Does batrachotoxin autoresistance coevolve with toxicity in Phyllobates poison‐dart frogs?

Toxicity is widespread among living organisms, and evolves as a multimodal phenotype. Part of this phenotype is the ability to avoid self‐intoxication (autoresistance). Evolving toxin resistance can involve fitness tradeoffs, so autoresistance is often expected to evolve gradually and in tandem with toxicity, resulting in a correlation between the degrees of toxicity and autoresistance among toxic populations. We investigate this correlation in Phyllobates poison frogs, notorious for secreting batrachotoxin (BTX), a potent neurotoxin that targets sodium channels, using ancestral sequence reconstructions of BTX‐sensing areas of the muscular voltage‐gated sodium channel. Reconstructions suggest that BTX resistance arose at the root of Phyllobates, coinciding with the evolution of BTX secretion. After this event, little or no further evolution of autoresistance seems to have occurred, despite large increases in toxicity throughout the history of these frogs. Our results, therefore, provide no evidence in favor of an evolutionary correlation between toxicity and autoresistance, which conflicts with previous work. Future research on the functional costs and benefits of mutations putatively involved in BTX resistance, as well as their prevalence in natural populations, should shed light on the evolutionary mechanisms driving the relationship between toxicity and autoresistance in Phyllobates frogs.     

Evidence of DNA replication in an arbuscular mycorrhizal fungus in the absence of the host plant

Summary This study provides evidence thatGigaspora margarita replicates its nuclear DNA, even in the absence of a host plant. Three experimental approaches were used: (i) static cytofluorimetry to quantify the DNA content, (ii) pulse treatments with bromodeoxyuridine (BrdU), which is an analogue of thymidine, to reveal nuclei undergoing DNA synthesis, and (iii) ultrastructural observations to study changes in chromatin morphology during the fungal cell cycle. A slight second peak of approximately twice the value of a major peak was found by cytofluorimetry, showing that a small number of nuclei had entered in cycle during in vitro development. Nuclei which had incorporated BrdU were observed after pulses of 24 h; nuclei with condensed chromatin were also apparent at this time. The results demonstrate thatG. margarita has all the metabolic pathways needed to replicate its nuclear DNA even in the absence of the host, suggesting that more complex mechanisms inhibit the extended growth in vitro of arbuscular mycorrhizal fungi.Abbreviations AM-fungi arbuscular mycorrhizal fungi - A.U. arbitrary units - BrdU 5-bromo-2-deoxyuridine - DAPI 4,6-diamidino-2-phenylindole - UV ultraviolet light     

Functional and structural interactions between osteoblastic and preosteoclastic cells in vitro

Osteoblasts are involved in the bone resorption process by regulating osteoclast maturation and activity. In order to elucidate the mechanisms underlying osteoblast/preosteoclast cell interactions, we developed an in vitro model of co-cultured human clonal cell lines of osteoclast precursors (FLG 29.1) and osteoblastic cells (Saos-2), and evaluated the migratory, adhesive, cytochemical, morphological, and biochemical properties of the co-cultured cells. In Boyden chemotactic chambers, FLG 29.1 cells exhibited a marked migratory response toward the Saos-2 cells. Moreover, they preferentially adhered to the osteoblastic monolayer. Direct co-culture of the two cell types induced: (1) positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells; (2) a decrease of the alkaline phosphatase activity expressed by Saos-2 cells; (3) the appearance of typical ultrastructural features of mature osteoclasts in FLG 29.1 cells; (4) the release into the culture medium of granulocyte-macrophage colony stimulating factor. The addition of parathyroid hormone to the co-culture further potentiated the differentiation of the preosteoclasts, the cells tending to fuse into large multinucleated elements. These in vitro interactions between osteoblasts and osteoclast precursors offer a new model for studying the mechanisms that control osteoclastogenesis in bone tissue.     

Distinct Morphological Phenotypes of Cell Fusion Mutants

Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Δ fus2Δ, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus⁻ zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.     

Rhizobium etli and Rhizobium gallicum Nodulate Common Bean (Phaseolus vulgaris) in a Traditionally Managed Milpa Plot in Mexico: Population Genetics and Biogeographic Implications

The stability of the genetic structure of rhizobial populations nodulating Phaseolus vulgaris cultivated in a traditionally managed milpa plot in Mexico was studied over three consecutive years. The set of molecular markers analyzed (including partial rrs, glnII, nifH, and nodB sequences), along with host range experiments, placed the isolates examined in Rhizobium etli bv. phaseoli and Rhizobium gallicum bv. gallicum. Cluster analysis of multilocus enzyme electrophoresis and plasmid profile data separated the two species and identified numerically dominant clones within each of them. Population genetic analyses showed that there was high genetic differentiation between the two species and that there was low intrapopulation differentiation of the species over the 3 years. The results of linkage disequilibrium analyses are consistent with an epidemic genetic structure for both species, with frequent genetic exchange taking place within conspecific populations but not between the R. etli and R. gallicum populations. A subsample of isolates was selected and used for 16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, nifH copy number determination, and host range experiments. Plasmid profiles and nifH hybridization patterns also revealed the occurrence of lateral plasmid transfer among distinct multilocus genotypes within species but not between species. Both species were recovered from nodules of the same plants, indicating that mechanisms other than host, spatial, or temporal isolation may account for the genetic barrier between the species. The biogeographic implications of finding an R. gallicum bv. gallicum population nodulating common bean in America are discussed.     

Lake Baikal: a unique place to study evolution of sponges and their stress response in an environment nearly unimpaired by anthropogenic perturbation.

Lake Baikal is considered as a unique place to study evolution. In this review, we report on recent data on the evolution of endemic freshwater sponges of this ancient lake. Nucleotide sequence data support the idea that these sponges are of monophyletic origin and evolved from Spongillidae. Baikalian sponges form the dominating biomass in the benthos of the lake. Data on the expression of the biomarker heat shock protein 70, revealed that the endemic sponge species of Lake Baikal are useful as bioindicators to assess the anthropogenic impact on the lake.     

phnE and glpT Genes Enhance Utilization of Organophosphates in Escherichia coli K-12

Wild-type Escherichia coli K-12 strain JA221 grows poorly on low concentrations (≤1 mM) of diisopropyl fluorophosphate and its hydrolysis product, diisopropyl phosphate (DIPP), as sole phosphorus sources. Spontaneous organophosphate utilization (OPU) mutants were isolated that efficiently utilized these alternate sources of phosphate. A genomic library was constructed from one such OPU mutant, and two genes were isolated that conferred the OPU phenotype to strain JA221 upon transformation. These genes were identified as phnE and glpT. The original OPU mutation represented phnE gene activation and corresponded to the same 8-bp unit deletion from the cryptic wild-type E. coli K-12 phnE gene that has been shown previously to result in phnE activation. In comparison, sequence analysis revealed that the observed OPU phenotype conferred by the glpT gene was not the result of a mutation. PCR clones of glpT from both the mutant and the wild type were found to confer the OPU phenotype to JA221 when they were present on the high-copy-number pUC19 plasmid but not when they were present on the low-copy-number pWSK29 plasmid. This suggests that the OPU phenotype associated with the glpT gene is the result of amplification and overproduction of the glpT gene product. Both the active phnE and multicopy glpT genes facilitated effective metabolism of low concentrations of DIPP, whereas only the active phnE gene could confer the ability to break down a chromogenic substrate, 5-bromo-4-chloro-3-indoxyl phosphate-p-toluidine (X-P_i). This result indicates that in E. coli, X-P_i is transported exclusively by the Phn system, whereas DIPP (or its metabolite) may be transported by both Phn and Glp systems.     

Na+/H+-exchange in A6 cells: Polarity and vasopressin regulation

Summary We have analyzed the mechanism of Na⁺-dependent pH_i; recovery from an acid load in A6 cells (an amphibian distal nephron cell line) by using the intracellular pH indicator 27-bis(2-carboxyethyl)5, 6 carboxyfluorescein (BCECF) and single cell microspectrofluorometry. A6 cells were found to express Na⁺/H⁺-exchange activity only on the basolateral membrane: Na⁺/H⁺-exchange activity follows simple saturation kinetics with an apparent K _mfor Na⁺ of approximately 11 mm; it is inhibited in a competitive manner by ethylisopropylamiloride (EIPA). This Na⁺/H⁺-exchange activity is inhibited by pharmacological activation of protein kinase A (PKA) as well as of protein kinase C (PKC). Addition of arginine vasopressin (AVP) either at low (subnanomolar) or at high (micromolar) concentrations inhibits Na⁺/H⁺-exchange activity; AVP stimulates IP₃ production at low concentrations, whereas much higher concentrations are required to stimualte cAMP formation. These findings suggest that in A6 cells (i) Na⁺/H⁺-exchange is located in the basolateral membrane and (ii) PKC activation (heralded by IP₃ turnover) is likely to be the mediator of AVP action at low AVP concentrations.This work was supported by the Swiss National Science Foundation (Grant No. 32-30785.91), the Stiftung für wissenschaftliche Forschung an der Universität Zürich, the Hartmann-Müller Stiftung, the Sandoz-Stiftung, the Roche Research Foundation, and the Geigy Jubiläumsstiftung. Prof. Dr. V. Casavola and Dr. R. Guerra were supported by a research grant, No. 91.02470.CT14 of the Consiglio Nazionale della Ricerche (C.N.R.) We are grateful to Prof. Dr. B.C. Rossier of the Institute of Pharmacology of Lausanne (Switzerland) for the gift of the A6 cells, to H.P. Gaeggeler for the supply of the necessary culture media and to Jutka Forgo for her excellent help in the day-to-day culturing of the A6 cells. The secretarial assistance of D. Rossi is gratefully acknowledged.