Biological Effects of Trichoderma harzianum Peptaibols on Mammalian Cells

Trichoderma species isolated from water-damaged buildings were screened for toxicity by using boar sperm cells as indicator cells. The crude methanolic cell extract from Trichoderma harzianum strain ES39 inhibited the boar sperm cell motility at a low exposure concentration (50% effective concentration, 1 to 5 μg [dry weight] ml of extended boar semen⁻¹). The same exposure concentration depleted the boar sperm cells of NADH₂. Inspection of the exposed boar sperm cells by transmission electron microscopy revealed damage to the plasma membrane. By using the black lipid membrane technique, it was shown that the semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 induced voltage-dependent conductivity. The high-performance liquid chromatography-purified metabolites of T. harzianum strain ES39 dissipated the mitochondrial membrane potential (Δψ_m) of human lung epithelial carcinoma cells (cell line A549). The semipurified metabolites (eluted from a SepPak C₁₈ cartridge) of T. harzianum strain ES39 were analyzed by mass spectrometry (MS). Matrix-assisted laser desorption ionization and nanoflow electrospray ionization MS revealed five major peptaibols, each of which contained 18 residues and had a mass ranging from 1,719 to 1,775 Da. Their partial amino acid sequences were determined by collision-induced dissociation tandem MS.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Genetic selection system for improving recombinant membrane protein expression in E. coli

A major barrier to the physical characterization and structure determination of membrane proteins is low yield in recombinant expression. To address this problem, we have designed a selection strategy to isolate mutant strains of Escherichia coli that improve the expression of a targeted membrane protein. In this method, the coding sequence of the membrane protein of interest is fused to a C‐terminal selectable marker, so that the production of the selectable marker and survival on selective media is linked to expression of the targeted membrane protein. Thus, mutant strains with improved expression properties can be directly selected. We also introduce a rapid method for curing isolated strains of the plasmids used during the selection process, in which the plasmids are removed by in vivo digestion with the homing endonuclease I‐CreI. We tested this selection system on a rhomboid family protein from Mycobacterium tuberculosis (Rv1337) and were able to isolate mutants, which we call EXP strains, with up to 75‐fold increased expression. The EXP strains also improve the expression of other membrane proteins that were not the target of selection, in one case roughly 90‐fold.     

New platinum-oxicam complexes as anti-cancer drugs. Synthesis, characterization, release studies from smart hydrogels, evaluation of reactivity with selected proteins and cytotoxic activity in vitro

The reaction of aqueous cis-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ with Na⁺HMEL⁻ (H₂MEL, meloxicam, 4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide), and Na⁺HISO⁻ (H₂ISO, isoxicam, 4-hydroxy-2-methyl-N-(5-methylisoxazol-3-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide) at pH 7 produced micro-crystalline cis-[Pt(NH₃)₂(N^1′-HMEL)₂], 5 and cis-[Pt(NH₃)₂(N^1′-HISO)₂], 6. The X-ray diffraction structure of 5 shows two HMEL⁻ anions donating through the thiazole nitrogen atoms and adopting a head-to-tail (HT) conformation. The ¹H NMR spectrum for 5 from DMSO-d₆ shows inertness of the complex up to at least 24 h. Delivery studies for 5 and 6 from vinyl hydrogel based on l-phenylalanine (pH 6.5, 25 °C) show that concentrations of complexes ranging between 2.5 and 5 μM can be reached after a day. Compounds 5 and 6 show strong anti-proliferative effects on CH1 cells (ovarian carcinoma, human) in vitro, IC₅₀ values being 0.60 and 0.37 μM, respectively (0.16 μM for reference, cis-diamminodichloridoplatinum(II), cisplatin). ESI-MS measurements clearly documented that both 5 and 6 form adducts with the three model proteins ubiquitin (UBI), cytochrome c (CYT C) and superoxide dismutase (SOD), the HISO⁻ complex being significantly more effective than the HMEL⁻ one. Density functional methods help in finding rationale for the easiest dissociation of Pt-H₂ISO/HISO bonds when compared to the Pt-N^1′-H₂MEL/N^1′-HMEL linkages.     

Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

Background

In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.

Results

Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 10³ to 10⁶ PFU/ml, could also be reliably detected.

Conclusions

This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.     

Cloning and characterization of the DNA region responsible for Megacin A-216 production in Bacillus megaterium 216

Upon induction, Bacillus megaterium 216 produces the bacteriocin megacin A-216, which leads to lysis of the producer cell and kills B. megaterium and a few other bacterial species. The DNA region responsible for megacinogeny was cloned in B. megaterium. The nucleotide sequence of a 5,494-bp-long subfragment was determined, and the function of the genes on this fragment was studied by generating deletions and analyzing their effects on MegA phenotypes. An open reading frame (ORF) encoding a 293-amino-acid protein was identified as the gene (megA) coding for megacin A-216. BLAST searches detected sequence similarity between megacin A-216 and proteins with phospholipase A2 activity. Purified biologically active megacin A-216 preparations contained three proteins. Mass spectrometry analysis showed that the largest protein is the full-length translation product of the megA gene, whereas the two shorter proteins are fragments of the long protein created by cleavage between Gln-185 and Val-186. The molecular masses of the three polypeptides are 32,855, 21,018, and 11,855 Da, respectively. Comparison of different megacin preparations suggests that the intact chain as well as the two combined fragments can form biologically active megacin. An ORF located next to the megA gene and encoding a 91-amino-acid protein was shown to be responsible for the relative immunity displayed by the producer strain against megacin A-216. Besides the megA gene, at least two other genes, including a gene encoding a 188-amino-acid protein sharing high sequence similarity with RNA polymerase sigma factors, were shown to be required for induction of megacin A-216 expression.     

Effect of prenatal treatment with methylazoxymethanol on carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in the neonatal,young, and adult offspring

Carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism was investigated in the neonatal, young and adult cerebral cortex slices of rats prenatally treated with methylazoxymethanol (MAM) on gestational day 15 (GD15) or GD19. In rat offspring treated on GD15 there was a significant reduction in the accumulation of [³H]inositol phosphates induced by carbachol and a significant increase in the accumulation of [³H]inositol phosphates induced by norepinephrine on day 7, whereas no changes were observed at the other ages. No significant changes, on the other hand, were observed for glutamate-stimulated phosphoinositide metabolism in GD15 treated rats and for carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in animals treated on GD19 at any of the different ages evaluated. These results indicate that treatment with MAM on GD15, which results in a marked microencephaly, causes a marked alteration of muscarinic and ₁-adrenergic receptor-stimulated phosphoinositide metabolism during brain development and that these alterations undergo adaptive changes in the adult brain.     

Myrtucommulone production by a strain of Neofusicoccum australe endophytic in myrtle (Myrtus communis)

Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC₅₀ of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.     

Proposed GSSP for the base of Cambrian Stage 7, coinciding with the first appearance of Lejopyge laevigata, Hunan, China

The Luoyixi section, exposed in a roadcut along the Youshui River (Fengtan Reservoir), Guzhang County, Hunan Province, China, is proposed as the stratotype for the base of an unnamed stage boundary (base of the Cambrian stage provisionally termed Stage 7). The proposed position of the GSSP is 121.3 m above the base of the Huaqiao Formation, at a horizon coinciding with the first appearance of the cosmopolitan agnostoid trilobite Lejopyge laevigata. The section fulfills all the requirements for a GSSP, and the horizon can be constrained not only with the primary stratigraphic marker (L. laevigata) but also with secondary biostratigraphic, sequence-stratigraphic, and chemostratigraphic correlation tools. The first appearance of L. laevigata is one of the most readily recognizable levels in the Cambrian, and can be correlated with precision to all paleocontinents.