Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

Background: Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity. Results: Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system. Conclusions: Our results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

Novel Primate-Specific Genes, RMEL 1, 2 and 3, with Highly Restricted Expression in Melanoma, Assessed by New Data Mining Tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

Videodensitometric analysis of advanced carotid plaque: correlation with MMP-9 and TIMP-1 expression

Background: Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of MMP (TIMP) promote derangement of the extracellular matrix, which is ultimately reflected in plaque images seen on ultrasound. Videodensitometry can identify structural disturbances in plaques. Objectives: To establish the correlations between values determined using videodensitometry in B-mode ultrasound images of advanced carotid plaques and the total expression of MMP-9 and TIMP-1 in these removed plaques. Methods: Thirty patients underwent ultrasonic tissue characterization of carotid plaques before surgery, using mean gray level (MGL), energy, entropy and homogeneity. Each patient was assigned preoperatively to one of 2 groups: group I, symptomatic patients (n = 16; 12 males; mean age 66.7 +/- 6.8 years), and group II, asymptomatic patients (n = 14; 8 males; mean age 67.6 +/- 6.81 years). Tissue specimens were analyzed for MMP-9 and TIMP-1 expression. Nine carotid arteries were used as normal tissue controls. Results: MMP-9 expression levels were elevated in group II and in normal tissues compared to group I (p < 0.001). TIMP-1 levels were higher in group II than in group I, and significantly higher in normal tissues than in group I (p = 0.039). The MGL was higher in group II compared to group I (p = 0.038). Energy had greater values in group II compared to group I (p = 0.02). There were no differences between patient groups in homogeneity and entropy. Energy positively correlated with MMP-9 and TIMP-1 expression (p = 0.012 and p = 0.031 respectively). Homogeneity positively correlated with MMP-9 and TIMP-1 expression (p = 0.034 and p = 0.047 respectively). There were no correlations between protein expression and MGL or entropy. Conclusions: Videodensitometric computer analysis of ultrasound scanning images can be used to identify stable carotid plaques, which have higher total expression levels of MMP-9 and TIMP-1 than unstable plaques.

Genotypic Characteristics of HIV Type 1 Based on gp120 Hypervariable Region 3 of Isolates from Southern Brazil

The aim of this study was to investigate HIV-1 molecular diversity and the epidemiological profile of HIV-1-infected patients from Ribeirao Preto, Brazil. A nested PCR followed by sequencing of a 302-base pair fragment of the env gene (C2-V3 region) was performed in samples from HIV-1-positive patients. A total of 45 sequences were aligned with final manual adjustments. The phylogenetic analyses showed a higher prevalence of HIV-1 subtype B in the studied population (97.8%) with only one sample yielding an F1 subtype. The viral genotyping prediction showed that CCR5 tropism was the most prevalent in the studied cohort. Geno2pheno analysis showed that R5 and CXCR4 prediction were 69% and 31%, respectively. There was no statistical significance, either in viral load or in CD4(+) T cell count when R5 and X4 prediction groups were compared. Moreover, the GPGR tetramer was the most common V3 loop core motif identified in the HIV-1 strains studied (34.1%) followed by GWGR, identified in 18.1% of the samples. The high level of B subtype in this Brazilian population reinforces the nature of the HIV epidemic in Brazil, and corroborates previous data obtained in the Brazilian HIV-infected population.

Caspase-1 is involved in the genesis of inflammatory hypernociception by contributing to peripheral IL-1 beta maturation

Background: Caspase-1 is a cysteine protease responsible for the processing and secretion of IL-1 beta and IL-18, which are closely related to the induction of inflammation. However, limited evidence addresses the participation of caspase-1 in inflammatory pain. Here, we investigated the role of caspase-1 in inflammatory hypernociception (a decrease in the nociceptive threshold) using caspase-1 deficient mice (casp1-/-). Results: Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. The production of cytokines, PGE(2) and neutrophil migration were evaluated by ELISA, radioimmunoassay and myeloperoxidase activity, respectively. The interleukin (IL)-1 beta and cyclooxygenase (COX)-2 protein expression were evaluated by western blotting. The mechanical hypernociception induced by intraplantar injection of carrageenin, tumour necrosis factor (TNF)alpha and CXCL1/KC was reduced in casp1-/- mice compared with WT mice. However, the hypernociception induced by IL-1 beta and PGE(2) did not differ in WT and casp1-/- mice. Carrageenin-induced TNF-alpha and CXCL1/KC production and neutrophil recruitment in the paws of WT mice were not different from casp1-/- mice, while the maturation of IL-1 beta was reduced in casp1-/- mice. Furthermore, carrageenin induced an increase in the expression of COX-2 and PGE(2) production in the paw of WT mice, but was reduced in casp1-/- mice. Conclusion: These results suggest that caspase-1 plays a critical role in the cascade of events involved in the genesis of inflammatory hypernociception by promoting IL-1 beta maturation. Because caspase-1 is involved in the induction of COX-2 expression and PGE(2) production, our data support the assertion that caspase-1 is a key target to control inflammatory pain.

Antiretroviral resistance among HIV type 1-infected women first exposed to antiretrovirals during pregnancy: Plasma versus PBMCs

Resistance-associated mutations (RAMs) in plasma samples from HIV-1-infected women who received antiretroviral (ARV) prophylaxis during pregnancy was assessed and correlated with the detection of RAMs in peripheral blood mononuclear cells (PMBCs). The study population was composed of HIV-1-infected women enrolled in a prospective cohort study in Latin America and the Caribbean (NISDI Perinatal Study) as of March 1, 2005, who were diagnosed with HIV-1 infection during the current pregnancy, who received ARVs during pregnancy for prevention of mother-to-child transmission of HIV-1, and who were followed through at least the 6-12 week postpartum visit. Plasma samples collected at enrollment during pregnancy and at 6-12 weeks postpartum were assayed for RAMs. Plasma results were compared to previously described PBMC results from the same study population. Of 819 enrolled subjects, 197 met the eligibility criteria. Nucleic acid amplification was accomplished in 123 plasma samples at enrollment or 6-12 weeks postpartum, and RAMs were detected in 22 (17.9%; 95% CI: 11.7-25.9%). Previous analyses had demonstrated detection of RAMs in PBMCs in 19 (16.1%). There was high concordance between RAMs detected in plasma and PBMC samples, with only eight discordant pairs. The prevalence of RAMs among these pregnant, HIV-1-infected women is high (>15%). Rates of detection of RAMs in plasma and PBMC samples were similar.

HLA-G 14-bp Insertion/Deletion Polymorphism Is a Risk Factor for HTLV-1 Infection

About 95% of HTLV-1 infected patients remain asymptomatic throughout life, and the risk factors associated with the development of related diseases, such as HAM/TSP and ATL, are not fully understood. The human leukocyte antigen-G molecule (HLA-G), a nonclassical HLA class I molecule encoded by MHC, is expressed in several pathological conditions, including viral infection, and is related to immunosuppressive effects that allow the virus-infected cells to escape the antiviral defense of the host. The 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene influences the stability of the transcripts and could be related to HTLV-1-infected cell protection and to the increase of proviral load. The present study analyzed by conventional PCR the 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene in 150 unrelated healthy subjects, 82 HTLV-1 infected patients with symptoms (33 ATL and 49 HAM), and 56 asymptomatic HTLV-1 infected patients (HAC). In addition, the proviral load was determined by quantitative real-time PCR in all infected groups and correlated with 14-bp insertion/deletion genotypes. The heterozygote genotype frequencies were significantly higher in HAM, in the symptomatic group, and in infected patients compared to control (p < 0.05). The proviral load was higher in the symptomatic group than the HAC group (p < 0.0005). The comparison of proviral load and genotypes showed that -14-bp/-14-bp genotype had a higher proviral load than +14-bp/-14-bp and +14-bp/+14-bp genotypes. Although HLA-G 14-bp polymorphism does not appear to be associated

Notes on Population Dynamics of Amblyomma Ticks (Acari: Ixodidae) in Brazil

Previous population dynamics data, generated for Amblyomma parvum Aragao and Aniblyomma cajennense (Fabricius) in Argentina and southeastern Brazil, have indicated that these ticks complete I generation per year, with larvae predominating in autumn, nymphs in winter, and mostly adults during spring and summer. The present study reports population dynamics data for free-living Amblyomma spp. ticks in northern Brazil (Amazon forest, latitude 10 degrees S, 63 degrees W), and for Amblyomma spp. ticks collected oil birds in Southeastern Brazil (latitude 23 degrees S, 45 degrees W). In northern Brazil, adult ticks predominated from mid-spring to mid-autumn, larvae predominated in early winter, and nymphs from mid-winter to mid-spring. Seven Amblyomma spp. were identified, although A. cajannense predominated in I of the 2 sites sampled. In southeastern Brazil, larval infestations on birds peaked in autumn, followed by a nymphal infestation peak in late winter. At least 32% and 75% of these larvae and nymphs, respectively, were identified as Amblyomma longirostre (Koch). Similar to previous work, the present study showed that Amblyomma spp. larvae and nymphs predominated during autumn-winter months, and mostly adults during spring-summer months, a pattern compatible with 1 genration/yr, even at latitude 10 degrees S in northern Brazil.

Alstonine as an Antipsychotic: Effects on Brain Amines and Metabolic Changes

Managing schizophrenia has never been a trivial matter. Furthermore, while classical antipsychotics induce extrapyramidal side effects and hyperprolactinaemia, atypical antipsychotics lead to diabetes, hyperlipidaemia, and weight gain. Moreover, even with newer drugs, a sizable proportion of patients do not show significant improvement. Alstonine is an indole alkaloid identified as the major component of a plant-based remedy used in Nigeria to treat the mentally ill. Alstonine presents a clear antipsychotic profile in rodents, apparently with differential effects in distinct dopaminergic pathways. The aim of this study was to complement the antipsychotic profile of alstonine, verifying its effects on brain amines in mouse frontal cortex and striatum. Additionally, we examined if alstonine induces some hormonal and metabolic changes common to antipsychotics. HPLC data reveal that alstonine increases serotonergic transmission and increases intraneuronal dopamine catabolism. In relation to possible side effects, preliminary data suggest that alstonine does not affect prolactin levels, does not induce gains in body weight, but prevents the expected fasting-induced decrease in glucose levels. Overall, this study reinforces the proposal that alstonine is a potential innovative antipsychotic, and that a comprehensive understanding of its neurochemical basis may open new avenues to developing newer antipsychotic medications.

Effect of Hormonal Supplementation Periods and In Vitro Maturation Media on Developmental Competence of Pig Oocytes

Background: The oocyte ability to undergo successful fertilization, cleavage and embryonic development depends on meiotic maturation and developmental competence acquisition. In vitro maturation (IVM) protocols currently use eCG, hCG or a combination of both, the effect of these gonadotrophins during IVM and subsequent embryonic development is still controversial. Several media have been used for IVM of porcine oocytes: TCM199, Whitten's and NCSU23 have also been shown to support pig oocyte IVM. This study was designed to determine the effect of hormonal supplementation period and maturation media during in vitro maturation of pig oocytes (1) and subsequent embryonic development (2). Materials, Methods & Results: Oocytes with intact cumulus oophurus layers and homogeneous cytoplasm were collected from prebubertal gilts. IVM was subjected in NCSU23, TCM199 or Whitten's media supplemented with 10 IU/mL eCG and 10 IU/mL hCG for the first 24 or 48 h of IVM. In each replicate the oocytes were fixed every 4 h from 32 to 48 h IVM or the past 48 h after IVM, oocytes were fertilized in vitro in mTBM medium for six hours and cultured in NCSU23 medium for nine days. Cleavage, blastocyst and hatching rates were evaluated at 48 h (day 2), 168 h (day 7) and 216 h (day 9), respectively. The addition of eCG and hCG during the first 24 h IVM increased the proportion of oocytes that reached MII stage at 44 h of maturation in NCSU23 medium. This effect was also observed in Whitten medium at 44 and 48 h (P < 0.05). However, it was not observed in the TCM199 medium. No effect of maturation medium on oocyte nuclear maturation (P > 0.05) was observed in oocytes matured in the presence of eCG and hCG during the first 24 h IVM or during 48 h IVM. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation in Whitten media for 24 h. Higher indexes were obtained at 44 and 48 h. When NCSU23 media was used, no difference after 36 h of maturation was observed. The same result was observed in TCM199. A progressive increase of maturation indexes was observed on oocytes matured with hormonal supplementation for 48 h in Whitten media. Higher indexes were obtained in 36 and 40 h. When NCSU23 or TCM199 were used, no difference was observed. No effect of IVM media on the percentage of fertilized oocytes and polyspermic oocytes or number of spermatozoa per fertilized oocytes was observed. Also, no effect of IVM media on cleavage and blastocyst rates was seen. However, the proportion of hatched blastocysts was lower in NCSU23 compared to Whitten or TCM199. Discussion: Similar results were reported by Marques et al. [13], that it no differences between hormonal supplementation for 22 or 44 h were observed. Therefore, more studies are needed to elucidate the role of these hormones in nuclear in vitro maturation in pig oocytes. In conclusion, no effect of maturation media on meiotic progression was observed. However, the proportion of oocytes that reached metaphase II (MII) stage was higher when eCG + hCG were added for 24 h than 48 h mainly at the 44 h of maturation. In addition, no differences were observed in cleavage and blastocyst rates of the cultured embryos. However, embryos cultured in NCSU23 showed lower rates of hatching compared to other media. These results indicated no effect of maturation media on the fertilization and embryonic development even in the presence of cysteine, PFF and EGF, except for hatched embryos that these rates were lower in NCSU23.

Effects of Er:YAG Laser Irradiation on the Microtensile Bond Strength to Bleached Enamel

Objective: The objective of this study was to evaluate the influence of different Er:YAG laser (lambda = 2.94 mu m) energy parameters on the microtensile bond strength (mu TBS) and superficial morphology of bovine enamel bleached with 16% carbamide peroxide. Background: Laser irradiation could improve adhesion to bleached enamel surfaces. Methods: Sixty bovine enamel blocks (7x3x3 mm(3)) were randomly assigned to six groups according to enamel preparation procedures (n = 10): G1-bleaching and Er:YAG laser irradiation with 25.52 J/cm(2) (laser A, LA); G2-bleaching and Er:YAG laser irradiation with 4.42J/cm(2) (laser B, LB); G3-bleaching; G4-Er:YAG laser irradiation with 25.52 J/cm(2); G5-Er:YAG laser irradiation with 4.42J/cm(2); G6-control, no treatment. G1 to G3 were bleached for 6 h during 21 days. Afterwards, enamel surfaces in all groups were slightly abraded with 600-grit SiC papers and G1, G2, G4 and G5 were irradiated according to each protocol. Enamel blocks were then restored with an etch-and-rinse adhesive system and a 4-mm thick composite buildup was made in two increments (n = 9). After 24 h, restored blocks were serially sectioned with a cross-section area of similar to 1 mm(2) at the bonded interface and tested in tension in a universal testing machine (1 mm/min). Failure mode was determined at a magnification of x100 using a stereomicroscope. One treated block of each group was selected for scanning electron microscopy (SEM) analysis. mu TBS data were analyzed by two-way ANOVA and no statistical differences were observed among groups. Results: Mean bond strengths (SD) in MPa were: G1-30.4(6.2); G2-27.9(8.5); G3-32.3(3.9); G4-23.7(5.8); G5-29.3(6.0); G6-29.1(6.1). A large number of adhesive failures was recorded for bleached and irradiated enamel surfaces. Conclusions: Bleached enamel surfaces mu TBS values were not significantly different from those of unbleached enamel. Even though Er:YAG laser irradiation with both parameters had no influence on mu TBS for bleached and unbleached enamel, SEM analysis revealed that Er:YAG laser irradiation with 25.52J/cm(2) should not be recommended, as enamel ablation was observed, whereas irradiation with 4.42J/cm(2) did not promote any remarkable changes on enamel surface.

Effect of Laser Phototherapy on the Release of TNF-alpha and MMP-1 by Endodontic Sealer-Stimulated Macrophages

Objective: Our aim was to analyze the effect of laser phototherapy on the secretory activity of macrophages activated by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and stimulated by substances leached from an epoxy resin-based sealer (AH-Plus) and a calcium hydroxide-based sealer (Sealapex). Background Data: Laser phototherapy can modulate the inflammatory process, improving wound healing. This type of therapy could be useful for modulating postoperative symptoms seen after endodontic treatment. Materials and Methods: Cytotoxicity was indirectly assessed by measuring mitochondrial activity. Macrophages were stimulated by the leached substances or not (controls), and the groups were then irradiated or not. The secretion of pro-inflammatory cytokines (TNF-alpha and MMP-1) was analyzed using ELISA. Two irradiations at 6-h intervals were done with an As-Ga-Al diode laser (780 nm, 70 mW, spot size 4.0 mm(2), 3 J/cm(2), for 1.5 sec) in contact mode. Results: The sealers were non-cytotoxic to macrophages. The production of TNF-alpha was significantly decreased by laser phototherapy, regardless of experimental group. The level of secretion of MMP-1 was similar in all groups. Conclusion: Based on the conditions of this study we concluded that in activated macrophages, laser phototherapy impairs the secretion of the pro-inflammatory cytokine TNF-alpha, but has no influence on MMP-1 secretion.

Differential expression of HIF-1 alpha in CD44(+)CD24(-/) (low) breast ductal carcinomas

Background: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1 alpha expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44(+)CD24(-/low) immunophenotype. Methods: Immunohistochemical analysis of CD44(+)CD24(-/low) expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results: In univariate analysis, CD44(+)CD24(-/low) expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44(+)CD24(-/low) immunophenotype showed relationship with HIF-1 alpha (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44(+)CD24(-/low) positive breast tumors.

Influence of Laser Photobiomodulation on Collagen IV During Skeletal Muscle Tissue Remodeling After Injury in Rats

Objective: The aim of the present study was to determine the effect of GaAlAs low-level laser therapy (LLLT) on collagen IV remodeling of the tibialis anterior (TA) muscle in rats after cryolesion. Background: Considerable interest exists in skeletal muscle regeneration in situations such as repair after exercise-induced muscle injury, after muscle transplantation, in muscular dystrophy, exercise-induced muscle injury, and the recovery of strength after atrophy due to disuse. A number of studies have demonstrated the potential of LLLT in facilitating the muscle-healing process; however, no consensus is found in the literature regarding the best laser-irradiation parameters. Methods: Adult male Wistar rats (n = 45) were used and randomly divided into three groups: control (n = 5); nontreated cryolesioned group (n = 20), and LLLT-cryolesioned group (n = 20). The cryolesioned groups were analyzed at 1, 7, 14, and 21 days after the injury procedure. Laser irradiation was performed 3 times per week on the injured region by using the GaAlAs laser (660 nm; beam spot of 0.04 cm(2), output power of 20 mW, power density of 500 mW/cm(2), and energy density of 5 J/cm(2), for 10 sec). The muscles were removed, frozen, cryosectioned, and then stained with hematoxylin-eosin for the visualization of general morphology or used for immunohistochemical analysis of collagen IV. Results: It was demonstrated that LLLT promotes an increase in collagen IV immunolabeling in skeletal muscle in the first 7 days after acute trauma caused by cryoinjury, but does not modify the duration of the tissue-repair process. Even with LLLT, the injured muscle tissue needs similar to 21 days to achieve the same state of organization as that in the noninjured muscle. Conclusion: The collagen IV content is modulated in regenerating skeletal muscle under LLLT, which might be associated with better tissue outcome, although the histologic analysis did not detect tissue improvement in the LLLT group.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.