[Senolytic effects on dental pulp stem cell's proliferation and differentiation during long-term expansion].

Objective: To investigate the impact of intermittent senescent cell clearance on the proliferation and differentiation of dental pulp stem cells (DPSC) in long-term, large-scale expansion, and to explore strategies for maintaining the youthful state of DPSC in vitro. Methods: Human-derived dental pulp stem cells were isolated from healthy permanent teeth extracted for orthodontic or impeding eruption reasons, provided by the Department of Oral and Maxillofacial Surgery at West China Hospital of Stomatology, Sichuan University. Long-term, large-scale in vitro expansion of DPSC was conducted. The study compared young DPSC (passage 5) with aged DPSC (passage 25) using cellular senescence-associated ß-galactosidase staining, colony formation assay, and Alizarin Red S staining for osteogenic differentiation induction. To assess the differences between the two cell populations in terms of senescence and amplification and differentiation ability. Medicine screening for the most effective senolytic was compared among 5 common senolytics [Navitoclax (ABT-263), curcumin, dasatinib, fisetin, and quercetin]. The clearance efficacy was compared using cellular senescence-associated ß-galactosidase staining to reflect the changes in senescent cell ratio. The senolytic with the highest efficacy was chosen for further experiments. The passage at which the proportion of senescent cells significantly increased was identified, and the selected senolytic was administered three times at three-generation intervals from that passage to remove senescent cells. Both the control and senolytic-treated groups were estimated by fluorescence cellular senescence-associated ß-galactosidase staining, real-time fluorescence quantitative PCR (RT-qPCR), colony formation assay, wound healing assay, and Alizarin Red S staining for osteogenic differentiation induction. Subcutaneous heterotopic osteogenesis was performed in nude mice and the grafts were analyzed by HE staining and alkaline phosphatase (ALP) immunohistochemical staining. Results: The proportion of senescent cells increased as the expansion extended, leading to decreased proliferation and osteogenic differentiation ability of senescent DPSC compared to young DPSC (P<0.05). Senescent DPSC exhibited altered mRNA expression levels of senescence-related genes, including p21, p16INK4a, IL-6, and Ki67 (P<0.001). Among the five senolytics, ABT-263 had the biggest decreases in the proportion of senescent cells. After intermittent ABT-263 treatment during expansion, the proportion of senescent cells in the senolytic-treated group [(6.72±2.34)%] was significantly lower than that in the control group [(31.82±0.57)%] (P<0.001). RT-qPCR confirmed that compared with the control group, mRNA expressions of p21, p16INK4a, and IL-6 in the senolytic-treated group were significantly decreased (P<0.05), while mRNA expressions of Ki67 were significantly increased (P<0.01). Furthermore, the cell healing ability and osteogenic differentiation ability of the senolytic-treated group were higher than those of the control group (P<0.05). In vivo experimental results indicated that the relative new bone area [(2.36±0.48)%] after DPSC transplantation in the senolytic-treated group was greater than that in the control group [(1.00±0.46)%] (P<0.05), and the expression of ALP was higher than that in the control group (P<0.01). Conclusions: ABT-263 can effectively eliminate senescent cells in long-term large-scale DPSC expansion. Continuous treatment with ABT-263 during cultivation can maintain the proliferation and differentiation ability of DPSC both in vivo and in vitro.

[Clinical translational research and application prospects of mesenchymal stem cells in periodontal tissue regeneration].

Periodontitis is an oral infectious disease that occurs in the tooth supporting tissues, which damages the soft and hard tissues of the periodontium and eventually results in tooth mobility and loss. Traditional clinical treatment can effectively control the periodontal infection and inflammation. However, as the therapeutic effect depending on the local condition of periodontal defect and the systemic factors of patients, it's hard to achieve satisfactory and stable periodontal tissue regeneration for damaged periodontal tissues. Recently, as a promising therapeutic strategy in periodontal regeneration, mesenchymal stem cells (MSC) play an important role in modern regenerative medicine. Combined with the clinical translational researches of MSC in periodontal tissue engineering and the research results of our group in the past decade, it is summarized and explained the mechanism of MSC promoting periodontal regeneration, the preclinical and the clinical transformation researches as well as the future application prospects of MSC in periodontal regeneration therapy in this paper.

[Exosomes-based strategies for dental pulp regeneration].

Researches on pulp regeneration based on odontogenic stem cells have made a breakthrough and some results have been applied clinically in recent years. It is demonstrated that exosomes secreted by stem cells play a significant role in tissue regeneration. Compared with stem cells, exosomes exhibit more advantages such as wider sources, more immediate effects, and less immune rejections, which are expected to be exploited in the pulp regeneration by cell-homing. In this review, the conventional dental pulp tissue engineering and exosomes-based dental pulp regeneration method are discussed, the new strategy for dental pulp regeneration by using exosomes is analyzed and proposed.

[Combined use of the Solitaire AB stent and Navien delivery catheter for mechanical thrombectory of acute vertebrobasilar artery occlusion].

Objective: To investigate the efficacy and safety of mechanical thrombectory with a tri-axial system of the solitaire AB stent through a Navien delivery catheter to treatment acute vertebrobasilar artery occlusion. Methods: The clinical data of 16 consecutive patients with acute ischemic stroke of vertebrobasilar artery occlusion treated with the Solitaire AB device from March 2016 to March 2018 in Department of Neurosurgery, the First Affiliated Hospital, Medical College of Shihezi University, were extracted and then retrospectively analyzed.Recanalization rate as well as complications after treatment were analysized. Also, neurological functions of the patients before and after operation 1 weeks, measured by National Institute of Health stroke scale (NIHSS) score were compared via t test and the clinical outcomes were assessed by modified Rankin score (mRS) at 90 days after operation. Results: There were 10 male and 6 female patients with a median age of 59 years (ranging from 45 to 78 years).Fourteen patients resulted in successfully recanalization, and 2 cases failed both of whose onset to sheath mean time were 8.5 hours.The NIHSS score at 7 days was 13±7, which was significantly decreased compared to the admission NIHSS score 24±4 (P=0.000).No symptomatic intracranial hemorrhage case was found after operation.At 90 days, 8 patients achieved good outcome (mRS 0 to 2), one patient died (mRS 6),two patients moderately seriously disabled (mRS 4).Three patients resulted in moderate outcome (mRS 3).In 3 cases of death,2 patients died due to failure in recanalization,another patient died of severe pulmonary infection. Conclusion: The results suggest that this technique of a tri-axial system used of the Solitaire AB stent through a Navien delivery catheter can effectively retrieve clots from the occlusive artery and improves functional outcome in patients with acute ischemic stroke of vertebrobasilar artery.

[Development of new technology in periodontal tissue engineering].

The loss of periodontal support tissues might cause movement or finally loss of the teeth affected, impair furthermore the pronunciation and mastication functions, and even result in a series of physiological and psychological problems. Tissue engineering, as a technology to remodel missing tissues or organs and functional reconstruction, has achieved gratifying progress in regeneration of periodontal tissues. However, conventional construction methods have some deficiencies for functional periodontal reconstruction. In recent years, with the progress of tissue engineering technology, a series of new techniques and methods, such as cell sheet technology, decellular technology, electrospinning technology and three-dimensional printing, has been applied in tissue engineering bringing new hope for the regeneration of periodontal tissues. In this review article, the recent progress achieved in the field of periodontal tissue engineering and application of modern technology are summarized to make a brief exposition and to explore the future development of periodontal regeneration.

Effect of Mg/Ca molar ratios on characteristics of anaerobic-anoxic denitrifying dephosphatation.

In this study, the effect of three Mg/Ca molar ratios (5.0, 3.8 and 1.7) on denitrifying phosphate removal performance, biomass morphology, and Extracellular Polymeric Substances (EPS) were examined. Results showed that when the influent Mg/Ca molar ratio was 3.8, the anaerobic-anoxic EBPR performed complete phosphate removal. The microbial bacterial population was a mixed culture comprised of 81±3% DPAO and 13±2% denitrifying glycogen accumulating organisms (DGAO). A higher influent Mg/Ca molar ratio (5.0) had a distinct impact on phosphate removal, biomass morphology, and EPS. This probably induced the deterioration of the anaerobic-anoxic Enhanced Biological Phosphorus Removal (EBPR). The results of this study may inform the proper operation of an anaerobic-anoxic EBPR, and contribute to its application in the real world.

[Tooth and its supporting tissues regeneration using functional tooth modules].

Stem cell based generation of functional tissue modules is becoming a hotspot in the field of tissue regeneration. These functional tissue modules, which can be prepared through a variety of ways, including self-organization of stem cells, cell sheets, electrospinning and bio-printing, can serve as the basic unit and be assembled to form larger tissues and organs. This new technology can be a break-through to address current issues in tissue regeneration and further promote the translational use of regenerated tissues/organs. Construction of functional tooth modules is still in its early stage, where the stem cell source, preparing method and strategy of translational use are not clearly defined yet. How to construct the functional tissue module through the existing technology still needs further study. Here, we would like to share our current understanding and thoughts on constructing strategies and potential clinical application of functional tooth modules including dental pulp, periodontal tissue and tooth root, and hope it could shed new lights on the regeneration of tooth.

[The expression and significance of pepsin in laryngeal carcinoma].

OBJECTIVE: To evaluate the relationship between pepsin induced by laryngopharyngeal reflux and laryngeal carcinoma. METHODS: Patients with vocal cord leukoplakia(n=18) and laryngeal carcinoma(n=21) encountered in Nanfang Hospital between December 2012 and April 2014 were included and sixteen healthy volunteers were recruited as control. Laryngeal biopsy specimens were taken from the patients with laryngeal carcinoma, or vocal cord leukoplakia and control subjects for the immunohistochemical study of pepsin. The correlation between pepsin expression and reflux events of 24 hour multichannel intraluminal impedance-pH monitoring (MII-pH) was analyzed. RESULTS: The patients with laryngeal carcinoma showed the highest expression of pepsin, followed by the patients with vocal cord leukoplakia and control subjects, with significant difference among the three groups (in strong positive expression, the constituent ratio of each group are 0/16、1/18 and 4/21, P<0.01). The presence of pepsin was associated with upright and total laryngopharyngeal acid reflux (P<0.05), including acid reflux episodes, the percentage of times that the pH was below four, the percentages of acid reflux time and average acid removal time. There was a significant correlation between the pepsin level and the esophageal acid reflux parameters (P<0.05) except supine the percentage of time that the pH was below four. CONCLUSIONS: Pepsin expression in laryngeal tissue increases in patients with vocal cord leukoplakia and laryngeal carcinoma, contributing to the development of laryngopharyngeal carcinogenesis.

Accuracy of three-dimensional facial soft tissue simulation in post-traumatic zygoma reconstruction.

The aim of this study was to evaluate the accuracy of novel software-CMF-preCADS-for the prediction of soft tissue changes following repositioning surgery for zygomatic fractures. Twenty patients who had sustained an isolated zygomatic fracture accompanied by facial deformity and who were treated with repositioning surgery participated in this study. Cone beam computed tomography (CBCT) scans and three-dimensional (3D) stereophotographs were acquired preoperatively and postoperatively. The 3D skeletal model from the preoperative CBCT data was matched with the postoperative one, and the fractured zygomatic fragments were segmented and aligned to the postoperative position for prediction. Then, the predicted model was matched with the postoperative 3D stereophotograph for quantification of the simulation error. The mean absolute error in the zygomatic soft tissue region between the predicted model and the real one was 1.42±1.56mm for all cases. The accuracy of the prediction (mean absolute error ≤2mm) was 87%. In the subjective assessment it was found that the majority of evaluators considered the predicted model and the postoperative model to be 'very similar'. CMF-preCADS software can provide a realistic, accurate prediction of the facial soft tissue appearance after repositioning surgery for zygomatic fractures. The reliability of this software for other types of repositioning surgery for maxillofacial fractures should be validated in the future.

Accumulibacter clades Type I and II performing kinetically different glycogen-accumulating organisms metabolisms for anaerobic substrate uptake.

The anaerobic acetate (HAc) uptake stoichiometry of phosphorus-accumulating organisms (PAO) in enhanced biological phosphorus removal (EBPR) systems has been an extensive subject of study due to the highly variable reported stoichiometric values (e.g. anaerobic P-release/HAc-uptake ratios ranging from 0.01 up to 0.93 P-mol/C-mol). Often, such differences have been explained by the different applied operating conditions (e.g. pH) or occurrence of glycogen-accumulating organisms (GAO). The present study investigated the ability of biomass highly enriched with specific PAO clades ('Candidatus Accumulibacter phosphatis' Clade I and II, hereafter PAO I and PAO II) to adopt a GAO metabolism. Based on long-term experiments, when Poly-P is not stoichiometrically limiting for the anaerobic VFA uptake, PAO I performed the typical PAO metabolism (with a P/HAc ratio of 0.64 P-mol/C-mol); whereas PAO II performed a mixed PAO-GAO metabolism (showing a P/HAc ratio of 0.22 P-mol/C-mol). In short-term batch tests, both PAO I and II gradually shifted their metabolism to a GAO metabolism when the Poly-P content decreased, but the HAc-uptake rate of PAO I was 4 times lower than that of PAO II, indicating that PAO II has a strong competitive advantage over PAO I when Poly-P is stoichiometrically limiting the VFA uptake. Thus, metabolic flexibility of PAO clades as well as their intrinsic differences are additional factors leading to the controversial anaerobic stoichiometry and kinetic rates observed in previous studies. From a practical perspective, the dominant type of PAO prevailing in full-scale EBPR systems may affect the P-release processes for biological or combined biological and chemical P-removal and recovery and consequently the process performance.

Antitumor effects of interferon-alpha on cell growth and metastasis in human nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a highly malignant and frequently metastasized tumor, and the prognosis is very poor when distant metastases occur. Recently, immunotherapy is becoming a promising therapeutic approach. Interferon-α (IFN-α) represents the cytokines exhibiting the longest record of use in clinical oncology. In this study, we examined the antitumor effects of IFN-α1b on NPC. The results showed that recombinant human IFN-α1b (hIFN-α1b) suppressed cell growth, induced a G1-phase cell cycle arrest in vitro, increased the expression of p16 and pRb, and decreased the expression of CCND1 and CDK6. In vivo analyses showed that either recombinant adeno-associated virus (rAAV)-IFN-α1b or hIFN-α1b treatment inhibited tumor growth and metastasis, reduced intratumoral microvessel density, increased cell apoptosis and necrosis, and induced prolonged survival. Notably, rAAV-IFN-α1b or hIFN-α1b treatment led to significantly higher serum levels of IL-12 and GM-CSF in mice compared to respective controls. Our findings suggest that IFN-α1b acts as a multifunctional antitumor agent in NPC, which may have important therapeutic implications.

Identification of osteo-adipo progenitor cells in fat tissue.

OBJECTIVES: In this study, a group of cells that expressed both osteogenic and adipogenic characters was identified from murine adipose stromal cells. MATERIALS AND METHODS: These cells could be enriched in the Sca-1-1 population and express both osteogenic and adipogenic genes. Osteogenic induction enhanced expression of osteogenic genes and inhibited expression of adipogenic genes, while adipogenic induction enhanced expression of adipogenic genes and inhibited expression of osteogenic genes. These cells have been called osteo-adipo progenitors (OAPs). RESULTS: OAPs expressed transcription factor runt-related transcription factor 2 (RUNX2) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) proteins in cytoplasm. When OAPs were cultured in adipogenic medium, PPAR-gamma moved to the nucleus and the cells differentiated into adipocytes, while the RUNX2 remained in the cytoplasm. In contrast, when OAPs were cultured in osteogenic medium, RUNX2 moved to the nucleus and the cells differentiated to osteocytes, while the PPAR-gamma remained in the cytoplasm. CONCLUSIONS: These experiments suggest that osteoblasts and adipocytes share a common predecessor, the OAP, in murine adipose stromal cells.

Improvement of peripheral nerve regeneration by a tissue-engineered nerve filled with ectomesenchymal stem cells.

Ectomesenchymal stem cells (EMSCs) originate from the cranial neural crest. They are a potential source of neuronal and Schwann cells (SCs) of the peripheral nervous system (PNS) during embryonic development. The third passage of EMSCs enzymatically isolated from the mandibular processes of Sprague-Dawley rats were cultured in forskolin and bovine pituitary extract for 6 days to generate functional Schwann cell phenotypes. Next, 10-mm defects in the sciatic nerves were bridged with an autograft, tissue-engineered nerve filled with differentiated cells in collagen, or a PLGA conduit alone in 18 rats, and the nerve defects of another four rats were left untreated. The regenerated nerves were evaluated by the sciatic functional index (SFI) monthly and by histological analysis 4 months after grafting. The recovery index of the sciatic nerve improved significantly in the autograft and tissue-engineered nerve groups, both of which were superior to the PLGA group. In animals transplanted with the EMSCs, there was greater regeneration than with conduit alone during the same period of implantation. These results show that when EMSCs are transplanted to a peripheral nerve defect they differentiate into supportive cells that contribute to the promotion of axonal regeneration.

Analysis of ingredient and heating value of municipal solid waste.

Great differences between municipal solid wastes (MSW) produced at different places and different times in terms of such parameters as physical ingredient and heating value lead to difficulty in effective handling of MSW. In this paper, ingredient, heating value and their temporal varying trends of typical MSW in Beijing were continuously measured and analyzed. With consideration of the process in pyrolysis and incineration, correlation between physical ingredients and heating values was induced, favorable for evaluation of heating value needed in handling of MSW from simple analysis of physical ingredients of it.

Internal circulating fluidized bed incineration system and design algorithm.

The internal circulating fluidized bed (ICFB) system is characterized with fast combustion, low emission, uniformity of bed temperature and controllability of combustion process. It is a kind of novel clean combustion system, especially for the low-grade fuels, such as municipal solid waste (MSW). The experimental systems of ICFB with and without combustion were designed and set up in this paper. A series of experiments were carried out for further understanding combustion process and characteristics of several design parameters for MSW. Based on the results, a design routine for the ICFB system was suggested for the calculation of energy balance, airflow rate, heat transfer rate, and geometry arrangement. A test system with ICFB combustor has been set up and the test results show that the design of the ICFB system is successful.

Aging and effects of ultraviolet A exposure may be quantified by fluorescence excitation spectroscopy in vivo.

The fluorescence properties of skin chromophores such as tryptophan and collagen cross-links might be useful markers of aging and photoaging. As the fluorescence of pepsin-digestible collagen cross-links was found to increase with aging and decrease with photoaging we investigated the characteristics of this dependence. In vivo fluorescence excitation spectra (emission at 380 nm) of SKH hairless mouse model skin are characterized by two bands centered near 295 nm and 335 nm due, respectively, to epidermal tryptophan moieties and pepsin-digestible collagen cross-links. Several groups of hairless mice were followed over a period of 18 mo to document changes in skin fluorescence with aging. Other groups of animals were exposed to either broad band or narrowband ultraviolet A radiation to determine the effects of ultraviolet A exposure on the fluorescence of the dermal collagen cross-links and to determine an action spectrum for the induced changes. We also found that the intensity of pepsin-digestible collagen cross-links in vivo increases linearly with age and that the fluorescence of epidermal tryptophan decreases linearly with age. We found that the fluorescence of pepsin-digestible collagen cross-links decreases immediately following exposure to ultraviolet A whereas epidermal tryptophan fluorescence increases. Both changes were dose dependent but the increase in tryptophan fluorescence occurred exclusively in young animals (2--6 mo old). We found that the ultraviolet-induced fluorescence decrease of pepsin-digestible collagen cross-links is wavelength specific. The action spectrum for the ultraviolet A effect on the in vivo fluorescence of pepsin-digestible collagen cross-links shows a distinct maximum at 335 nm that corresponds to the maximum in the fluorescence excitation spectrum due to pepsin-digestible collagen cross-links. Our results seem to indicate that in vivo fluorescence of epidermal tryptophan moieties and collagen cross-links in the dermal matrix may serve as markers for skin aging, for photoaging, and for immediate assessment of exposure to ultraviolet A radiation.

Probing heme protein conformational equilibration rates with kinetic selection.

Double-pulse flash photolysis experiments on solutions of carbonmonoxymyoglobin (MbCO) are used to determine the time scale for protein conformational averaging. The interconversion times for transitions between the "open" and "closed" subpopulations of MbCO are found to be 10(-6)-10(-4)s, depending on solvent composition and temperature. In aqueous solution at 273 K, the interconversion rate is found to be 1.4 x 10(6)s. Since the interconversion rate is comparable to or slower than the geminate rebinding rate, we describe the geminate phase of the kinetics as a superposition of contributions from the open and closed states. Although geminate kinetics remain intrinsically nonexponential for both open and closed states near room temperature, we find that substates within these two subpopulations interconvert more rapidly than the geminate rebinding. These observations cannot be explained by a superposition of contributions from a quasicontinuous conformational distribution (Steinbach et al., 1991) and are probably due to the long-time tail of the relaxation of the protein (Tian et al., 1992). Bimolecular rebinding takes place at a statistically averaged rate, since the interconversion and relaxation rates are faster than the bimolecular kinetics. The geminate and bimolecular kinetics are analyzed quantitatively as a function of pH using this approach and the spectroscopically determined populations of the open and closed states. The analysis accounts for the observed kinetics and also successfully predicts the kinetic response observed in the double-pulse experiments. In aqueous solution at 273 K, the geminate amplitudes and rates are found to be I(0)g = 32% and k(0)g = 1.3 x 10(7)s(-1) for the open state and I(1)g = 9.3% and k(1)g = 1.4 x 10(6)s(-1) for the closed state. In 75% glycerol solution at 264 K, the dominant component of the geminate rebinding is characterized by I(0)g1 = 89% and k(0)g1 = 3.1 x 10(6)s(-1) for the open state and I(1)g1 = 26% and k(1)g1 = 3.1 x 10(6)s(-1) for the closed state. The fact that the interconversion rate is comparable to the geminate rate of the closed state in aqueous solution is consistent with the idea that the open state provides an important pathway for ligand escape from (or entry to) the heme pocket (Tian et al., 1993). The increased viscosity of 75% glycerol solution delays the closed--> open interconversion until the end of the geminate phase, which forces the ligand to find alternative pathways to the solution. This observation, in conjunction with the near equivalence of the geminate rates for the open and closed states in 75% glycerol solution, suggests that the solvent composition fundamentally alters the protein-ligand dynamics.

Measurements of CO geminate recombination in cytochromes P450 and P420.

The kinetics of CO geminate recombination in cytochrome P450cam are studied at room temperature subsequent to laser photolysis. The geminate rebinding kinetics of P450 are strongly affected by the presence of the camphor substrate. We observe a approximately 2% geminate yield for substrate-bound P450 and a 90% geminate yield when the substrate is absent. The drastic difference in the geminate kinetics suggests that the presence of camphor significantly alters the CO rebinding and escape rates by modifying the heme pocket environment. Two geminate phases and two bimolecular rebinding phases in the substrate free protein were observed, which could arise from slowly interconverting protein conformations. When the temperature or the viscosity of the solution is changed, the fast geminate rate remains the same, whereas the slow geminate rate and the two bimolecular rates change significantly. The geminate rebinding yield of substrate-free P420 is smaller than that of substrate free P450, but its geminate rebinding rate is faster. This demonstrates that in the absence of substrate, CO escapes from the pocket of P420 much more rapidly than from P450 and suggests that the distal pocket environment is altered in the P420 form.

Investigations of ligand association and dissociation rates in the "open" and "closed" states of myoglobin.

Kinetic and Raman spectroscopic studies are combined to analyze ligand association and dissociation rates as a function of pH in aqueous solutions of myoglobin. A double-pulse flash photolysis protocol is used to kinetically select a rapidly rebinding (open pocket) fraction of the myoglobin ensemble and determine the timescale for averaging (approximately 1 to 10 microseconds) between the "open" and "closed" distal pocket protein conformations. Since this timescale is fast compared to the rate of ligand migration from the solution to the heme pocket (approximately 10(-4)s), a time-averaged population analysis, rather than a superposition of states, can be used to describe the ligand association and dissociation kinetics. Raman spectroscopy provides the relative populations of the open and closed distal pocket states as a function of pH which, in parallel with kinetics measurements, are used to determine the rates for ligand association and dissociation specific to these states. In aqueous solution at 293 K (1 mM CO) we find kon0 = 5.6 x 10(3) s-1, koff0 = 8.5 x 10(-2) s-1 for the open state and kon1 = 5.0 x 10(2) s-1, koff1 = 1.3 x 10(-2) s-1 for the closed state. The order of magnitude increase in the dissociation and association rates of the open form suggests that it may play a significant role in the ligand binding process, even though it comprises only approximately 5% of the time-averaged population at pH 7. For oxygen binding at 293 K (1.36 mM O2) we find kon0 = 4.6 x 10(4) s-1, koff0 approximately 10(4 +/- 2) s-1 for the open state and kon1 = 2.0 x 10(4) s-1, koff1 = 13 s-1 for the closed state. The dramatic increase in the dissociation rate of the open form is probably due to the loss of the hydrogen bond with the distal histidine, which stabilizes the bound O2 in the closed state. Overall, these results demonstrate that the open conformation plays a significant role in determining the ligand association and dissociation rates and suggest that environmentally induced modulations of the open population could be used as a biomolecular control mechanism for the uptake and delivery of oxygen in muscle cells.