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A comparison between two brine shrimp assays to detect in vitro cytotoxicity in marine natural products.

BackgroundThe brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity. It has also been suggested for screening pharmacological activities in plant extracts. However, we think that it is necessary to evaluate the suitability of the brine shrimp methods before they are used as a general bio-assay to test natural marine products for pharmacological activity.Material and MethodsThe bioactivity of the isopropanolic (2-PrOH) extracts of 14 species of marine invertebrates and 6 species of macroalgae was evaluated with the shrimp lethality assay (lethality assay), as well as with another assay based on the inhibition of hatching of the cyst (hatchability assay). The extracts were also assayed for cytotoxicity against two human cell lines, lung carcinoma A-549 and colon carcinoma HT-29, in order to assess the sensitivity of the shrimp assays to detect cytotoxic activity.ResultsTwo sponges (Hyatella sp, Dysidea sp.), two gorgonians (Pacifigorgia adamsii, Muricea sp.), one tunicate (Polyclinum laxum), and three echinoderms (Holothuria impatiens, Pseudoconus californica and Pharia pyramidata) showed a strong cytostatic (growth inhibition) and cytotoxic effect. The hatchability assay showed a strong activity in 4 of the species active against the two human cell lines tested (Hyatella sp, Dysidea sp., Pacifigorgia adamsii and Muricea sp.), and the lethality assay also showed a high lethality in 4 of them (Pacifigorgia adamsii, Muricea sp., Polyclinum laxum, and Pharia pyramidata). Each bioassay detected activity in 50% of the species that were considered active against the two human cell lines tested. However, the simultaneous use of both bioassays increased the percentage to 75%.ConclusionsOur results seem consistent with the correlation previously established between cytotoxicity and brine shrimp lethality in plant extracts. We suggest using both bioassays simultaneously to test natural marine products for pharmacological activity.

Rho 1 GTPase activates the (1-3)beta-D-glucan synthase and is involved in Schizosaccharomyces pombe morphogenesis.

The Schizosaccharomyces pombe Cdc42 and Rho1 GTPases were tested for their ability to complement the cwg2‐1 mutant phenotype of a decrease in (1‐3)beta‐D‐glucan synthase activity when grown at the non‐permissive temperature. Only Rho1 is able to partly complement the defect in glucan synthase associated with the cwg2–1 mutation. Moreover, overexpression of the rho1 gene in wild‐type S.pombe cells causes aberrant morphology with loss of polarity and cells with several septa. Under this condition (1–3)beta‐D‐glucan synthase activity is increased four times, but is still dependent on GTP. When S.pombe is transformed with constitutively active rho1 mutant alleles (rho1‐G15V or rho1‐Q64L), cells stop growing and show a very thick cell wall with hardly any septum. Under this condition the level of (1–3)beta‐D‐glucan synthase activity is at least 20 times higher than wild‐type and is independent of GTP. Neither cdc42+ nor the cdc42‐V12G or cdc42‐Q61L constitutively active mutant alleles affect (1–3)beta‐D‐glucan synthase activity when overexpressed in S.pombe. Cells overproducing Rho1 are hypersensitive to inhibitors of cell wall biosynthesis or to cell wall degrading enzymes. We conclude that Rho1 GTPase directly activates (1–3)beta‐D‐glucan synthase and regulates S.pombe morphogenesis.

A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae

Using a hierarchical approach, 620 non-essential single-gene yeast deletants generated by EUROFAN I were systematically screened for cell-wall-related phenotypes. By analyzing for altered sensitivity to the presence of Calcofluor white or SDS in the growth medium, altered sensitivity to sonication, or abnormal morphology, 145 (23%) mutants showing at least one cell wall-related phenotype were selected. These were screened further to identify genes potentially involved in either the biosynthesis, remodeling or coupling of cell wall macromolecules or genes involved in the overall regulation of cell wall construction and to eliminate those genes with a more general, pleiotropic effect. Ninety percent of the mutants selected from the primary tests showed additional cell wall-related phenotypes. When extrapolated to the entire yeast genome, these data indicate that over 1200 genes may directly or indirectly affect cell wall formation and its regulation. Twenty-one mutants with altered levels of β1,3-glucan synthase activity and five Calcofluor white-resistant mutants with altered levels of chitin synthase activities were found, indicating that the corresponding genes affect β1,3-glucan or chitin synthesis. By selecting for increased levels of specific cell wall components in the growth medium, we identified 13 genes that are possibly implicated in different steps of cell wall assembly. Furthermore, 14 mutants showed a constitutive activation of the cell wall integrity pathway, suggesting that they participate in the modulation of the pathway either directly acting as signaling components or by triggering the Slt2-dependent compensatory mechanism. In conclusion, our screening approach represents a comprehensive functional analysis on a genomic scale of gene products involved in various aspects of fungal cell wall formation.

NGF binding to the trk tyrosine kinase receptor requires the extracellular immunoglobulin-like domains

Neurotrophins initiate their biological effects by activating members of the trk tyrosine kinase subfamily. The extracellular region of trk receptors is distinguished by several common structural features, including leucine-rich repeats, clusters of cysteine-rich domains, and two immunoglobulin-like domains. However, the receptor sequences required for ligand binding have not been localized. In order to define the domains involved in NGF binding, a series of chimeric receptors was constructed using cDNA sequences from rat trkA and trkB. The chimeric constructs were expressed after transient transfection in 293 cells and the expression of each receptor was verified by immunoprecipitation and immunoblot analysis. Equilibrium binding of transfected cells revealed that the two IgG domains of trkA are essential for NGF binding. The requirement for the two IgG domains was further confirmed by Scatchard analysis and affinity crosslinking with 125I-NGF. These results indicate that NGF binding is crucially dependent upon interactions with the IgG domains of the trkA receptor.

TrkA Immunoglobulin-Like Ligand Binding Domains Inhibit Spontaneous Activation of the Receptor

ABSTRACT The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.

Does ear C sink strength contribute to overcoming photosynthetic acclimation of wheat plants exposed to elevated CO2

Wheat plants (Triticum durum Desf., cv. Regallo) were grown in the field to study the effects of contrasting [CO2] conditions (700 versus 370 μmol mol−1) on growth, photosynthetic performance, and C management during the post-anthesis period. The aim was to test whether a restricted capacity of sink organs to utilize photosynthates drives a loss of photosynthetic capacity in elevated CO2. The ambient 13C/12C isotopic composition (δ13C) of air CO2 was changed from –10.2‰ in ambient [CO2] to –23.6‰ under elevated [CO2] between the 7th and the 14th days after anthesis in order to study C assimilation and partitioning between leaves and ears. Elevated [CO2] had no significant effect on biomass production and grain filling, and caused an accumulation of C compounds in leaves. This was accompanied by up-regulation of phosphoglycerate mutase and ATP synthase protein content, together with down-regulation of adenosine diphosphate glucose pyrophosphatase protein. Growth in elevated [CO2] negatively affected Rubisco and Rubisco activase protein content and induced photosynthetic down-regulation. CO2 enrichment caused a specific decrease in Rubisco content, together with decreases in the amino acid and total N content of leaves. The C labelling revealed that in flag leaves, part of the C fixed during grain filling was stored as starch and structural C compounds whereas the rest of the labelled C (mainly in the form of soluble sugars) was completely respired 48 h after the end of labelling. Although labelled C was not detected in the δ13C of ear total organic matter and respired CO2, soluble sugar δ13C revealed that a small amount of labelled C reached the ear. The 12CO2 labelling suggests that during the beginning of post-anthesis the ear did not contribute towards overcoming flag leaf carbohydrate accumulation, and this had a consequent effect on protein expression and photosynthetic acclimation.

Stress-induced Response, Localization, and Regulation of the Pmk1 Cell Integrity Pathway in Schizosaccharomyces pombe *

Mitogen-activated protein kinase (MAPK) signaling pathways are critical for the sensing and response of eukaryotic cells to extracellular changes. In Schizosaccharomyces pombe, MAPK Pmk1/Spm1 has been involved in cell wall construction, morphogenesis, cytokinesis, and ion homeostasis, as part of the so-called cell integrity pathway together with MAPK kinase kinase Mkh1 and MAPK kinase Pek1. We show that Pmk1 is activated in multiple stress situations, including hyper- or hypotonic stress, glucose deprivation, presence of cell wall-damaging compounds, and oxidative stress induced by hydrogen peroxide or pro-oxidants. The stress-induced activation of Pmk1 was completely dependent on Mkh1 and Pek1 function, supporting a nonbranched pathway in the regulation of MAPK activation. Fluorescence microscopy revealed that Mkh1, Pek1, and Pmp1 (a protein phosphatase that inactivates Pmk1) are cytoplasmic proteins. Mkh1 and Pek1 were also found at the septum, whereas Pmk1 localized in both cytoplasm and nucleus as well as in the mitotic spindle and septum during cytokinesis. Interestingly, Pmk1 subcellular localization was unaffected by stress or the absence of Mkh1 and Pek1, suggesting that its activation by the Mkh1-Pek1 cascade takes place at the cytoplasm and/or septum and that the active and inactive forms of this kinase cross the nuclear membrane. Cdc42 GTPase and its effectors, p21-activated kinases Pak2 and Pak1, are not upstream elements controlling the basal level or the stress-induced activation of Pmk1. However, Sty1 MAPK was essential for proper Pmk1 deactivation after hypertonic stress in a process regulated by Atf1 transcription factor. These results provide the first evidence for the existence of cross-talk between two MAPK cascades during the stress response in fission yeast.

A Novel p75 Neurotrophin Receptor-Related Protein, NRH2, Regulates Nerve Growth Factor Binding to the TrkA Receptor

Nerve growth factor (NGF) functions as a ligand for two receptors, the TrkA tyrosine kinase receptor and the p75 neurotrophin receptor (p75NTR). The Ig-like domains of Trk receptors and the cysteine-rich repeats of p75NTR are involved in binding to the neurotrophins. Recently, a closely related gene to p75NTR called neurotrophin receptor homolog-2 (NRH2) was identified; however, the function of NRH2 and its relevance to neurotrophin signaling are unclear. NRH2 contains a similar transmembrane and intracellular domain as p75NTR but lacks the characteristic cysteine-rich repeats in the extracellular domain. Here we show that NRH2 is expressed in several neuronal populations that also express p75NTR and Trk receptors. NRH2 does not bind to NGF; however, coimmunoprecipitation experiments demonstrate that NRH2 is capable of interacting with TrkA receptors. Coexpression of NRH2 with TrkA receptors resulted in the formation of high-affinity binding sites for NGF. These results indicate that a transmembrane protein related to p75NTR is capable of modulating Trk receptor binding properties.

Evidence for the Mechanism of Action of the Antifungal Phytolaccoside B Isolated from Phytolacca tetramera Hauman

Phytolaccoside B (1), an antifungal monodesmoside triterpenoid glycoside isolated from berries of Phytolacca tetramera Hauman (Phytolaccaceae), alters the morphology of yeasts and molds. The malformations were similar to those produced by enfumafungin, a known inhibitor of (1-->3)-beta-D-glucan synthase, an enzyme that catalyzes the synthesis of (1-->3)-beta-D-glucan, one of the major polymers of the fungal cell wall. However, enzymatic assays revealed that 1 did not inhibit (1-->3)-beta-D-glucan synthase, but it did produce a notable enhancement of the chitin synthase 1 activity and, concomitantly, a rise in chitin, another important polymer of the fungal cell walls. This finding was corroborated by fluorescence microscopy and also by quantification of the chitin. In addition, a 2-fold increase in the thickness of the fungal cell wall was observed with transmission electronic microscopy. On the other hand, 1 neither bound to ergosterol nor caused hemolysis of red blood cells, although some fungal membrane damage was observed at the MIC of 1.

The Schizosaccharomyces pombe cwg2+ gene codes for the beta subunit of a geranylgeranyltransferase type I required for beta-glucan synthesis.

The product of the Schizosaccharomyces pombe cwg2+ gene is involved in the biosynthesis of beta‐D‐glucan. When grown at the non‐permissive temperature, cwg2‐1 mutant cells lyse in the absence of an osmotic stabilizer and display a reduced (1‐3) beta‐D‐glucan content and (1‐3) beta‐D‐glucan synthase activity. The cwg2+ gene was cloned by the rescue of the cwg2‐1 mutant phenotype using an S. pombe genomic library and subsequently verified by integration of the appropriate insert into the S. pombe genome. Determination of the nucleotide sequence of this gene revealed a putative open reading frame of 1065 bp encoding a polypeptide of 355 amino acids with a calculated M(r) of 40,019. The cwg2+ DNA hybridizes to a main transcript, the 5′ end of which maps to a position 469 bp upstream of the predicted start of translation. The sequence between the transcription and the translation start sites is unusually long and has several short open reading frames which suggest a translational control of the gene expression. Comparative analysis of the predicted amino acid sequence shows that it possesses significant similarity to three Saccharomyces cerevisiae proteins, encoded by the DPR1/RAM1, CDC43/CAL1 and ORF2/BET2 genes respectively, which are beta subunits of different prenyltransferases. When grown at 37 degrees C, cwg2‐1 mutant extracts were specifically deficient in geranylgeranyltransferase type I activity, as measured in vitro. Multiple copies of the CDC43 gene can partially suppress the growth and (1‐3) beta‐D‐glucan synthase defect of the cwg2‐1 mutant at the restrictive temperature. In a similar manner, the cwg2+ gene can partially suppress the cdc43‐2 growth defect. These results indicate that cwg2+ is the structural gene for the beta subunit of geranylgeranyltransferase type I in S. pombe and that this enzyme is required for (1‐3) beta‐D‐glucan synthase activity. The functional homology of Cwg2 with Cdc43, which has been implicated in the control of cell polarity, suggests a link between two morphogenetic events such as establishment of cell polarity and cell wall biosynthesis.