Contribution to the determination of the chemotherapeutic drug G1 in biological fluids

A sensitive gas chromatographic method for the quantitative determination of the new antibacterial and antifungal drug G1, 1-(5-bromofuran-2-yl)-2-bromo-2-nitroethene, has been optimized. The method involves a fast and single extraction step from spiked serum and urine samples. The G1 drug was quantified using an internal standard method and by means of a nitrogen-selective detector. The results are statistically significant and show that mean levels of G1 as low as 1 μg ml⁻¹ can be measured accurately.     

A single nucleotide polymorphism in the matrix metalloproteinase-1 (MMP-1) promoter influences amnion cell MMP-1 expression and risk for preterm premature rupture of the fetal membranes.

Interstitial collagen gives fetal membranes tensile strength, and membrane rupture has been attributed to collagen degradation. A polymorphism at -1607 in the matrix metalloproteinase-1 (MMP-1) promoter (an insertion of a guanine (G)) creates a core Ets binding site and increases promoter activity. We investigated whether this polymorphism is functionally significant for MMP-1 expression in amnion cells and whether it is associated with preterm premature rupture of the membranes (PPROM). The 2G promoter had >2-fold greater activity than the 1G allele in amnion mesenchymal cells and WISH amnion cells. Phorbol 12-myristate 13-acetate (PMA) increased mesenchymal cell nuclear protein binding with greater affinity to the 2G allele. Induction of MMP-1 mRNA by PMA was significantly greater in cells with a 1G/2G or 2G/2G genotype compared with cells homozygous for the 1G allele. When treated with PMA, the 1G/2G and 2G/2G cells produced greater amounts of MMP-1 protein than 1G/1G cells. A significant association was found between fetal carriage of a 2G allele and PPROM. We conclude that the 2G allele has stronger promoter activity in amnion cells, that it confers increased responsiveness of amnion cells to stimuli that induce MMP-1, and that this polymorphism contributes to the risk of PPROM.     

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.     

Promoter polymorphism MMP-1 (-1607 2G/1G) and MMP-3 (-1612 5A/6A) in development of HAND and modulation of pathogenesis of HAND

The pathogenesis of HIV-associated neurocognitive disorder (HAND) is modulated by host genetic susceptibility factors such as Matrix metalloproteinases (MMPs). Promoter polymorphism of MMP-1 and MMP-3 may modify the expression of the gene. Hence, we evaluated the association of MMP-1-16072G/1G and MMP-3-1612 5A/6A polymorphisms with development of HAND and the modulation of pathogenesis of HAND. We enrolled a total of 180 individuals, 50 HIV-infected individuals with HAND, 130 without HAND, and 150 healthy controls. Polymorphism of MMP-1 and MMP-3 were genotyped by PCR-RFLP. MMP-1-1607 2G1G, -16071G/2G-1G/1G genotypes and -1607 1G allele were associated with the development of HAND (OR = 1.64, P = 0.05; OR = 1.45, P = 0.04; OR = 1.69, P = 0.05). MMP-1-16071G1G, MMP-3-16125A5A genotypes increased the risk for the development of HAND (OR = 1.78, P = 0.25; OR = 2.39, P = 0.13). MMP-3-1612 5A5A, -1612 6A/5A-5A/5A genotypes and -1612 5A allele were associated with the reduced risk of HAND (OR = 0.40, P = 0.05; OR = 0.53, P = 0.04; OR = 0.40, P = 0.01). Haplotype 5A1G increased the risk of development of HAND (OR = 1.93, P = 0.05). As observed in advanced HIV disease stage, MMP-1-1607 1G1G genotype enhance the risk for advancement of HIV disease (OR = 1.69, P = 0.89). MMP-3-1612 6A5A genotype showed higher risk for development of HAND in alcohol users (0R = 1.65, P = 0.44). MMP-1 genotype may have an influence on development of HAND whereas MMP3-1612 5A5A genotype may reduce risk for pathogenesis of HAND.     

Joint Effect of MCP-1 Genotype GG and MMP-1 Genotype 2G/2G Increases the Likelihood of Developing Pulmonary Tuberculosis in BCG-Vaccinated Individuals

We previously reported that the – 2518 MCP-1 genotype GG increases the likelihood of developing tuberculosis (TB) in non-BCG-vaccinated Mexicans and Koreans. Here, we tested the hypothesis that this genotype, alone or together with the – 1607 MMP-1 functional polymorphism, increases the likelihood of developing TB in BCG-vaccinated individuals. We conducted population-based case-control studies of BCG-vaccinated individuals in Mexico and Peru that included 193 TB cases and 243 healthy tuberculin-positive controls from Mexico and 701 TB cases and 796 controls from Peru. We also performed immunohistochemistry (IHC) analysis of lymph nodes from carriers of relevant two-locus genotypes and in vitro studies to determine how these variants may operate to increase the risk of developing active disease. We report that a joint effect between the – 2518 MCP-1 genotype GG and the – 1607 MMP-1 genotype 2G/2G consistently increases the odds of developing TB 3.59-fold in Mexicans and 3.9-fold in Peruvians. IHC analysis of lymph nodes indicated that carriers of the two-locus genotype MCP-1 GG MMP-1 2G/2G express the highest levels of both MCP-1 and MMP-1. Carriers of these susceptibility genotypes might be at increased risk of developing TB because they produce high levels of MCP-1, which enhances the induction of MMP-1 production by M. tuberculosis-sonicate antigens to higher levels than in carriers of the other two-locus MCP-1 MMP-1 genotypes studied. This notion was supported by in vitro experiments and luciferase based promoter activity assay. MMP-1 may destabilize granuloma formation and promote tissue damage and disease progression early in the infection. Our findings may foster the development of new and personalized therapeutic approaches targeting MCP-1 and/or MMP-1.     

Genotyping of Macrophage Migration Inhibitory Factor (MIF) CATT5–8 Repeat Polymorphism by Denaturing High-Performance Liquid Chromatography (DHPLC)

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine expressed in many different cell types and implicated in the pathogenesis of numerous acute and chronic inflammatory diseases. Variable Number of Tandem Repeat (VNTR) CATT_5–8 at position ?794 in the promoter of the MIF gene has been associated with several human pathological conditions. Different methods for genotyping the CATT tetranucleotide repeats have been described. Here, we report, for the first time, the complete characterization of the CATT_5–8 repeat polymorphism using exclusively the denaturing high-performance liquid chromatography (DHPLC) technique under partially denaturing conditions. This approach, based on a step-by-step DHPLC protocol, allowed the accurate determination of all the homozygous and heterozygous genotypes in 350 DNA samples from control subjects. The results were validated by comparison to DNA sequencing, and the DHPLC approach was accurate, sensitive, and highly reproducible. Data from the current study demonstrate that this method of analysis by DHPLC may represent a powerful and sensitive alternative tool for a rapid and efficient genotyping of short tandem repeats presenting a limited number of alleles.     

Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes.     

Matrix metalloproteinase-1 activates a pertussis toxin-sensitive signaling pathway that stimulates the release of matrix metalloproteinase-9

The matrix metalloproteinases (MMPs) are a family of structurally related metalloendopeptidases so named due to their propensity to target extracellular matrix (ECM) proteins. Accumulating evidence, however, suggests that these proteases cleave numerous non-ECM substrates including enzymes and cell surface receptors. MMPs may also bind to cell surface receptors, though such binding has typically been thought to mediate internalization and degradation of the bound protease. More recently, it has been shown that MMP-1 coimmunoprecipitates with the alpha2beta1 integrin, a receptor for collagen. This association may serve to localize the enzymatic activity of MMP-1 so that collagen is cleaved and cell migration is facilitated. In other studies, however, it has been shown that integrin engagement may be linked to the activation of signaling cascades including those mediated by Gialpha containing heterotrimers. As an example, alpha2beta1 can form a complex with CD47 that may associate with Gialpha. In the present study we have therefore investigated the possibility that MMP-1 may affect intracellular changes that are linked to the activation of a Gi protein-coupled receptor. We show that treatment of neural cells with MMP-1 is followed by a rapid reduction in cytosolic levels of cAMP. Moreover, MMP-1 potentiates proteinase activated receptor-1 (PAR-1) agonist-linked increases in intracellular calcium, an effect which is often observed when an agonist of a Gi protein-coupled receptor is administered in association with an agonist of a Gq coupled receptor. In addition, MMP-1 stimulates pertussis toxin sensitive release ofMMP-9 both from cultured neural cells and monocyte/macrophages. Together, these results suggest that MMP-1 signals through a pertussis toxin-sensitive G protein-coupled receptor.     

Rapid detection of CAA/CAG repeat polymorphism in the AIB1 gene using DHPLC

Denaturing high-performance liquid chromatography (DHPLC) has been used for rapid and accurate DNA mutation analysis; to extend the DNA fragment lengths analysis. Recently, polymorphism in polyglutamine-coding region of Amplified In Breast cancer gene 1 (AIB1) was analyzed as an independent genetic risk factor influencing breast cancer onset in carriers of mutation in breast cancer predisposing gene 1 (BRCA1). We have implemented efficient, cost-effective and rapid method for analysis of the AIB1 polyglutamine repeat polymorphism based on DHPLC analysis (WAVE system) of unlabeled PCR products. This strategy can be useful for genotyping of other trinucleotide repeat polymorphisms using DHPLC in medium/high throughput settings.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Assessment of DHPLC usefulness in the genotyping of GSTP1 exon 5 SNP: comparison to the PCR-RFLP method

Single-nucleotide polymorphism (SNP) analysis can be performed by several methods such as PCR-RFLP, real time PCR and mass spectrometry. Denaturating High Pressure Liquid Chromatography (DHPLC) analysis allows the detection of DNA mutations in heteroduplex samples. GSTP1 exon 5 gene presents a single-nucleotide polymorphism (a to g) that results into an amino-acid substitution (Ile to Val). Ile and Val variants are identified respectively by a and b alleles. This polymorphism affects enzyme activity and is highly frequent within Caucasian populations and therefore widely studied in the context of SNP related to cancer susceptibility. Our goal was to evaluate DHPLC usefulness in detecting a well-known SNP in comparison to PCR-RFLP, in the field of molecular epidemiological studies. Fifty Caucasian people were genotyped by both methods. Heterozygous samples were identified easily at two temperatures using the DHPLC method. Discrimination between a/a and b/b homozygous genotypes was done by pooling every homozygous sample with a known a/a sample. Our genotyping using both methods resulted in the characterisation of 32 (64%) a/a homozygous, 18 (36%) a/b heterozygous and 5 (10%) b/b homozygous. All samples were also identically genotyped by the two methods. Our results show that DHPLC is a good alternative to classical PCR-RFLP method in genotyping SNPs. Advantages of this chromatographic method were no restriction site needed and a reduced technical time thanks to an automated injection. Moreover, unlike classical RFLP gel analysis, DHPLC chromatograms provided objective criteria for sample classification.     

Estrogen receptor beta and truncated variants enhance the expression of transfected MMP-1 promoter constructs in response to specific mechanical loading

Background

Joint diseases such as osteoarthritis (OA) predominantly afflict post-menopausal women, suggesting a pertinent role for female hormones. Estrogen receptor beta (ER-β) has been detected in connective tissues of the knee joint suggesting that these tissues are responsive to the hormone estrogen. Matrix metalloproteinase-1 (MMP-1) activity contributes to cartilage degradation, a key factor leading to OA development in synovial joints. Two polymorphic forms of MMP-1 exist due to a deletion/insertion of the guanine residue in the promoter, and the 2G allelic variant of MMP-1 exhibits more activity than the 1G allele. Previous studies have demonstrated that the polymorphic forms of the human MMP-1 are influenced by the modulating effects of estrogen receptor isoforms. In addition to hormonal influences, physiological factors such as altered mechanical loading are also contributory features of OA. In the present study, the combined influence of biomechanical and hormonal variables on the activity of MMP-1 isoforms was evaluated. We hypothesized that the combined effects of ER-β and sheer stress will differentially activate the two allelic forms of MMP-1 in a hormone-independent manner.

Methods

HIG-82 synoviocytes were transiently transfected with 1G or 2G alleles (±) ER-β and subjected to either shear or equibiaxial stress. Next, 1G/2G promoter activity was measured to determine the combined influence of physiological stimuli. Truncated ER-β constructs were used to determine the importance of different domains of ER-β on 1G/2G activation.

Results

The 2G allele exhibited a constitutively higher activity than the 1G allele, which was further increased when the transfected cells were subject to shear stress, but not equibiaxial stress. Moreover, the combination of ER-β and shear stress further increased the activity levels of the 1G/2G allelic variants. Additionally, select AF-2 truncated ER-β variants led to increased activity levels for the 2G allele, indicating the AF-1 domain was likely involved in the response to mechanical stimulation.

Conclusions

These results suggest that the 1G/2G alleles of MMP-1 are influenced by specific mechanical stimuli like shear stress, as well as the ER-β receptor. These findings contribute to the potential allelic involvement in connective tissue diseases such as OA in females compared to males.

     

Single-cell A3243G mitochondrial DNA mutation load assays for segregation analysis.

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification-based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.