Transcriptional responses of Escherichia coli during recovery from inorganic or organic mercury exposure.

BACKGROUND: The protean chemical properties of mercury have long made it attractive for diverse applications, but its toxicity requires great care in its use, disposal, and recycling. Mercury occurs in multiple chemical forms, and the molecular basis for the distinct toxicity of its various forms is only partly understood. Global transcriptomics applied over time can reveal how a cell recognizes a toxicant and what cellular subsystems it marshals to repair and recover from the damage. The longitudinal effects on the transcriptome of exponential phase E. coli were compared during sub-acute exposure to mercuric chloride (HgCl2) or to phenylmercuric acetate (PMA) using RNA-Seq. RESULTS: Differential gene expression revealed common and distinct responses to the mercurials throughout recovery. Cultures exhibited growth stasis immediately after each mercurial exposure but returned to normal growth more quickly after PMA exposure than after HgCl2 exposure. Correspondingly, PMA rapidly elicited up-regulation of a large number of genes which continued for 30 min, whereas fewer genes were up-regulated early after HgCl2 exposure only some of which overlapped with PMA up-regulated genes. By 60 min gene expression in PMA-exposed cells was almost indistinguishable from unexposed cells, but HgCl2 exposed cells still had many differentially expressed genes. Relative expression of energy production and most metabolite uptake pathways declined with both compounds, but nearly all stress response systems were up-regulated by one or the other mercurial during recovery. CONCLUSIONS: Sub-acute exposure influenced expression of ~45% of all genes with many distinct responses for each compound, reflecting differential biochemical damage by each mercurial and the corresponding resources available for repair. This study is the first global, high-resolution view of the transcriptional responses to any common toxicant in a prokaryotic model system from exposure to recovery of active growth. The responses provoked by these two mercurials in this model bacterium also provide insights about how higher organisms may respond to these ubiquitous metal toxicants.

Methylmercury degradation by Pseudomonas putida V1.

Environmental contamination of mercury (Hg) has caused public health concerns with focuses on the neurotoxic substance methylmercury, due to its bioaccumulation and biomagnification in food chains. The goals of the present study were to examine: (i) the transformation of methylmercury, thimerosal, phenylmercuric acetate and mercuric chloride by cultures of Pseudomonas putida V1, (ii) the presence of the genes merA and merB in P. putida V1, and (iii) the degradation pathways of methylmercury by P. putida V1. Strain V1 cultures readily degraded methylmercury, thimerosal, phenylmercury acetate, and reduced mercuric chloride into gaseous Hg(0). However, the Hg transformation in LB broth by P. putida V1 was influenced by the type of Hg compounds. The merA gene was detected in P. putida V1, on the other hand, the merB gene was not detected. The sequencing of this gene, showed high similarity (100%) to the mercuric reductase gene of other Pseudomonas spp. Furthermore, tests using radioactive (14)C-methylmercury indicated an uncommon release of (14)CO2 concomitant with the production of Hg(0). The results of the present work suggest that P. putida V1 has the potential to remove methylmercury from contaminated sites. More studies are warranted to determine the mechanism of removal of methylmercury by P. putida V1.

Type IV collagen α1-chain noncollagenous domain blocks MMP-2 activation both in-vitro and in-vivo.

α1(IV)NC1 inhibits angiogenesis by regulating MAPK activation, this biological function was partly attributed α1(IV)NC1 binding to α1ß1-integrin. However, its potent antiangiogenic activity and the molecular targets of α1(IV)NC1 has not been investigated. In the present study, the regulation of MMP-2 activation by α1(IV)NC1 was evaluated. α1ß1-integrin which is required for inhibition of angiogenesis is not playing a role in cellular invasion and inhibition of MMP-2 activation by α1(IV)NC1. We found that α1(IV)NC1 binds the CBD of MMP-2 and forming a stable complex that prevents activation of MMP-2. The antiangiogenic activity of α1(IV)NC1 is mediated, in part, by this binding activity. In addition, up-regulation of TIMP-2 by α1(IV)NC1 led to saturation of MT1-MMP binding sites, which in turn led to inhibition of MMP-2 activation. In-vivo studies using α1-integrin null-mice treated with higher doses of α1(IV)NC1 showed integrin independent inhibition of tumor growth and active-MMP-2, without affecting MMP-9, MMP-7 and angiostatin.

Enhanced activity of an angiotensin-(1-7) neuropeptidase in glucocorticoid-induced fetal programming.

We previously identified angiotensin converting enzyme (ACE) and an endopeptidase activity that degraded angiotensin-(1-7) [Ang-(1-7)] to Ang-(1-5) and Ang-(1-4), respectively, in the cerebrospinal fluid (CSF) of 6-month old male sheep. The present study undertook a more comprehensive analysis of the CSF peptidase that converts Ang-(1-7) to Ang-(1-4) in control and in utero betamethasone-exposed sheep (BMX). Characterization of the Ang-(1-7) peptidase revealed that the thiol agents 4-aminophenylmercuric acetate (APMA) and p-chloromercuribenzoic acid (PCMB), as well as the metallo-chelators o-phenanthroline and EDTA essentially abolished the enzyme activity. Additional inhibitors for serine, aspartyl, and cysteine proteases, as well as selective inhibitors against the endopeptidases neprilysin, neurolysin, prolyl and thimet oligopeptidases did not attenuate enzymatic activity. Competition studies against the peptidase revealed similar IC50s for Ang-(1-7) (5µM) and Ang II (3µM), but lower values for Ala(1)-Ang-(1-7) and Ang-(2-7) of 1.8 and 2.0µM, respectively. In contrast, bradykinin exhibited a 6-fold higher IC50 (32µM) than Ang-(1-7) while neurotensin was a poor competitor. Mean arterial pressure (78±1 vs. 94±2mmHg, N=4-5, P<0.01) and Ang-(1-7) peptidase activity (14.2±1 vs 32±1.5fmol/min/ml CSF, N=5, P<0.01) were higher in the BMX group, and enzyme activity inversely correlated with Ang-(1-7) content in CSF. Lower Ang-(1-7) expression in brain is linked to baroreflex impairment in hypertension and aging, thus, increased activity of an Ang-(1-7) peptidase may contribute to lower CSF Ang-(1-7) levels, elevated blood pressure and impaired reflex function in this model of fetal programming.

Amyloid-ß-induced matrix metalloproteinase-9 secretion is associated with retinal pigment epithelial barrier disruption.

Local and chronic inflammation induced by amyloid-ß (Aß) plays a central role in the development of age-related macular degeneration. The retina is an immune-privileged site due to local tissue barrier. Yet, the manner by which immune cells pass through this barrier and accumulate in the retina remains unclear. Matrix metalloproteinases (MMPs) induce barrier disruption via proteolysis of epithelial tight junction (TJ) proteins. We hypothesized that Aß-induced MMP secretion causes disruption of epithelial barrier integrity. To test this hypothesis, human adult retinal pigment epithelial (haRPE) cells were exposed to Aß, and the expression of MMP-2 and MMP-9 was detected using gelatin zymography. To demonstrate the key role of MMPs in modulating epithelial barrier structure, the MMP agonist 4-aminophenylmercuric acetate (APMA), an MMP inhibitor (GM6001) and siRNA against MMP-9 were employed for comparison. We found that MMP-9, secreted by Aß- or APMA-stimulated cells, mediated low transepithelial electrical resistance (TER) and high transepithelial permeability by disrupting TJ proteins. However, these alterations were reduced by the MMP inhibitor GM6001 or by silencing of the MMP-9 gene. Our findings suggest that the degradation of TJ proteins such as zonula occludens-1, occludin and F-actin by MMP-9 secreted by Aß-stimulated cells constitutes an important mechanism in the breakdown of the barrier which contributes to chronic inflammation in the retina of age-related macular degeneration.

Effect of phosphoric acid on the degradation of human dentin matrix.

This study determined if dentin proteases are denatured by phosphoric acid (PA) used in etch-and-rinse dentin adhesives. Dentin beams were completely demineralized with EDTA for 30 days. We "acid-etched" experimental groups by exposing the demineralized dentin beams to 1, 10, or 37 mass% PA for 15 sec or 15 min. Control beams were not exposed to PA but were incubated in simulated body fluid for 3 days to assay their total endogenous telopeptidase activity, by their ability to solubilize C-terminal crosslinked telopeptides ICTP and CTX from insoluble dentin collagen. Control beams released 6.1 ± 0.8 ng ICTP and 0.6 ± 0.1 ng CTX/mg dry-wt/3 days. Positive control beams pre-incubated in p-aminophenylmercuric acetate, a compound known to activate proMMPs, released about the same amount of ICTP peptides, but released significantly less CTX. Beams immersed in 1, 10, or 37 mass% PA for 15 sec or 15 min released amounts of ICTP and CTX similar to that released by the controls (p > 0.05). Beams incubated in galardin, an MMP inhibitor, or E-64, a cathepsin inhibitor, blocked most of the release of ICTP and CTX, respectively. It is concluded that PA does not denature endogenous MMP and cathepsin activities of dentin matrices.

In vitro activity of phenylmercuric acetate against ocular pathogenic fungi.

OBJECTIVES: To determine the antifungal activity of phenylmercuric acetate against ocular pathogenic fungi in vitro and develop new antifungal eye drops to combat keratomycosis. METHODS: The in vitro activity of phenylmercuric acetate was assessed against 261 isolates of ocular pathogenic fungi that included 136 Fusarium spp. isolates, 98 Aspergillus spp. isolates, 10 Alternaria alternata isolates and 17 other pathogens. The activity of phenylmercuric acetate was compared with the activities of amphotericin B and natamycin. In vitro susceptibility testing was performed by broth microdilution assay, in accordance with the CLSI (formerly NCCLS) M38-A guidelines for filamentous fungi. RESULTS: MIC90s of phenylmercuric acetate were 0.0156, 0.0156, 0.0156 and 0.0156 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. MIC90s of amphotericin B were 2, 2, 1 and 1 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. MIC90s of natamycin were 8, 32, 4 and 4 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. CONCLUSIONS: Phenylmercuric acetate has promising antifungal activity, which is significantly superior to the activities of amphotericin B and natamycin against a wide variety of ocular pathogenic fungi based on comparative MIC values. Additional evaluation is required to determine its clinical utility.

'Sexual chemistry' before the pill: science, industry and chemical contraceptives, 1920-1960.

The history of contraceptives met the history of drugs long before the invention of the contraceptive pill. In the first half of the twentieth century, numerous pharmaceutical laboratories, including major ones, manufactured and marketed chemical contraceptives: jellies, suppositories, creams, powders and foams applied locally to prevent conception. Efforts to put an end to the marginal status of these products and to transform them into 'ethical' drugs played an important role in the development of standardized laboratory tests of efficacy of contraceptive preparations; debates on the validity of such tests; evaluation of the long-term toxicity of chemical compounds; and the rise of collaborations between activists, non-profit organizations and the pharmaceutical industry. Chemical contraceptives were initially associated with quack medicine, shady commercial practices and doubtful morality. Striving to change the status of contraceptives and to promote safe and efficient products that reduced fertility in humans shaped some of the key features of the present-day production and regulation of pharmaceuticals.

Activation of MMP-9 by human lung epithelial cells in response to the cystic fibrosis-associated pathogen Burkholderia cenocepacia reduced wound healing in vitro.

Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.

Contact sensitization pattern in 172 atopic subjects.

BACKGROUND: Some authors have claimed a decreased cell-mediated immunity among atopic individuals, which would lead to observations of decreased rates of allergic contact dermatitis (ACD). OBJECTIVES: The purpose of our study was to investigate contact sensitization patterns in atopic subjects compared with non-atopic subjects. METHODS: Patch test data for 1247 patients undergoing patch testing at Massachusetts General Hospital between 1990 and 2006 were reviewed. Using accepted criteria, 172 subjects were classified as atopic individuals (AIs), and 1075 were classified as non-atopic individuals (NAIs). Sensitization rates were compared between these two groups. RESULTS: Sensitization rates (65.0% and 57.4% in the AI and NAI groups, respectively) and average numbers of positive responses (1.5 and 1.2 in the AI and NAI groups, respectively) were higher in AIs. Leading allergens observed were similar for both groups. Sensitization to potassium dichromate and phenylmercuric acetate was significantly greater in the AI group. The most frequent diagnosis in both groups was ACD (41.9% and 45.5% in the AI and NAI groups, respectively). In addition, more NAIs who were employed in occupations with exposure to wet and/or irritant conditions had hand eczema (P < 0.005). CONCLUSIONS: Atopic individuals were shown to be at least as likely to have ACD as NAIs. The most common sensitizers were similar in both groups, suggesting common sources of sensitization.

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein.

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu(373-374)Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as "aggrecanase" (ADAMTS) cleavage sites, while cleavage between Ser(341-342)Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu(2047-2048)Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.

Genital contact dermatitis: a retrospective analysis.

BACKGROUND: many topical products are used in the genital region. Allergic contact dermatitis (ACD) may develop from product use or due to treatment of an underlying dermatosis. OBJECTIVES: our goal was to identify the frequency of ACD and identify top allergens in the genital region. METHODS: data were analyzed for 1,238 patients tested between January 1990 and December 2006. Fifteen allergens caused reactions at rates greater than 1%. Thirteen anatomic regions were assessed. Statistical analyses were by chi-square test and Fisher exact test. Adjusted level of significance due to multiple testing was α  =  .002. RESULTS: of individuals with genital dermatitis (n  =  37; aged 24-77 years, 48.6% female), 41% (15 of 37) had at least one positive patch-test result although only 30% (11 of 37) had a final diagnosis of relevant ACD. Mean age was 46 years for males and 41 years for females. The top five allergens were balsam of Peru (10.8%), fragrance mix I (8.1%), tolu balsam (8.1%), phenylmercuric acetate (8.1%), and neomycin (5.4%). Females were more often allergic (50%) compared to males (37%); 59.5% of patients had no positive reactions. CONCLUSION: genital dermatitis is rare; the minority tested positively for ACD. The top five allergens were present in toiletries and cosmetics used on genital skin. The top three allergens are fragrance related, underscoring the importance of using fragrance-free products on mucosal skin.

Utility of combining MMP-9 enzyme-linked immunosorbent assay and MMP-9 activity assay data to monitor plasma enzyme specific activity.

The aim of this study was to combine matrix metalloproteinase-9 (MMP-9) protein (enzyme-linked immunosorbent assay [ELISA]) and MMP-9 activity (fluorescence resonance energy transfer [FRET] assay) data to generate units of specific activity in endogenous and p-aminophenylmercuric acetate (APMA)-activated lithium heparin plasma. The results indicate that specific activity is constant in APMA-activated plasma (mean value=1359.4 pmol/min/microg) and approximately 12% plasma MMP-9 is endogenously active. Exogenous tissue inhibitor of metalloproteinase-1 (TIMP-1) has a greater inhibitory effect on endogenously active MMP-9 than on APMA-activated MMP-9. In conclusion, specific activity can be used as a tool to monitor MMP-9 inhibition. APMA activation affects natural enzyme inhibition, possibly by chemical modification of the C-terminal portion of the enzyme containing the TIMP-1 binding site.

Methods for studying activation of matrix metalloproteinases.

The degradation of the extracellular matrix during development and in disease is thought to result from the combined action of several proteolytic enzyme systems, including the matrix metalloproteinases (MMPs), serine proteinases, and cysteine proteinases. The majority of the soluble MMPs are synthesized as proenzymes which require extracellular activation in order to gain proteolytic activity and the analysis of their activation mechanism is a prerequisite for understanding MMP-mediated proteolysis.The emphasis of this chapter is the provision of the experimental tools to study MMP activation in vitro and in cellular model systems. Hence, we use the activation of procollagenase-3 (proMMP-13) and progelatinase A (proMMP-2) as examples of the methods used.

Assay of matrix metalloproteinases against matrix substrates.

The assays described allow the activity of members of the matrix metalloproteinase (MMP) family that degrade collagen, gelatin and casein substrates to be measured. The protocols described include the preparation of radiolabeled substrates, methods for the separation of degraded product from undegraded substrate, and methods for the activation of MMPs. The advantages and disadvantages of these methods are discussed in relation to immunoassays that measure the amount of individual MMPs.

Walker 256 cancer cells secrete tissue inhibitor of metalloproteinase-free metalloproteinase-9.

Walker 256 (W256) cancer cells, developed as ascites in rats, in response to endogenous unidentified stimuli, secrete a gelatinase of apparent molecular mass of 94 kDa, immunologically homologous to the zymogen of matrix metalloproteinase-9 (proMMP-9). After treatment with the activating agent 4-aminophenylmercuric acetate (APMA), affinity-purified W256 gelatinase is converted to a final processed form of 66 kDa in a similar fashion to TIMP-free human proMMP-9. It is demonstrated that although being capable of binding TIMP-1, W256 proMMP-9 is secreted from W256 cells in TIMP-free forms (monomers or oligomers). Moreover, using biochemical and immunological methods, it is established that the W256 cells do not express or secrete TIMP-1 protein, although RT-PCR analysis indicated low-level TIMP-1 mRNA expression. W256 cancer cells displayed high metastatic ability in rats that may be attributed in part to secretion of TIMP-free proMMP-9.

Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells.

Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype.

Phenyl mercuric acetate (PMA): mercury-bearing flexible gymnasium floors in schools--evaluation of hazards and controlled abatement.

Phenyl mercuric acetate (PMA) historically has been used as a catalyst in polyurethane systems. In the 1950s-1970s, PMA was used as a catalyst in the 3M Tartan brand polyurethane flexible floors that were installed commonly in school gymnasiums. Mercury vapor is released into air above the surface of these floors. Sampling mercury in bulk flooring material and mercury vapor in air was conducted in nine Idaho schools in the spring of 2006. These evaluations were conducted in response to concerns by school officials that the floors could contain mercury and could release the mercury vapor into the air, presenting a potential health hazard for students, staff, and visitors. Controlled abatement was conducted in one school where remodeling would impact the mercury-bearing flexible gym floors ( approximately 9,000 ft(2) total). The controlled abatement consisted of containment of the work area with negative air technology; worker protection, including mercury-specific training, use of personal protective equipment, and biological and exposure monitoring; and environmental protection, including proper disposal of mercury-bearing hazardous waste material.

[The value of allergy survey in a retrospective series of 40 patients with burning-mouth syndrome (stomatodynia)]. / Stomatodynies: intérêt de l'enquête allergologique dans une série rétrospective de 40 malades.

BACKGROUND: By definition, stomatodynia or burning-mouth syndrome involves oral pain with no causes being found on history taking or examination. An allergic origin is often suspected by doctors and patients alike. In this study, we attempted to assess the value of epicutaneous tests in demonstrating allergic causes for patients presenting stomatodynia. PATIENTS AND METHODS: This was a single-centre retrospective study of patients undergoing epicutaneous tests between 1996 and 2003 to screen for allergic causes of mouth pain not accounted for by any abnormalities seen during examination performed at consultations for mouth disease. RESULTS: Forty patients were included (11 male, 29 female; mean age: 58 years), and 39 were excluded. Sixteen patients presented at least one positive test, with a total of 35 positive tests in all. In decreasing order of frequency, the causes were metals, mercury derivatives (nickel salts: n=5; chrome salts: n=3; palladium salts: n=2; phenylmercuric acetate: n=2; thiomersal: n=2; cobalt salts: n=1; gold salts: n=1; mercury: n=1) and resins (acrylates: n=4). The relevance of these test results was considered probable in three cases and possible in five cases, associated with the existence of metals or resins in patients' mouths. The Peru balm test was positive in four cases but was not relevant. Tests for personal products were negative in all cases, with the exception of one case of resin from a prosthesis and one case of tixocortol pivalate. COMMENTS: Type I stomatodynia (daily occurrence with gradually increase in discomfort throughout the day) and type II stomatodynia (permanent) are not normally attributable to allergies. However, for type III stomatodynia (non-permanent, with acute episodes followed by remission), an allergy survey guided by questioning may be undertaken to determine the cause, primarily prostheses or diet. The relevance of positive test results must be interpreted with caution in view of the incidence of positive epicutaneous tests for metals and Peru balm among the general population studied.

Generation of novel, secreted epidermal growth factor receptor (EGFR/ErbB1) isoforms via metalloprotease-dependent ectodomain shedding and exosome secretion.

Exosomes are small membrane vesicles derived from intracellular multivescicular bodies (MVBs) that can undergo constitutive and regulated secretion from cells. Exosomes can also secrete soluble proteins through metalloprotease-dependent ectodomain shedding. In this study, we sought to determine whether ErbB1 receptors are present within exosomes isolated from the human keratinocyte cell line, HaCaT, and whether exosome-associated ErbB1 receptors can undergo further proteolytic processing. We show that full-length transmembrane ErbB1 is secreted in HaCaT exosomes. EGF treatment and calcium flux stimulated the release of phosphorylated ErbB1 in exosomes but only ligand-stimulated release was blocked by the ErbB1 kinase inhibitor, AG1478, indicating that ligand-dependent ErbB1 receptor activation can initiate ErbB1 secretion into exosomes. In addition, other immunoreactive but truncated ErbB1 isoforms were detected in exosomes suggestive of additional proteolytic processing. We demonstrate that cellular and exosomal ErbB1 receptors can undergo ectodomain shedding to generate soluble N-terminal ectodomains and membrane-associated C-terminal remnant fragments (CTFs). ErbB1 shedding was activated by calcium flux and the metalloprotease activator APMA (4-aminophenylmercuric acetate) and was blocked by a metalloprotease inhibitor (GM6001). Soluble ErbB1 ectodomains shed into conditioned medium retained the ability to bind exogenous ligand. Our results provide new insights into the proteolysis, trafficking and fate of ErbB1 receptors and suggest that the novel ErbB1 isoforms may have functions distinct from the plasma membrane receptor.