Exploring the mycobiota of bromeliads phytotelmata in Brazilian Campos Rupestres.

The phytotelmata is a water-filled tank on a terrestrial plant, and it plays an important role in bromeliad growth and ecosystem functioning. Even though previous studies have contributed to elucidate the composition of the prokaryotic component of this aquatic ecosystem, its mycobiota (fungal community) is still poorly known. In the present work, ITS2 amplicon deep sequencing was used to examine the fungal communities inhabiting the phytotelmata of two bromeliads species that coexist in a sun-exposed rupestrian field of Southeastern Brazil, namely Aechmea nudicaulis (AN) and Vriesea minarum (VM). Ascomycota was the most abundant phylum in both bromeliads (57.1 and 89.1% in AN and VM respectively, on average), while the others were present in low abundance (< 2%). Mortierellomycota and Glomeromycota were exclusively observed in AN. Beta-diversity analysis showed that samples from each bromeliad significantly clustered together. In conclusion, despite the considerable within-group variation, the results suggested that each bromeliad harbor a distinct fungi community, what could be associated with the physicochemical characteristics of the phytotelmata (mainly total nitrogen, total organic carbon, and total carbon) and plant morphological features.

Antifungal activity of bacterial strains from maize silks against Fusarium verticillioides.

Fusarium verticillioides is pathogenic to maize and mycotoxin-producer, causing yield losses, feed and food contamination, and risks to human and animal health. Endophytic (ISD04 and IPR45) and epiphytic (CT02 and IM14) bacteria from maize silks were tested in vitro and greenhouse against F. verticillioides and for hydrolytic enzyme production (cellulase, pectinase, protease, lipase, and chitinase). The strains preliminarily identified as Achromobacter xylosoxidans (ISD04), Pseudomonas aeruginosa (IPR45), and Bacillus velezensis (CT02 and IM14) by 16S gene sequencing. All strains showed antifungal activity in vitro with inhibition values from 58.5 to 100%; they changed hyphae morphology and inhibited the conidial germination by up to 100% (IPR45). The four strains produced at least one enzyme with antifungal activity. The microbiolized seeds reduced the fungal development in stored grains and stalk rot severity in the greenhouse by 72.6% (ISD04). These results highlight the potential of these strains as biocontrol agents against F. verticillioides.

Synthetic Peptides Derived From Lycosa Erythrognatha Venom: Interaction With Phospholipid Membranes and Activity Against Resistant Bacteria.

Superbugs are a public health problem, increasing the need of new drugs and strategies to combat them. Our group has previously identified LyeTxI, an antimicrobial peptide isolated from Lycosa erythrognatha spider venom. From LyeTxI, we synthesized and characterized a derived peptide named LyeTxI-b, which has shown significant in vitro and in vivo activity. In this work, we elucidate the interaction of LyeTxI-b with artificial membranes as well as its effects on resistant strains of bacteria in planktonic conditions or biofilms. Isothermal titration calorimetry revealed that LyeTxI-b interacts more rapidly and with higher intensity with artificial vesicles, showing higher affinity to anionic vesicles, when compared to synthetic LyeTxI. In calcein experiments, LyeTxI-b caused greater levels of vesicle cleavage. Both peptides showed antibacterial activity at concentrations of µmol L-1 against 12 different clinically isolated strains, in planktonic conditions, in a concentration-dependent manner. Furthermore, both peptides elicited a dose-dependent production of reactive oxygen species in methicillin-resistant Staphylococcus aureus. In S. aureus biofilm assay, LyeTxI-b was more potent than LyeTxI. However, none of these peptides reduced Escherichia coli biofilms. Our results show LyeTxI-b as a promising drug against clinically resistant strains, being a template for developing new antibiotics.

Plant growth-promoting traits of yeasts isolated from the tank bromeliad Vriesea minarum L.B. Smith and the effectiveness of Carlosrosaea vrieseae for promoting bromeliad growth.

Yeasts can play important roles in promoting plant growth; however, little information is available in this regard for yeasts in water of bromeliad tanks. Here, we characterize the ability of 79 yeast isolates from tank bromeliad Vriesea minarum, an endangered species, to solubilize phosphate, secrete siderophores, and synthesize indole-3-acetic acid (IAA). The results showed that 67.8% of all assayed yeast isolates mobilized inorganic phosphate; 40.0% secreted siderophores; and 89.9% synthetized IAA and IAA-like compounds. Among the species studied, Carlosrosaea vrieseae UFMG-CM-Y6724 is highlighted for producing IAA (76.1 µg mL-1) and siderophores, and solubilizing phosphate. In addition, evaluation of the effects of filtrate containing IAA-like compounds produced by the C. vrieseae on the development and photosynthetic performance of V. minarum seedlings found it to improve seedling growth equal to that of commercial IAA. These results demonstrate that C. vrieseae can produce compounds with great potential for future use as biofertilizer agents.

Diversity of Metal-Resistant and Tensoactive-Producing Culturable Heterotrophic Bacteria Isolated from a Copper Mine in Brazilian Amazonia.

Bacterial extracellular polymeric substances (EPSs) present diverse properties of biotechnological interest, such as surface modification, metal adsorption and hydrophobic substances solubilization through surface tension reduction. Thus, there is a growing demand for new producing strains and structurally variable biomolecules with different properties. One approach for scanning this biodiversity consists of exploring environments under selective pressures. The aim of this study was to evaluate the composition of culturable heterotrophic bacterial communities from five different sites from a copper mine in the Amazon biome by an enrichment technique to obtain metal resistant bacteria (lead, arsenic, cadmium, copper and zinc) capable of producing EPSs. The bacterial densities at the sites varied from 2.42 × 103 to 1.34 × 108 NMP mL-1 and the 77 bacterial isolates obtained were classified in four divisions, ß-Proteobacteria (16.88%), γ-Proteobacteria (7.29%), Firmicutes (61%) and Actinobacteria (12.98%). Bacillus, Alcaligenes, and Lysinibacillus were the most dominant among the 16 observed genera, but the relative frequency of each varied according to the sample and the metal used in the enrichment culture. 58% of the bacterial strains (45) could produce EPSs. From these, 33 strains showed emulsifying activity (E24), and 9 of them reached values higher than 49%. Only Actinomyces viscosus E3.Pb5 and Bacillus subtilis group E3.As2 reduced the medium surface tension to values lower than 35 mN m-1. It was possible to confirm the high presence of bacteria capable of producing EPSs with tensoactive properties in Amazon copper mines and the evolutionary pressure exerted by the heavy metals during enrichment. These molecules can be tested as an alternative for use in processes that involve the removal of metals, such as the bioremediation of contaminated environments.

The wound healing effect of aqueous extract from Piper amalago L. in diabetic patient.

Diabetes patients present a complex healing process due to several factors directly linked to their pathology. The use of medicinal plants that aid in tissue repair can bring great benefits to such individuals. This case report describes how the topical application of the aqueous extract produced from the leaves of Piper amalago L. was used to aid the healing of a lacerated wound in the left thumb of a patient with type 2 diabetes mellitus. The aqueous extract of the leaves of Piper amalago L. was prepared in boiling water. During the boiling process the dried leaves were submerged in the boiling water and left for five min. The injured thumb was submerged in the solution and the leaves were placed on the injury. The action of the aqueous extract obtained from the leaves of P. amalago was shown to be promising in the healing of a wound in a patient with type 2 diabetes mellitus. The topical application of the aqueous extract produced from the leaves of P. amalago assisted in the healing of a lacerated wound in the left thumb of a patient with type 2 diabetes mellitus over a period of 15 days.

Metataxonomic analyses reveal differences in aquifer bacterial community as a function of creosote contamination and its potential for contaminant remediation.

Metataxonomic approach was used to describe the bacterial community from a creosote-contaminated aquifer and to access the potential for in situ bioremediation of the polycyclic aromatic hydrocarbons (PAHs) by biostimulation. In general, the wells with higher PAH contamination had lower richness and diversity than others, using the Shannon and Simpson indices. By the principal coordinate analysis (PCoA) it was possible to observe the clustering of the bacterial community of most wells in response of the presence of PAH contamination. The significance analysis using edgeR package of the R program showed variation in the abundance of some Operational Taxonomic Units (OTUs) of contaminated wells compared to uncontaminated ones. Taxons enriched in the contaminated wells were correlated positively (p < 0.05) with the hydrocarbons, according to redundancy analysis (RDA). All these enriched taxa have been characterized as PAH degrading agents, such as the genus Comamonas, Geobacter, Hydrocarboniphaga, Anaerolinea and Desulfomonile. Additionally, it was possible to predict, with the PICRUSt program, a greater proportion of pathways and genes related to the degradation of PAHs in the wells with higher contamination levels. We conclude that the contaminants promoted the enrichment of several groups of degrading bacteria in the area, which strengthens the feasibility of applying biostimulation as an aquifer remediation strategy.

Evaluation of the interaction between polymyxin B and Pseudomonas aeruginosa biofilm and planktonic cells: reactive oxygen species induction and zeta potential.

BACKGROUND: Although the most widely accepted mechanism of action for polymyxins is related to bacterial lysis via disruption, we hypothesized that this antimicrobial drug class could have other effects on Pseudomonas aeruginosa planktonic and sessile cells. Little is known regarding oxidative burst and zeta potential (ZP) data associated with the interaction between polymyxin B and P. aeruginosa cells. The present study evaluated endogenous reactive oxygen species (ROS) production and changes in the net charges of biofilm and planktonic cells in response to polymyxin B. RESULTS: Polymyxin B induced concentration-dependent killing at all concentrations tested in planktonic and sessile cells from P. aeruginosa strains. Sublethal concentrations of polymyxin B induced oxidative burst. ROS production was higher in resistant planktonic cells than in biofilm cells but this was not observed for susceptible cells. Moreover, no net surface charge alterations were observed in planktonic cells from a susceptible strain treated with polymyxin B, but a significant increase of ZP was noted in planktonic cells from a resistant strain. CONCLUSION: Oxidative burst generated by planktonic and sessile cells from P. aeruginosa strains against polymyxin B indicates that ROS may have an important role in the mechanism of action of this drug. ZP data revealed that electrostatic interactions of the cationic peptide with the anionic surface of the cells are strain-dependent. Therefore, we suggested that the intracellular effects of polymyxin B should be further investigated to understand polymyxin B-induced stress in P. aeruginosa.

Bacterial Small RNAs in the Genus Herbaspirillum spp.

The genus Herbaspirillum includes several strains isolated from different grasses. The identification of non-coding RNAs (ncRNAs) in the genus Herbaspirillum is an important stage studying the interaction of these molecules and the way they modulate physiological responses of different mechanisms, through RNA⁻RNA interaction or RNA⁻protein interaction. This interaction with their target occurs through the perfect pairing of short sequences (cis-encoded ncRNAs) or by the partial pairing of short sequences (trans-encoded ncRNAs). However, the companion Hfq can stabilize interactions in the trans-acting class. In addition, there are Riboswitches, located at the 5' end of mRNA and less often at the 3' end, which respond to environmental signals, high temperatures, or small binder molecules. Recently, CRISPR (clustered regularly interspaced palindromic repeats), in prokaryotes, have been described that consist of serial repeats of base sequences (spacer DNA) resulting from a previous exposure to exogenous plasmids or bacteriophages. We identified 285 ncRNAs in Herbaspirillum seropedicae (H. seropedicae) SmR1, expressed in different experimental conditions of RNA-seq material, classified as cis-encoded ncRNAs or trans-encoded ncRNAs and detected RNA riboswitch domains and CRISPR sequences. The results provide a better understanding of the participation of this type of RNA in the regulation of the metabolism of bacteria of the genus Herbaspirillum spp.

Bioética: análise da compreensão de biodireito por meio de cases / Bioética: análisis de la comprensión del bioderecho por medio de casos / Bioethics: Analysis of the Understanding of Biolaw Using Cases

Resumo As ações de saúde contra o Estado tornam-se cada vez mais comuns. A discussão gira em torno dos medicamentos e tratamentos que não estão no rol de serviços do Sistema Único de Saúde (SUS). O objetivo deste estudo é analisar o comportamento ético dos estudantes de pós-graduação da área de saúde, diante de situações que envolvem a ética e o direito dos cidadãos. Os pesquisadores utilizaram a metodologia de pesquisa de campo, por meio da aplicação de um questionário online que continha um texto com dilemas éticos e de biodireito. Os resultados apontaram que a maioria dos entrevistados demonstrou empatia pelas ações requeridas pelos cidadãos; segundo os alunos, o Estado tem condições financeiras para isso, além de ser seu dever. Finalmente, pode-se perceber que os princípios bioéticos são analisados de acordo com a sua percepção de vida, mesmo diante de situações conflituosas em que o coletivo pode ser prejudicado ante a individualização.

Resumen Las acciones de salud en contra del Estado se vuelven cada vez más comunes. La discusión es acerca de medicamentos y tratamientos que no están en la lista de servicios del Sistema Único de Salud de Brasil. El objetivo del artículo es analizar la conducta ética de los estudiantes de posgrado en el área de la salud, en situaciones que implican la ética y los derechos de los ciudadanos. Los investigadores utilizaron la metodología de investigación de campo mediante la aplicación de un cuestionario en línea que contenía un texto con dilemas éticos y de bioderechos. Los resultados permiten observar que la mayoría de los entrevistados demostró empatía por las acciones requeridas por los ciudadanos; según los estudiantes, el Estado tiene las condiciones financieras para ello, además es su deber. Por último, se puede notar que los principios bioéticos se analizan en función de su percepción de la vida, incluso ante situaciones de conflicto en que el colectivo puede ser perjudicado ante la individualización.

Abstract Healthcare-related legal actions against the state have become increasingly common. The discussion revolves around drugs and treatments that are not in the SUS service list. The aim of this article is to analyze the ethical behavior of graduate health sciences students in situations involving ethics and citizen rights. Researchers used the field research method by administering an online questionnaire which contained a text with ethical and biolaw dilemmas. Results showed that the majority of respondents felt empathy for the actions brought by citizens; according to the students, the state has the financial conditions and is obliged to take care of them. Finally, it can be noted that bioethical principles are analyzed based on their perception of life, even in the face of conflicting circumstances in which the community may be harmed by individualization.

Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

BACKGROUND: New Delhi Metallo-b-lactamase (NDM-1) is an enzyme emerging around the world conferring resistance to a wide range of ß-lactams agents and whose early detection is extremely important. We proposed to standardize the detection of the blaNDM-1 gene using the LOOP-mediated isothermal amplification technique (LAMP). METHODS: In all, 14 Gram-negative bacterial strains isolated from patients presenting pneumonia associated with mechanical ventilation were used for the blaNDM-1 standardization by LAMP. Klebsiella pneumoniae ATCC BAA-2473 and two clinical strains were used as a positive control. All results were compared to the reaction in polymerase chain reaction (PCR), considered gold standard for this detection. RESULTS: There was an excellent correlation between the two techniques employed, since all measured clinical strains were negative in both employed tests and two clinical, and a reference strains were positive. CONCLUSIONS: The lamp technique seems to be an excellent option for the rapid detection of blaNDM-1. The amplification time is much shorter than other molecular techniques, the PCR machine is not necessary, it is easy of implementation and costs is low.

Comparative study of free and liposome-entrapped chloramphenicol against biofilms of potentially pathogenic bacteria isolated from cooling towers.

This study aimed to investigate for the first time the in vitro antibiofilm effectiveness of two chloramphenicol liposome formulations against biofilms of potentially pathogenic bacteria associated to corrosion isolated from the water of cooling towers from a Brazilian industry. Antibiofilm assays with liposomes were performed in 96-wells microtiter plates, and data was compared to free chloramphenicol treatment. Chloramphenicol-loaded liposomes were successfully produced using the dehydration-rehydration method, with vesicle diameters of 131 nm (100 nm membrane extrusion) and 182 nm (200 nm membrane extrusion) assessed by dynamic light scattering. The liposomes obtained by 100 nm membrane extrusion were more effective than 200 nm membrane extrusion vesicles against the biofilms after overnight exposure, and the free drug had no antibiofilm effect. Our study open doors for more investigations on liposome entrapment of antimicrobial compounds such as biocides of industrial use, for controlling biofilm formation in aquatic environments.

Comparative morphoanatomical analysis of Mikania species

ABSTRACT Mikania belongs to the Asteraceae family and includes a wide range of promising pharmacological activities. Several species of Mikania, which is popularly known in Brazil as “guaco”, occur in Southern Brazil and their external morphology is similar. The aim of this study was to investigate the morpho-anatomical characteristics of the leaf and stem of Mikania campanulata, Mikania cordifolia, Mikania glomerata, Mikania hastato-cordata, Mikania microptera and Mikania sessilifolia as a means of providing additional support for differentiating these taxa. The leaves and stems were investigated by employing scanning electron microscopy and light microscopy techniques. The morphological features of Mikania spp. leaves make it possible to differentiate between the species; nevertheless, when the plants were fragmented or pulverized the anatomical features of the leaves and stems supplied additional helpful data in this regard. The main anatomical characteristics were presence of hypodermis and lens shaped epidermal cells, set of trichomes; midrib, petiole and stem shape and vascular pattern; sclerenchymatous ring in the cortex, sclerenchymatous cells and secretory ducts in the pith.

Antibacterial Effect of Synthetic Peptide LyeTxI and LyeTxI/ß-Cyclodextrin Association Compound Against Planktonic and Multispecies Biofilms of Periodontal Pathogens.

BACKGROUND: Antimicrobial peptides (AMPs) have shown rapid and potent effect against planktonic bacteria. However, control of periodontopathic biofilms is a challenge even for AMPs. Thus, the present study evaluates in vitro antimicrobial activity of synthetic peptide LyeTxI and association compound LyeTxI/ß-cyclodextrin (ßCD) against multispecies biofilms. METHODS: Sensibility to LyeTxI and LyeTxI/ßCD was determined for planktonic Gram-negative periodontopathogens. Time-kill kinetic assay was performed at minimum inhibitory concentrations (MICs) in all planktonic strains. Multispecies biofilms were grown on pegs using a biofilm device and studied by scanning electron microscopy at 2, 5, and 10 days. Minimal biofilm eradication concentration (MBEC) was determined for 2- and 4-day multispecies biofilms. Metabolic activity of biofilms was determined by fluorometry study. RESULTS: Biofilms showed reproducible cell density on pegs of the biofilm device. LyeTxI and LyeTxI/ßCD were active against all strains tested at concentrations ≤62.5 µg/mL. Kinetic assays showed rapid bactericidal effect of LyeTxI against all periodontopathogens. MBECs of LyeTxI and LyeTxI/ßCD against multispecies 2-day biofilms were two-fold higher than MICs of cells shed from biofilms. LyeTxI was able to reduce multispecies 2-day metabolic activity by 90%. Multispecies 4-day biofilms were tolerant to all agents tested. CONCLUSIONS: LyeTxI and LyeTxI/ßCD are active against periodontopathic bacteria, showing rapid bactericidal effect and may be used to prevent biofilm development. In the future, AMPs could be therapeutic tools for treatment of periodontitis.

Reuse of refinery's tertiary-treated wastewater in cooling towers: microbiological monitoring.

The study was planned to quantify the distribution of bacteria between bulk water and biofilm formed on different materials in an industrial scale cooling tower system of an oil refinery operating with clarified and chlorinated freshwater (CCW) or chlorinated tertiary effluent (TRW) as makeup water. The sessile and planktonic heterotrophic bacteria and Pseudomonas aeruginosa densities were significantly higher in the cooling tower supplied with clarified and chlorinated freshwater (CTCW) (p < 0.05). In the two towers, the biofilm density was higher on the surface of glass slides and stainless steel coupons than on the surface of carbon steel coupons. The average corrosion rates of carbon steel coupons (0.4-0.8 millimeters per year (mpy)) and densities of sessile (12-1.47 × 10(3) colony-forming unit (CFU) cm(-1)) and planktonic (0-2.36 × 10(3) CFU mL(-1)) microbiota remained below of the maximum values of reference used by water treatment companies as indicative of efficient microbial control. These data indicate that the strategies of the water treatment station (WTS) (free chlorine) and industrial wastewater treatment station (IWTS) followed by reverse electrodialysis system (RES) (free chlorine plus chloramine) were effective for the microbiological control of the two makeup water sources.

Bioconversion of biodiesel refinery waste in the bioemulsifier by Trichosporon mycotoxinivorans CLA2.

BACKGROUND: The microbial bioemulsifiers was surface active compounds, are more effective in stabilizing oil-in-water emulsions. The yeasts have been isolated to produce bioemulsifiers from vegetable oils and industrial wastes. RESULTS: Trichosporon mycotoxinivorans CLA2 is bioemulsifier-producing yeast strain isolated from effluents of the dairy industry, with ability to emulsify different hydrophobic substrates. Bioemulsifier production (mg/L) and the emulsifying activity (E24) of this strain were optimized by response surface methodology using mineral minimal medium containing refinery waste as the carbon source, which consisted of diatomaceous earth impregnated with esters from filters used in biodiesel purification. The highest bioemulsifier production occurred in mineral minimal medium containing 75 g/L biodiesel residue and 5 g/L ammonium sulfate. The highest emulsifying activity was obtained in medium containing 58 g/L biodiesel refinery residue and 4.6 g/L ammonium sulfate, and under these conditions, the model estimated an emulsifying activity of 85%. Gas chromatography and mass spectrometry analysis suggested a bioemulsifier molecule consisting of monosaccharides, predominantly xylose and mannose, and a long chain aliphatic groups composed of octadecanoic acid and hexadecanoic acid at concentrations of 48.01% and 43.16%, respectively. The carbohydrate composition as determined by GC-MS of their alditol acetate derivatives showed a larger ratio of xylose (49.27%), mannose (39.91%), and glucose (10.81%). 1 H NMR spectra confirmed by COSY suggested high molecular weight, polymeric pattern, presence of monosaccharide's and long chain aliphatic groups in the bioemulsifier molecule. CONCLUSIONS: The biodiesel residue is an economical substrate, therefore seems to be very promising for the low-cost production of active emulsifiers in the emulsification of aromatics, aliphatic hydrocarbons, and kerosene.

Phenol degradation by Aureobasidium pullulans FE13 isolated from industrial effluents.

The degradation of phenol (2-30 mM) by free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13 isolated from stainless steel effluents was studied in batch cultures with saline solution not supplemented with nutrients or yeast extract. The rate at which the immobilized cells degrade phenol was similar to the rate at which the suspended cells could degrade phenol, for a concentration of up to 16 mM of phenol. The maximum phenol volumetric degradation rate for 16 mM phenol was found to be 18.35 mg l(-1)h(-1) in the assays with free cells and 20.45 mg l(-1)h(-1) in the assays with alginate-immobilized cells, 18 mM phenol and cellular concentration of 0.176 g/l. At concentrations higher than this, an inhibitory effect was observed, resulting in the lowering of the phenol degradation rates. The immobilization was detrimental to the catechol 1,2-dioxygenase activity. However, the immobilized cells remained viable for a longer period, increasing the efficiency of phenol degradation. The yeast showed catechol 1,2-dioxygenase activity only after growth in the phenol, which was induced at phenol concentrations as low as 0.05 mM and up to 25 mM at 45 h of incubation at 30 degrees C. Phenol concentrations higher than 6mM were inhibitory to the enzyme. Addition of glucose, lactate, succinate, and benzoate reduced the rate at which phenol is consumed by cells. Our results suggest that inoculants based on immobilized cells of A. pullulans FE13 has potential application in the biodegradation of phenol and possibly in the degradation of other related aromatic compounds.

Experimental evidence for a direct cytotoxicity of Loxosceles intermedia (brown spider) venom in renal tissue.

Brown spider (Loxosceles genus) venom causes necrotic lesions often accompanied by fever, hemolysis, thrombocytopenia, and acute renal failure. Using mice exposed to Loxosceles intermedia venom, we aimed to show whether the venom directly induces renal damage. The experimental groups were composed of 50 mice as controls and 50 mice that received the venom. Light microscopic analysis of renal biopsy specimens showed alterations including hyalinization of proximal and distal tubules, erythrocytes in Bowman's space, glomerular collapse, tubule epithelial cell blebs and vacuoles, interstitial edema, and deposition of eosinophilic material in the tubule lumen. Electron microscopic findings indicated changes including glomerular epithelial and endothelial cell cytotoxicity as well as disorders of the basement membrane. Tubule alterations include epithelial cell cytotoxicity with cytoplasmic membrane blebs, mitochondrial changes, increase in smooth endoplasmic reticulum, presence of autophagosomes, and deposits of amorphous material in the tubules. We also found that the venom caused azotemia with elevation of blood urea levels but did not decrease C3 complement concentration or cause hemolysis in vivo. Confocal microscopy with antibodies against venom proteins showed direct binding of toxins to renal structures, confirmed by competition assays. Double-staining immunofluorescence reactions with antibodies against type IV collagen or laminin, antibodies to venom toxins, and fluorescent cytochemistry with DAPI revealed deposition of toxins in glomerular and tubule epithelial cells and in renal basement membranes. Two-dimensional electrophoresis showed venom rich in low molecular mass and cationic toxins. By immunoblotting with antibodies to venom toxins on renal extracts from venom-treated mice, we detected a renal binding toxin at 30 kD. The data provide experimental evidence that L. intermedia venom is directly involved in nephrotoxicity.