Effect of ultrasonic activation on the reduction of bacteria and endotoxins in root canals: a randomized clinical trial.

AIM: This randomized clinical trial aimed to compare the effectiveness of ultrasonic activation with that of nonactivated irrigation on the removal of bacteria and endotoxin from root canals. METHODOLOGY: Fifty patients with necrotic pulps and asymptomatic apical periodontitis were randomly allocated into two groups according to the final irrigation protocol after root canal preparation: Group UI - ultrasonic irrigation (n = 25) and Group NI - needle irrigation (n = 25). The root canals were medicated with calcium hydroxide for 14 days. Microbiological sampling was performed before (S1) and after the root canal preparation (S2), after the irrigation protocols (S3) and after the removal of the intracanal medication (S4). Total bacteria counts were determined by qPCR and the endotoxin levels by the limulus amebocyte lysate assay. Intragroup analyses were performed using the Wilcoxon test for related samples, whereas intergroup analyses were performed using the Mann-Whitney U-test (P < 0.05). RESULTS: All S1 samples were positive for bacteria, with median numbers of 1.49 × 106 and 8.55 × 105 bacterial cells for the UI and NI groups, respectively. This number significantly decreased in S2 samples (UI: 1.41 × 104 ; NI: 3.53 × 104 ; both with P < 0.001). After final irrigation protocols, there was a significant decrease in bacterial load from S2 to S3 samples in both groups (UI: 4.29 × 103 ; NI: 1.08 × 104 ; P < 0.01). Intergroup analysis revealed a significant difference between irrigation methods regarding bacterial counts in S3 samples (P < 0.05). In contrast, no significant differences were observed between groups for endotoxin levels (P > 0.05). CONCLUSIONS: Ultrasonic activation was more effective than nonactivated irrigation for reducing the number of bacteria but not the endotoxin levels in root canals of teeth with apical periodontitis.

Single Low-Dose Ionizing Radiation Induces Genotoxicity in Adult Zebrafish and its Non-Irradiated Progeny.

This study investigated to what extent a single exposure to low doses of ionizing radiation can induce genotoxic damage in irradiated adult zebrafish (Danio rerio) and its non-irradiated F1 progeny. Four groups of adult zebrafish were irradiated with a single dose of X-rays at 0 (control), 100, 500 and 1000 mGy, respectively, and couples of each group were allowed to reproduce following irradiation. Blood of parental fish and whole-body offspring were analysed by the comet assay for detection of DNA damage. The level of DNA damage in irradiated parental fish increased in a radiation dose-dependent manner at day 1 post-irradiation, but returned to the control level thereafter. The level of DNA damage in the progeny was directly correlated with the parental irradiation dose. Results highlight the genotoxic risk of a single exposure to low-dose ionizing radiation in irradiated individuals and also in its non-irradiated progeny.

Effects of trichlorfon on ecotoxicological biomarkers in farmed Colossoma macropomum (tambaqui).

Producers of fish have been looking for viable alternatives for the management of Colossoma macropomum (tambaqui) in confinement systems in order to avoid the harm and subsequent losses caused by parasitic diseases. One alternative used by farmers is pesticides, such as trichlorfon, which has a genotoxic effect. Thus, this study aimed to evaluate the changes in gene expression due to the side effects of trichlorfon in tambaqui. Two treatments were used based on LC50-96h of 0.870 mg/L using 30% and 50% trichlorfon with exposure periods of 48, 72 and 96 h. For differential expression of the genes in the liver, real-time PCR was performed for the AChE, GST, CYP2J6, CYP2C8, 18S and GAPDH genes. After 96 h of exposure to trichlorfon, an alteration in the gene expression profile of the antioxidant defense system (GST) of the tambaqui was observed. It was also observed that this organophosphate did not affect the expression of genes related to the isoenzymes that are responsible for the biotransformation of xenobiotics in phase I (2J6 and 2C8) and cholinesterase AChE. It was concluded that the reduction in gene expression of GST suggests a decrease in metabolization capacity in phase II.

Carbamazepine, venlafaxine, tramadol, and their main metabolites: Toxicological effects on zebrafish embryos and larvae.

Pharmaceutical compounds and their metabolites are found in natural and wastewater. However, investigation of their toxic effects on aquatic animals has been neglected, especially for metabolites. This work investigated the effects of the main metabolites of carbamazepine, venlafaxine and tramadol. Zebrafish embryos were exposed (0.1-100 µg/L) for 168hpf exposures to each metabolite (carbamazepine-10,11-epoxide, 10,11-dihydrocarbamazepine, O-desmethylvenlafaxine, N-desmethylvenlafaxine, O-desmethyltramadol, N-desmethyltramadol) or the parental compound. A concentration-response relationship was found for the effects of some embryonic malformations. Carbamazepine-10,11-epoxide, O-desmethylvenlafaxine and tramadol elicited the highest malformation rates. All compounds significantly decreased larvae responses on a sensorimotor assay compared to controls. Altered expression was found for most of the 32 tested genes. In particular, abcc1, abcc2, abcg2a, nrf2, pparg and raraa were found to be affected by all three drug groups. For each group, the modelled expression patterns showed differences in expression between parental compounds and metabolites. Potential biomarkers of exposure were identified for the venlafaxine and carbamazepine groups. These results are worrying, indicating that such contamination in aquatic systems may put natural populations at significant risk. Furthermore, metabolites represent a real risk that needs more scrutinising by the scientific community.

Evaluation of the Potential of Marine Algae Extracts as a Source of Functional Ingredients Using Zebrafish as Animal Model for Aquaculture.

Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry.

Effects of the microcystin profile of a cyanobacterial bloom on growth and toxin accumulation in common carp Cyprinus carpio larvae.

A 12 day growth trial was conducted to compare the effect of the variation in microcystins (MC) composition of two bloom samples of Microcystis aeruginosa on the growth performance and microcystin accumulation in common carp Cyprinus carpio larvae. Two M. aeruginosa natural bloom samples with different MC profiles were collected and larvae were exposed to cyanobacterial cells through their diet. Three diets, a basal control diet and two diets prepared from the basal diet plus the same toxins content (60 ng MC g(-1) diet) of each cyanobacterial bloom, were given at the same ration level to three groups of larvae during the experimental period. Larval mass and standard length from day 9 were significantly different between cyanobacterial treatments and in both cases lower than that of the control. The MC accumulation by larvae, inversely correlated with the growth performance, was also significantly different between cyanobacterial treatments (26.96 v. 17.32 ng g(-1) at the end of the experimental period). These results indicate that MC variants profile may have effects on the toxin uptake and toxicity. To date, this is the first laboratory study to show that fish accumulate MC depending on the toxin profile of the cyanobacterial bloom.

Effects of cyanobacterial extracts containing anatoxin-a and of pure anatoxin-a on early developmental stages of carp.

This study compares the effects of pure anatoxin-a and cyanobacterial extracts of an anatoxin-a producing strain on early stages of development of carp. Carp eggs were exposed from 2:30 h to 4 days post-fertilization to different ecologically relevant concentrations of anatoxin-a, provided as pure toxin or contained in the cyanobacterial extracts. Data on time to mortality, mortality rate, time to hatching, hatching rate, skeletal malformations rate, and larval standard length were registered until 8 days post-fertilization. At any tested concentration of anatoxin-a, the pure toxin was almost harmless to carp early stages of development, contrarily to cell extracts that were highly toxic. Only an adverse effect on the larval length was found at the highest concentration of pure toxin, while increasing concentrations of cell extracts caused increasing adverse effects in all the analyzed parameters. Anatoxin-a producing cyanobacteria should be regarded as putative modulators of aquatic ecosystems communities.

Changes in calcium dynamics following the reversal of the sodium-calcium exchanger have a key role in AMPA receptor-mediated neurodegeneration via calpain activation in hippocampal neurons.

Proteolytic cleavage of the Na(+)/Ca(2+) exchanger (NCX) by calpains impairs calcium homeostasis, leading to a delayed calcium overload and excitotoxic cell death. However, it is not known whether reversal of the exchanger contributes to activate calpains and trigger neuronal death. We investigated the role of the reversal of the NCX in Ca(2+) dynamics, calpain activation and cell viability, in alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor-stimulated hippocampal neurons. Selective overactivation of AMPA receptors caused the reversal of the NCX, which accounted for approximately 30% of the rise in intracellular free calcium concentration ([Ca(2+)](i)). The NCX reverse-mode inhibitor, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea (KB-R7943), partially inhibited the initial increase in [Ca(2+)](i), and prevented a delayed increase in [Ca(2+)](i). In parallel, overactivation of AMPA receptors strongly activated calpains and led to the proteolysis of NCX3. KB-R7943 prevented calpain activation, cleavage of NCX3 and was neuroprotective. Silencing of NCX3 reduced Ca(2+) uptake, calpain activation and was neuroprotective. Our data show for the first time that NCX reversal is an early event following AMPA receptor stimulation and is linked to the activation of calpains. Since calpain activation subsequently inactivates NCX, causing a secondary Ca(2+) entry, NCX may be viewed as a new suicide substrate operating in a Ca(2+)-dependent loop that triggers cell death and as a target for neuroprotection.

[Surgical treatment of complete penile duplication]. / Tratamiento quirúrgico de duplicidad peneana completa.

Penile duplication is a rare anomaly with an incidence of 1 in 5,500,000. It is almost associated with other malformations like double bladder, presence of the cloaca, imperforate anus, duplication of the recto sigmoid and vertebral deformities. The authors present the surgical technique to resolve a rare case of complete penile duplication in a 4 years old child, without any other malformation.

Excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors involves the AP-1 transcription factor.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

Bayesian network analysis of resistance pathways against HIV-1 protease inhibitors.

Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.

Genotoxic effects of binary mixtures of xenoandrogens (tributyltin, triphenyltin) and a xenoestrogen (ethinylestradiol) in a partial life-cycle test with Zebrafish (Danio rerio).

A partial life-cycle test with the model fish Danio rerio was performed in order to evaluate the genotoxic potential of binary mixtures of xenoandrogenic (tributyltin--TBT; triphenyltin--TPT) and an estrogenic compound (ethinylestradiol--EE2). Five days post-fertilisation larvae were diet-exposed to environmental relevant concentrations of TBT and TPT (25 ng/g-100 ng/g), and water-exposed to ethinylestradiol (3.5 ng/L) for a four-month period; binary mixtures of TBT plus EE2 and TPT plus EE2 were run in parallel. The erythrocytic nuclear abnormalities (ENA) assay in circulating erythrocytes was used to evaluate genotoxicity in the end of the four-month exposure period. A significant increase (p<0.05, Kruskall-Wallis non-parametric ANOVA) in ENA frequency, in comparison with control animals, was observed in those animals exposed to TBT and TPT (the highest doses only), and to EE2 and the binary mixtures, although neither synergistic nor additive effects of the tested compounds were evident. Overall, the results clearly indicate that chronic exposure to low levels of TBT, TPT, EE2 and binary mixtures of TBT plus EE2 and TPT plus EE2 are genotoxic to zebrafish, which may suggest that wild fish populations may be under increased DNA damage in areas contaminated by these endocrine disrupting chemicals.

Variation of age and total length in Sotalia guianensis (Van Bénéden, 1864) (Cetacea, Delphinidae), on the coast of Espírito Santo state, Brazil.

Variations of age and total length of Sotalia guianensis from the state of Espírito Santo, Brazil, were evaluated. Specimens were found stranded. Age and total length of 44 Guiana dolphins were assessed based on tooth analysis. Age varied between 0.5 year and 33 years (mean = 8.23 years). Most specimens were between zero and 6 years old (47%). Total length varied from 119 cm to 198 cm, with mean of 172.52 cm. Asymptotic length was reached at 185 cm and approximately 5-6 years of age. Mean total length and age were higher than in other regions of the distribution range of the species. Nevertheless, more studies have to be carried out to evaluate the morphological variations in S. guianensis populations in the study area and Brazil.

Neuroprotection by BDNF against glutamate-induced apoptotic cell death is mediated by ERK and PI3-kinase pathways.

Neurotrophins protect neurons against glutamate excitotoxicity, but the signaling mechanisms have not been fully elucidated. We studied the role of the phosphatidylinositol 3-kinase (PI3-K) and Ras/mitogen-activated protein kinase (MAPK) pathways in the protection of cultured hippocampal neurons from glutamate induced apoptotic cell death, characterized by nuclear condensation and activation of caspase-3-like enzymes. Pre-incubation with the neurotrophin brain-derived neurotrophic factor (BDNF), for 24 h, reduced glutamate-evoked apoptotic morphology and caspase-3-like activity, and transiently increased the activity of the PI3-K and of the Ras/MAPK pathways. Inhibition of the PI3-K and of the Ras/MAPK signaling pathways abrogated the protective effect of BDNF against glutamate-induced neuronal death and similar effects were observed upon inhibition of protein synthesis. Moreover, incubation of hippocampal neurons with BDNF, for 24 h, increased Bcl-2 protein levels. The results indicate that the protective effect of BDNF in hippocampal neurons against glutamate toxicity is mediated by the PI3-K and the Ras/MAPK signaling pathways, and involves a long-term change in protein synthesis.

Ca2(+)-dependent binding of tamoxifen to calmodulin isolated from bovine brain.

The interaction of the antiestrogen tamoxifen (Tx) with calmodulin (CaM) was investigated by cross-linking between the protein and [3H] tamoxifen aziridine. We observed that CaM binds Tx in a Ca2(+)-dependent manner and that two components are involved in the binding, with apparent dissociation constants (Kd) of about 6 nM and 9 microM. The high affinity binding site has a maximal capacity of 25 pmol/mg protein, whereas the low affinity binding site has a Bmax value of 120 nmol/mg protein. The stimulatory effect of Ca2+ is maximal at the pCa value of 5, and it is noncompetitively inhibited by Mg2+. In the micromolar range, the cation-dependent interaction of Tx with CaM exhibits positive cooperativity (nH = 1.4) and it is specific in the sense that it is inhibited by unlabeled Tx and by the CaM antagonist trifluoperazine. In contrast, no specificity was observed for the Tx binding, which is cation independent. Tx in the nanomolar range forms complexes with CaM which can be visualized by fluorography after electrophoretic separation in a polyacrylamide gel. Furthermore, CaM antagonism of Tx was observed with respect to inhibition of the CaM effect on the RBC membrane (Ca2(+) + Mg2+)-ATPase. The results indicate that Tx may alter Ca2(+)-dependent processes by interacting directly with CaM.

Effects of dietary exposure to herbicide and of the nutritive quality of contaminated food on the reproductive output of Daphnia magna.

Risk assessment of pesticides has been based on direct toxic effects on aquatic organisms. Indirect effects data are taken into account but with limitations, as it is frequently difficult to predict their real impacts in the ecosystems. In this context the main aim of this work was to assess how the exposure to the herbicide pendimethalin (Prowl(®)), under environmentally relevant concentrations, may compromise the nutritional composition of food for a relevant group of primary consumers of freshwater food webs-the daphnids, thus affecting their reproduction performance and subsequently the long-term sustainability of active populations of this grazer. Therefore, Daphnia magna individuals were chronically exposed in a clean medium to a control diet (NCF - i.e., non-contaminated green algae Raphidocelis subcapitata) and to a contaminated diet (CF - i.e., the same monoalgal culture grown in a medium enriched with pendimethalin in a concentration equivalent to the EC20 for growth inhibition of algae), during which reproductive endpoints were assessed. The algae were analysed for protein, carbohydrate and fatty acid content. The chemical composition of R. subcapitata in the CF revealed a slight decrease on total fatty acid levels, with a particular decrease of essential ω9 monounsaturated fatty acids. In contrast, the protein content was high in the CF. D. magna exposed to CF experienced a 16% reduction in reproduction, measured as the total number of offspring produced per female. Additionally, an internal pendimethalin body burden of 4.226µgg(-1) was accumulated by daphnids fed with CF. Hence, although it is difficult to discriminate the contribution of the pesticide (as a toxic agent transferred through the food web) from that of the food with a poor quality-compromised by the same pesticide, there are no doubts that, under environmentally relevant concentrations of pesticides, both pathways may compromise the populations of freshwater grazers in the long term, with consequences in the control of the primary productivity of these systems.

Interaction of chemical probes with sarcoplasmic reticulum membranes.

The labelling of the sarcoplasmic reticulum membranes by the chemical probes, trinitrobenzenesulfonate (TNBS) and fluorodinitrobenzene (FDNB) has been investigated. The incorporation of TNBS, but not of FDNB, depends on the binding of Ca2+ or Mg2+ to the membranes. The labelling of lipids and of the various reticulum proteins by TNBS is increased by those agents, but the effect is not uniform for all membrane proteins. The Ca2+ -ATPase contributes only 2.2% for the total labelling of the sarcoplasmic reticulum proteins, whereas the proteins of molecular weight 90 000 and 30 000 contribute about 34 and 56%, respectively. However, the Ca2+-ATPase isolated from the membrane reacts with an amount of TNBS 5-fold higher than that which reacts with the enzyme in situ. Both probes, TNBS and FDNB, inhibit the Ca2+-ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum, whereas the Mg2+-ATPase remains unaffected. The results indicate that FDNB is maximally incorporated into the sarcoplasmic reticulum membrane, whereas only some of the membrane amino groups are accessible to TNBS in the absence of Ca2+, Mg2+ or ATP which, when present, make additional amino groups available to TNBS. The highest degree of TNBS incorporation takes place into proteins, other than the ATPase, but sufficient reaction occurs with the enzyme to inhibit its activity.

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum.

The Ca2+ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca2+ and another represented by Ca2+ selectively bound to the membranes. Both of these Ca2+ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca2+ fraction. The existence of the membrane-bound Ca2+ induced by ATP is clearly shown in experiments in which the Ca2+ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca2+ ionophore, which makes the membrane permeable to Ca2+. Thus, in the presence of X-537A all Ca2+ accumulated due to ATP is bound to the membranes. This membrane-bound Ca2+ represents about 30 nmol/mg protein in the range of external pCa values of 7 to 3.5. The magnitude of this Ca2+ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca2+ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca2+ as a distinct fraction from that which exists intravesicularly.

Interaction of clathrin with large unilamellar phospholipid vesicles at neutral pH. Lipid dependence and protein penetration.

The interaction of clathrin with large unilamellar vesicles of various lipid compositions has been examined at neutral pH. Clathrin induces leakage of contents of vesicles that contain the acidic phospholipid phosphatidylserine. Leakage is greatly enhanced by the presence of a relatively minor amount of cholesterol, but is inhibited by phosphatidylcholine. Resonance energy transfer measurements between tryptophan residues of the protein and a fluorescent lipid analog, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine incorporated into the liposomal bilayer, suggests a dynamic interaction of clathrin with the bilayer at neutral pH. This interaction includes a (partial) penetration of the protein into the lipid bilayer, as revealed by hydrophobic photoaffinity labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine. The interaction of clathrin with lipid vesicles at neutral pH is inhibited when the protein is pretreated with trypsin or with the reducing agent dithiothreitol, suggesting that structural requirements govern clathrin-membrane interaction at these conditions. The physiological relevance of the present observations in light of vesiculation and endosomal maturation is discussed.