peptide substitution on neutralization and ADE of DENV infection, the rabbit anti-prM polyclonal antibodies (pAbs) and anti-JEVpr/DENV-M pAbs were prepared, and the neutralization and ADE of these two pAbs were further compared. Here, we report that both anti-JEVpr/DENV-M and anti-prM pAbs exhibited broad cross-reactivity and only partial neutralization with four DENV serotypes and immature DENV. Rabbit anti-prM pAbs showed a significant enhancement in a broad range of serum dilutions. However, there was no statistically significant difference in the enhancing activity of rabbit anti-JEVpr/DENV-M pAbs at various levels of dilution. These results demonstrate that anti-prM antibody-mediated ADE can be prevented by JEV pr peptide replacement. The present study contribute further to research on the pathogenesis of DENV infection.

Effect of C-terminal truncation on the molecular chaperone function and dimerization of Escherichia coli trigger factor.

To examine the role of the C-terminal domain in the chaperone function of trigger factor (TF), a number of truncation mutants were constructed, namely: TF419, TF389, TF380, TF360, TF344, and TF251, in which the C-terminal 13, 43, 52, 72, 88 residues or the entire C-domain were deleted, respectively. Co-expression of mutant chicken adenylate kinase (AK) with TF and the C-terminal truncation mutants was achieved using a plasmid pBVAT that allows expression of TF and AK from a single plasmid. The results show that truncation of the C-terminus of TF has only minor effect on its ability to assist AK refolding in vivo. Further, ribosome-binding experiments indicate that C-terminal truncation mutants can still bind to the ribosome and the presence of the C-terminus may in fact lower the affinity of TF for the ribosome in vivo. This indicates that the C-domain of trigger factor may not be essential for the ribosome-associated molecular chaperone function of TF. However, the purified TF C-terminal truncation mutants had a dramatically reduced ability to assist rabbit muscle GAPDH refolding in vitro and a reduced tendency to dimerize. This shows that the structural integrity of the C-terminus contributes to both the chaperone function of TF and the stability of the dimeric form.

Expanding the pressure technique: insights into protein folding from combined use of pressure and chemical denaturants.

The fundamental principles derived from in vitro protein folding experiments have practical application in understanding the pathology of diseases of protein misfolding and for the development of industrial processes to produce proteins as pharmaceuticals and biotechnological reagents. High pressure as a tool to denature or disaggregate proteins offers a number of unique advantages. The emphasis of this review is on how low concentrations of chemical denaturants can be used in combination with high pressure to extend the range and scope of this useful technique. This approach has already been used in a number of studies, which are discussed here in the context of the questions they address. These include: the origin of the volume change observed on protein unfolding, pressure-induced formation of partially structured intermediates, pressure-induced dissociation of oligomeric and aggregated proteins, and the use of volume changes to probe the structure of the transition state. Wider use of hydrostatic pressure as a denaturation tool, facilitated by combination with chemical denaturants, is likely to bring significant advances to our understanding of protein structure, stability and folding, particularly in relation to proteins associated with the amyloid and prion diseases.

Unraveling multistate unfolding of rabbit muscle creatine kinase.

GdmCl-induced unfolding of rabbit muscle creatine kinase, CK, has been studied by a variety of physico-chemical methods including near and far UV CD, SEC, intrinsic fluorescence (intensity, anisotropy and lifetime) as well as intensity and lifetime of bound ANS fluorescence. The formation of several stable unfolding intermediates, some of which were not observed previously, has been established. This was further confirmed by representation of fluorescence data in terms of "phase diagram", i.e. I(lambda1) versus I(lambda2) dependence, where I(lambda1) and I(lambda2) are fluorescence intensity values measured on wavelengths lambda(1) and lambda(2) under the different experimental conditions for a protein undergoing structural transformations. The unfolding behavior of CK was shown to be strongly affected by association of partially folded intermediates. A model of CK unfolding, which takes into account both structural perturbations and association of partially folded intermediates has been elaborated.

Two distinct intermediates of trigger factor are populated during guanidine denaturation.

Trigger factor (TF) is an important catalyst of nascent peptide folding and possesses both peptidyl-prolyl cis-trans isomerase (PPIase) and chaperone activities. TF has a modular structure, containing three domains with distinct structural and functional properties. The guanidine hydrochloride (GuHCl) induced unfolding of TF was investigated by monitoring Trp fluorescence, far-UV CD, second-derivative UV absorption, enzymatic and chaperone activities, chemical crosslinking and binding of the hydrophobic dye, 1-anilinonaphthalene-8-sulfonate (ANS); and was compared to the urea induced unfolding. The native state of TF was found to bind ANS in 1:1 stoichiometry with a K(d) of 84 microM. A native-like state, N', is stable around 0.5 M GuHCl, and shows increased ANS binding, while retaining PPIase activity and most secondary and tertiary structure, but loses chaperone and dimerization activities, consistent with slight conformational rearrangement. A compact denatured state, I, is populated around 1.0 M GuHCl, is inactive and does not show significant binding to ANS. The data suggest that TF unfolds in a stepwise manner, consistent with its modular structure. The ability of TF to undergo structural rearrangement to maintain enzymatic activity while reducing chaperone and dimerization abilities may be related to the physiological function of TF.