Regulation of lung injury and fibrosis by p53-mediated changes in urokinase and plasminogen activator inhibitor-1.

Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.

Early secreted antigenic target of 6-kDa protein of Mycobacterium tuberculosis primes dendritic cells to stimulate Th17 and inhibit Th1 immune responses.

Early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis is a T cell Ag that is a potential vaccine candidate, but it is also a virulence factor that mediates pathogenicity. To better understand the effects of ESAT-6 on the immune response, we studied the effect of ESAT-6 on human dendritic cells (DCs). Peripheral blood monocytes were treated with GM-CSF and IL-4 to yield immature DCs, which were matured by addition of LPS and CD40 ligand (CD40L), with or without ESAT-6. ESAT-6 inhibited LPS/CD40L-induced DC expression of costimulatory molecules, reduced DC-stimulated allogeneic T cell proliferation and IL-2 and IFN-γ production, and enhanced IL-17 production. ESAT-6-treated DCs also increased IL-17 and reduced IFN-γ production by M. tuberculosis-specific autologous T cells. ESAT-6 inhibited LPS/CD40L-induced DC production of IL-12 and enhanced that of IL-23 and IL-1ß, without affecting secretion of TNF-α, IL-6, or IL-8 through specific interaction with immature DCs. The effects of ESAT-6 were not mediated through cAMP or p38 MAPK. Medium from ESAT-6-conditioned DCs increased IL-17 and reduced IFN-γ production by T cells stimulated with anti-CD3 plus anti-CD28, and ESAT-6-induced IL-17 production was blocked by neutralizing both IL-23 and IL-1ß. ESAT-6 reduced LPS/CD40L-stimulated transcription of IL-12p35 and enhanced that of IL-23p19 through inhibition of IFN regulatory factor-1 and upregulation of activating transcription factor-2 and c-Jun, transcriptional regulators of IL-12p35 and IL-23p19, respectively. We conclude that ESAT-6 increases DC production of IL-23 and IL-1ß while inhibiting that of IL-12, thus enhancing Th17 at the expense of protective Th1 responses.

Induction of tissue factor by urokinase in lung epithelial cells and in the lungs.

RATIONALE: Urokinase-type plasminogen activator (uPA) regulates extracellular proteolysis in lung injury and repair. Although alveolar expression of uPA increases, procoagulant activity predominates. OBJECTIVES: This study was designed to investigate whether uPA alters the expression of tissue factor (TF), the major initiator of the coagulation cascade, in lung epithelial cells (ECs). METHODS: Bronchial, primary airway ECs and C57B6 wild-type, uPA-deficient (uPA(-/-)) mice were exposed to phosphate-buffered saline, uPA, or LPS. Immunohistochemistry, protein, cellular, and molecular techniques were used to assess TF expression and activity. MEASUREMENTS AND MAIN RESULTS: uPA enhanced TF mRNA and protein expression, and TF-dependent coagulation in lung ECs. uPA-induced expression of TF involves both increased synthesis and enhanced stabilization of TF mRNA. uPA catalytic activity had little effect on induction of TF. By contrast, deletion of the uPA receptor binding growth factor domain from uPA markedly attenuated the induction of TF, suggesting that uPA receptor binding is sufficient for TF induction. Lung tissues of uPA-deficient mice expressed less TF protein and mRNA compared with wild-type mice. In addition, intratracheal instillation of mouse uPA increased TF mRNA and protein expression and accelerated coagulation in lung tissues. uPA(-/-) mice exposed to LPS failed to induce TF. CONCLUSIONS: uPA increased TF expression and TF-dependent coagulation in the lungs of mice. We hypothesize that uPA-mediated induction of TF occurs in lung ECs to promote increased fibrin deposition in the airways during acute lung injury.

Inhibition of Platelet Adhesion from Surface Modified Polyurethane Membranes.

Coronary thrombosis is one of the leading causes of mortality and morbidity in cardiovascular diseases, and patients who received vascular stent treatments are likely to suffer from restenosis due to tissue damage from stenting procedures (extrinsic pathway) and/or presence of unregulated factor XII (intrinsic pathway). Regardless of the pathway, coagulation factors and exposed collagen activate the G-protein-coupled receptors located at the plasma membrane of the resting platelets resulting in the change of their shapes with protrusions of filopodia and lamellipodia for surface adhesion. In this mini review, we discussed the mechanisms involved in platelet activation, adhesion, and aggregation. More importantly, we reviewed the use of polyurethane membranes with modified surface functional groups to down-regulate platelet adhesion and aggregation activities. Polyurethane membranes with hydrophilic and negatively charged surface properties showed a reduced αIIb-ß3 signaling from the activated platelets, resulting in the decrease of platelet adhesion and aggregation. The use of polyurethane membranes with modified surface properties as coatings on vascular stents provides an engineering approach to mitigate blood clotting associated with restenosis.

Engineering Approaches to Prevent Blood Clotting from Medical Implants.

Injectable and/or Implantable medical devices are widely used in the treatment of diseases. Among them, vascular stents provide the medical solution to treat blood clotting. However, traditional metallic stents, even with current improvements in anticoagulation properties, have potential drawbacks in local inflammation when first implanted into the body and undesirable protein adsorption and cell adhesion after a prolonged period of time in the body. In this perspective, we discuss several engineering approaches, including drug-eluting materials, polymeric and non-polymeric coatings, and surface modifications to coating materials that can be applied to the surface of medical implants to significantly improve the hemocompatibility. These coatings are expected to have a slow degradation rate with the ability to either load drugs or attach biomacromolecules to form an architecture that mimics the surrounding cells. In general, our perspective provides a current view on the achievements of hemo-compatible coatings and future trends in coating materials that will extend the life of the medical implants.

Thermal and Physico-Mechanical Characterizations of Thromboresistant Polyurethane Films.

Hemocompatibility remains a challenge for injectable and/or implantable medical devices, and thromboresistant coatings appear to be one of the most attractive methods to down-regulate the unwanted enzymatic reactions that promote the formation of blood clots. Among all polymeric materials, polyurethanes (PUs) are a class of biomaterials with excellent biocompatibility and bioinertness that are suitable for the use of thromboresistant coatings. In this work, we investigated the thermal and physico-mechanical behaviors of ester-based and ether-based PU films for potential uses in thromboresistant coatings. Our results show that poly(ester urethane) and poly(ether urethane) films exhibited characteristic peaks corresponding to their molecular configurations. Thermal characterizations suggest a two-step decomposition process for the poly(ether urethane) films. Physico-mechanical characterizations show that the surfaces of the PU films were hydrophobic with minimal weight changes in physiological conditions over 14 days. All PU films exhibited high tensile strength and large elongation to failure, attributed to their semi-crystalline structure. Finally, the in vitro clotting assays confirmed their thromboresistance with approximately 1000-fold increase in contact time with human blood plasma as compared to the glass control. Our work correlates the structure-property relationships of PU films with their excellent thromboresistant ability.

Nanostructure-Enabled and Macromolecule-Grafted Surfaces for Biomedical Applications.

Advances in nanotechnology and nanomaterials have enabled the development of functional biomaterials with surface properties that reduce the rate of the device rejection in injectable and implantable biomaterials. In addition, the surface of biomaterials can be functionalized with macromolecules for stimuli-responsive purposes to improve the efficacy and effectiveness in drug release applications. Furthermore, macromolecule-grafted surfaces exhibit a hierarchical nanostructure that mimics nanotextured surfaces for the promotion of cellular responses in tissue engineering. Owing to these unique properties, this review focuses on the grafting of macromolecules on the surfaces of various biomaterials (e.g., films, fibers, hydrogels, and etc.) to create nanostructure-enabled and macromolecule-grafted surfaces for biomedical applications, such as thrombosis prevention and wound healing. The macromolecule-modified surfaces can be treated as a functional device that either passively inhibits adverse effects from injectable and implantable devices or actively delivers biological agents that are locally based on proper stimulation. In this review, several methods are discussed to enable the surface of biomaterials to be used for further grafting of macromolecules. In addition, we review surface-modified films (coatings) and fibers with respect to several biomedical applications. Our review provides a scientific update on the current achievements and future trends of nanostructure-enabled and macromolecule-grafted surfaces in biomedical applications.

The effects of compounded bioidentical transdermal hormone therapy on hemostatic, inflammatory, immune factors; cardiovascular biomarkers; quality-of-life measures; and health outcomes in perimenopausal and postmenopausal women.

Menopause impacts 25 million women world wide each year, and the World Health Organization estimates 1.2 billion women will be postmenopausal by 2030. Menopause has been associated with symptoms of hot flashes, night sweats, dysphoric mood, sleep disturbance, and conditions of cardiovascular disease, depression, osteoporosis, osteoarthritis, depression, dementia, and frailty. Conventional hormone replacement therapy results in increased thrombotic events, and an increased risk of breast cancer and dementia as evidenced in large prospective clinical trials including Heart and Estrogen/Progestin Replacement Study I and the Women's Health Initiative. A possible mechanism for these adverse events is the unfavorable net effects of conjugated equine estrogens and medroxyprogesterone acetate on the hemostatic balance and inflammatory and immune factors. Physiologic sex steroid therapy with transdermal delivery for peri/postmenopausal women may offer a different risk/benefit profile, yet long-term studies of this treatment model are lacking. The objective of this study was to examine the long-term effects of compounded bioidentical transdermal sex steroid therapy including estriol, estradiol, progesterone, DHEA, and testosterone on cardiovascular biomarkers, hemostatic, inflammatory, immune signaling factors; quality-of-life measures; and health outcomes in peri/postmenopausal women within the context of a hormone restoration model of care. A prospective, cohort, closed-label study received approval from the Human Subjects Committee. Recruitment from outpatient clinics at an academic medical center and the community at large resulted in three hundred women giving signed consent. Seventy-five women who met strict inclusion/exclusion criteria were enrolled. Baseline hormone evaluation was performed along with baseline experimental measures. Following this, women received compounded transdermal bioidentical hormone therapy of BiEst (80%Estriol/20%Estradiol), and/or Progesterone for eight weeks to meet established physiologic reference ranges for the luteal phase in premenopausal women. The luteal phase hormone ratios were selected based on animal and epidemiologic studies demonstrating favorable outcomes related to traumatic, ischemic, or neuronal injury. Follow-up testing was performed at eight weeks and adjustment to hormone regimens were made including addition of androgens of DHEA and Testosterone if indicated. Experimental subjects were monitored for 36 months. Baseline, 2-month, and annual values were obtained for: blood pressure, body mass index, fasting glucose, Homeostasis Metabolic Assessment of Insulin Resistance (HOMA-IR), fasting triglycerides, total Factor VII, Factor VIII, fibrinogen, Antithrombin III, Plasminogen Activator Inhibitor1(PAL-1), C-reactive protein (CRP), Interleukin-6 (IL-6), Matrix Metalloproteinase-9 (MMP-9), Tumor Necrosis Factor-alpha (TNF), Insulin-like Growth Factor (IGF-1), and sex steroid levels. Psychosocial measures included: Greene Climacteric Scale, Visual Analog Pain Scale, Hamilton Anxiety Scale, Hamilton Depression Scale, Holmes Rahe Stress Scale, Job Strain, and Home Strain. Health outcome measures included the number of prescribed medications used, number of co-morbidities, and endometrial thickness in postmenopausal women with intact uteri. Subjects receiving compounded transdermal bioidentical hormone therapy showed significant favorable changes in: Greene Climacteric Scale scores, Hamilton Anxiety Scale, Hamilton Depression Scale, Visual Analog Pain Scale, fasting glucose, fasting triglycerides, MMP-9, C-reactive Protein, fibrinogen, Factor VII, Factor VIII, Insulin-Like Growth Factor 1, and health outcomes of co-morbidities and a number of prescribed medications. Antithrombin III levels were significantly decreased at 36 months. All other measures did not exhibit significant effects. Administration of compounded transdermal bioidentical hormone therapy in doses targeted to physiologic reference ranges administered in a daily dose significantly relieved menopausal symptoms in peri/postmenopausal women. Cardiovascular biomarkers, inflammatory factors, immune signaling factors, and health outcomes were favorably impacted, despite very high life stress, and home and work strain in study subjects. The therapy did not adversely alter the net prothrombotic potential, and there were no associated adverse events. This model of care warrants consideration as an effective and safe clinical therapy for peri/postmenopausal women especially in populations with high perceived stress and a history of stressful life events prior to, or during the menopausal transition.

The ß-propeller gene Rv1057 of Mycobacterium tuberculosis has a complex promoter directly regulated by both the MprAB and TrcRS two-component systems.

The ß-propeller gene Rv1057 of Mycobacterium tuberculosis is activated by envelope stress and was first characterized as a regulatory target of the TrcRS two-component system (TCS). Rv1057 expression is repressed by TrcRS, and the Rv1057 proximal promoter contains a TrcR binding site. In this study, we determined that Rv1057 is also directly regulated by MprAB, a TCS associated with envelope stress. Multiple potential MprA binding sites (MprA boxes) were identified in the 1 kb intergenic region upstream of Rv1057, and four sites were shown to bind MprA. Although MprA boxes were found in the proximal promoter, analyses suggest that MprA and TrcR do not compete for binding in this region. An MprAB-dependent, detergent-inducible transcriptional start point for Rv1057 was identified downstream of the MprA boxes, and a second TrcR binding site and small ORF of the 13E12 family were discovered in the distal promoter. MprAB was required for activation of Rv1057 during growth in macrophages and under detergent stress, and lacZ promoter constructs suggest the entire intergenic region is utilized during MprAB-dependent activation of Rv1057. These findings indicate that Rv1057 has an extensive and complex promoter, and provide evidence for coordinated regulation of stress response genes by TCSs.

Analysis of factor VIIa binding to relipidated tissue factor by surface plasmon resonance.

Kinetic analysis of the tissue factor (TF)-factor VIIa (FVIIa) binding interaction is helpful in investigating the structure-function relationships of TF-FVIIa. However, a wide variation exists among the reported binding affinities of FVIIa to TF, particularly when comparing KD values obtained from functional activity assays versus ligand binding studies. Surface plasmon resonance (SPR) technique was used frequently to investigate binding kinetics of FVIIa to TF in a lipid-free environment. In the present study we used TF embedded in a phospholipid bilayer for determining binding kinectis using SPR. The data revealed that FVIIa had a much higher binding affinity (>100-fold) for TF embedded in the phospholiid bilayer than TF in a lipid-free environment, approaching the KD values that were noted in the enzymatic activity assays. The present data suggest that SPR binding studies using TF embedded in phospholipids is more appropriate for investigating how FVIIa (or FVIIa mutants/derivatives) may interact with TF in physiological settings.

Transdermal progesterone: effects on menopausal symptoms and on thrombotic, anticoagulant, and inflammatory factors in postmenopausal women.

Conventional hormone replacement therapy increases a woman's risk of thrombotic events as evidenced in large prospective clinical trials, including HERS I and the Women's Health Initiative. A possible mechanism for this is the unfavorable net effects of conjugated equine estrogens and medroxyprogesterone acetate on factors involved in hemostatic balance and inflammation. The objective of this study was to examine the short-term effects of transdermal progesterone on menopausal symptoms and serum levels of hemostatic, inflammatory, and immune signaling factors. In a prospective, randomized, double-blinded, placebo-controlled, crossover study, 30 healthy postmenopausal women received either 20 mg/day of transdermal progesterone or placebo for 4 weeks, followed by a 4-week washout period, and were then crossed over to receive either placebo or active drug for an additional 4 weeks. Baseline, 4-week follow-up, and end-of-study values were obtained for the Greene Climacteric Scale, and for serum levels of total factor VII:C, factor VIIa, factor V, fibrinogen, antithrombin III, plasminogen activator inhibitor-1, C-reactive protein, interleukin-6, matrix metalloproteinase-9, and tumor necrosis factor-a. Transdermal progesterone significantly improved Greene Climacteric Scale scores. In sharp contrast to previous studies of conventional hormone replacement therapy, no detrimental effect was observed on any of the hemostatic or inflammatory components examined. Administration of transdermal progesterone at a daily dose of 20 mg significantly relieves menopausal symptoms in postmenopausal women without adversely altering prothrombotic potential. We suggest, therefore, that this treatment be seriously considered as an effective and safe alternative clinical therapy for women suffering from menopausal symptoms.

Heparin modulates the 99-loop of factor IXa: effects on reactivity with isolated Kunitz-type inhibitor domains.

Reactivity of factor IXa with basic pancreatic trypsin inhibitor is enhanced by low molecular weight heparin (enoxaparin). Previous studies by us have suggested that this effect involves allosteric modulation of factor IXa. We examined the reactivity of factor IXa with several isolated Kunitz-type inhibitor domains: basic pancreatic trypsin inhibitor, the Kunitz inhibitor domain of protease Nexin-2, and the first two inhibitor domains of tissue factor pathway inhibitor. We find that enhancement of factor IXa reactivity by enoxaparin is greatest for basic pancreatic trypsin inhibitor (>10-fold), followed by the second tissue factor pathway inhibitor domain (1.7-fold) and the Kunitz inhibitor domain of protease Nexin-2 (1.4-fold). Modeling studies of factor IXa with basic pancreatic trypsin inhibitor suggest that binding of this inhibitor is sterically hindered by the 99-loop of factor IXa, specifically residue Lys(98). Slow-binding kinetic studies support the formation of a weak initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of Lys(98) to Ala in factor IXa results in enhanced reactivity with all inhibitors examined, whereas almost completely abrogating the enhancing effects of enoxaparin. The results implicate Lys(98) and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite.

The intrinsic ATPase activity of Mycobacterium tuberculosis DnaA promotes rapid oligomerization of DnaA on oriC.

Oligomerization of the initiator protein, DnaA, on the origin of replication (oriC) is crucial for initiation of DNA replication. Studies in Escherichia coli (Gram-negative) have revealed that binding of DnaA to ATP, but not hydrolysis of ATP, is sufficient to promote DnaA binding, oligomerization and DNA strand separation. To begin understanding the initial events involved in the initiation of DNA replication in Mycobacterium tuberculosis (Gram-positive), we investigated interactions of M. tuberculosis DnaA (DnaA(TB)) with oriC using surface plasmon resonance in the presence of ATP and ADP. We provide evidence that, in contrast to what is observed in E. coli, ATPase activity of DnaA(TB) promoted rapid oligomerization on oriC. In support, we found that a recombinant mutant DnaA(TB) proficient in binding to ATP, but deficient in ATPase activity, did not oligomerize as rapidly. The corresponding mutation in the dnaA gene of M. tuberculosis resulted in non-viability, presumably due to a defect in oriC-DnaA interactions. Dimethy sulphate (DMS) footprinting experiments revealed that DnaA(TB) bound to DnaA boxes similarly with ATP or ADP. DnaA(TB) binding to individual DnaA boxes revealed that rapid oligomerization on oriC is triggered only after the initial interaction of DnaA with individual DnaA boxes. We propose that ATPase activity enables the DnaA protomers on oriC to rapidly form oligomeric complexes competent for replication initiation.

Exosite occupation by heparin enhances the reactivity of blood coagulation factor IXa.

Blood coagulation factor IXa (fIXa) is a trypsin-like serine protease with low inherent activity that is greatly enhanced in the factor X activation complex. Molecular details of the conversion of fIXa from an inactive enzyme into a fully functional procoagulant are unclear. Recent studies have identified a heparin-binding exosite in the protease domain of fIXa. Effects of exosite occupation on fIXa activity are unclear. We used the Kunitz-type inhibitor bovine pancreatic trypsin inhibitor (BPTI) to probe fIXa reactivity in the absence and in the presence of heparin. While fIXa alone was poorly reactive with BPTI (K(i) approximately 0.7 mM), this reactivity was increased roughly 20-fold (K(i) = 37 +/- 6 microM) by heparin. This was reproducible with low-molecular-weight heparin (enoxaparin; K(i) = 70 +/- 12 microM). Surface plasmon resonance studies of the interaction between heparin and BPTI indicated an unstable interaction with very low affinity (K(d) = 172 microM). In contrast, kinetic studies revealed a high-affinity interaction between heparin and fIXa (K(d) = 128 +/- 26 nM) and showed that the enhancement of BPTI inhibition of fIXa by heparin was well described by a competitive inhibition model where heparin acts as an affecter of fIXa reactivity with inhibitor. Fluorescence studies with dansyl-EGR-fIXa supported the high-affinity interaction between heparin and fIXa and suggested an altered environment in the fIXa active-site region upon heparin binding. This modulating effect of heparin was supported by the observation of a heparin-induced increase in reactivity of fIXa toward a pentapeptide substrate. When viewed together, the results imply that specific physiological exosite interactions with heparin can induce alterations in the environment of the extended fIXa active site that can result in increased reactivity.

Tissue factor alters the pK(a) values of catalytically important factor VIIa residues.

Blood coagulation is triggered when the serine protease factor VIIa (fVIIa) binds to cell surface tissue factor (TF) to form the active enzyme-cofactor complex. TF binding to fVIIa allosterically augments the enzymatic activity of fVIIa toward macromolecular substrates and small peptidyl substrates. The mechanism of this enhancement remains unclear. Our previous studies have indicated that soluble TF (sTF; residues 1-219) alters the pH dependence of fVIIa amidolytic activity (Neuenschwander et al. (1993) Thromb. Haemostasis 70, 970), indicating an effect of TF on critical ionizations within the fVIIa active center. The pKa values and identities of these ionizable groups are unknown. To gain additional insight into this effect, we have performed a detailed study of the pH dependence of fVIIa amidolytic activity. Kinetic constants of Chromozym t-PA (MeSO(2)-D-Phe-Gly-Arg-pNA) hydrolysis at various pH values were determined for fVIIa alone and in complex with sTF. The pH dependence of both enzymes was adequately represented using a diprotic model. For fVIIa alone, two ionizations were observed in the free enzyme (pK(E1) = 7.46 and pK(E2) = 8.67), with at least a single ionization apparent in the Michaelis complex (pK(ES1) similar 7.62). For the fVIIa-TF complex, the pK(a) of one of the two important ionizations in the free enzyme was shifted to a more basic value (pK(E1) = 7.57 and pK(E2) = 9.27), and the ionization in the Michaelis complex was possibly shifted to a more acidic pH (pK(ES1) = 6.93). When these results are compared to those obtained for other well-studied serine proteases, K(E1) and K(ES1) are presumed to represent the ionization of the overall catalytic triad in the absence and presence of substrate, respectively, while K(E2) is presumed to represent ionization of the alpha-amino group of Ile(153). Taken together, these results would suggest that sTF binding to fVIIa alters the chemical environment of the fVIIa active site by protecting Ile(153) from deprotonation in the free enzyme while deprotecting the catalytic triad as a whole when in the Michaelis complex.

Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity.

To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.