Chromosomal gfp labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon: application for studies of bacteria-host interactions

Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.     

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.     

Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO₄²⁻ conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.     

Mycorrhizal synthesis between <Emphasis Type="Italic">Boletus edulis</Emphasis> species complex and rockroses (<Emphasis Type="Italic">Cistus</Emphasis> sp.)

Ectomycorrhizas of Boletus aereus, Boletus edulis, and Boletus reticulatus were synthesized with Cistus sp. under laboratory conditions using synthesis tubes filled with a mixture of sterilized peat-vermiculite and nutrient solution. The fungal strains isolated from sporocarps were identified by molecular techniques. The inoculated seedlings were grown for 4–5 months. The ectomycorrhizas formed were described based on standard morphological and anatomical characters. The three ectomycorrhizas described were very similar, with white monopodial-pinnate morphology, a three-layered plectenchymatous mantle on plan view and boletoid rhizomorphs.     

Partial persistence of exercise-induced myocardial angiogenesis following 4-week detraining in the rat

Enhanced angiogenesis, or capillary growth, has a prominent role among the various beneficial effects of exercise training on the myocardium. The aim of the present study is to assess if training-induced increases in capillarity and vascularization persist after 4 weeks of detraining. Adult male rats were trained to run on a treadmill for 10 weeks at ∼60% VO_2max, which did not induce cardiac hypertrophy, but increased (P < 0.05) the soleus/body weight ratio, left ventricle capillarity and von Willebrand-positive cell density (n = 6). In another group of animals (n = 6) subjected to training followed by 4-week detraining, the soleus/body weight ratio returned to normal, with only partial reversal of left ventricle capillarity and von Willebrand-positive cell density. Markers of angiogenesis (VEGF, KDR/VEGF-R2 and HIF-1α mRNA, studied by real-time RT-PCR) were upregulated at the end of training, and returned to baseline value after detraining. Electron microscopy highlighted some morphological features in trained hearts (endothelial cell sprouting and bridges and pericyte detachment), suggestive of endothelial cell proliferation and capillary growth that were absent in untrained and detrained hearts. We conclude that the training-induced increase in cardiac capillarity and vascularization are retained for some time upon cessation of the training program even in the absence of angiogenic stimuli.     

Thin semitransparent gels containing phenylboronic acid: porosity, optical response and permeability for sugars

Radical copolymerization of acrylamide (Am) (90 mol%) with N-acryloyl-m-aminophenylboronic acid (NAAPBA) (10 mol%) carried out on the surface of glass slides in aqueous solution and in the absence of chemical cross-linkers, resulted in the formation of thin semitransparent gels. The phenylboronic acid (PBA) ligand density was ca. 160 micromol/ml gel. The gels exhibited a macroporous structure and displayed optical response to sucrose, lactose, glucose and fructose in 50 mM sodium phosphate buffer, in the pH range from 6.5 to 7.5. The response was fairly reversible and linearly depended on glucose concentration in the wide concentration range from 1 to 60 mM at pH 7.3. The character of response was explained by the balance of two competing equilibrium processes: binding of glucose to phenylboronate anions and binary hydrophobic interactions of neutral PBA groups. The apparent diffusion coefficient of glucose in the gels was ca. 2.5 x 10(-7) cm(2)/s. A freshly prepared gel can be used daily for at least 1 month without changes in sensitivity. Autoclaving (121 degrees C, 1.2 bar, 10 min) allows for the gels sterilization, which is important for their use as glucose sensors in fermentation processes.     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.     

Production of recombinant proteins in clonal root cultures using episomal expression vectors

We have developed a fully contained system for expressing recombinant proteins that is based on clonal root cultures and episomal expression vectors. Clonal root lines expressing green fluorescent protein (GFP) or human growth hormone were generated from Nicotiana benthamiana leaves infected with the tobacco mosaic virus-based vector 30B after exposure to Agrobacterium rhizogenes. These lines accumulated GFP at over 50 mg per kg fresh tissue, a level that is comparable with other plant production systems in early stage development. Accumulation of both hGH and GFP in the clonal root lines was sustained over a 3-year period, and in the absence of antibiotic selection. This technology shows promise for commercial production of vaccine antigens and therapeutic proteins in contained facilities.     

FAK-independent alphavbeta3 integrin-EGFR complexes rescue from anoikis matrix-defective fibroblasts

Extracellular matrix (ECM) binding to integrin receptors regulates cell cycle progression and survival. In adherent cells, ECM disassembly induces anoikis, the apoptotic pathway switched on by loss of adhesion. ECM-deficient Ehlers-Danlos syndrome (EDS) fibroblasts, to adhere to rare fibronectin (FN) fibrils, and to proliferate, only organize, as FN receptor, the alphavbeta3 integrin. We report that in EDS cells the alphavbeta3 integrin is bound to talin and vinculin, but not to tensin, and that actin cytoskeleton is disorganized. Furthermore, in EDS cells Bcl-2 is down-regulated and caspases are active. We provide evidence that the antibody-mediated alphavbeta3 integrin or the FN inhibition induces anoikis in EDS cells. The alphavbeta3 integrin transduces survival signals to pp60src-mediated tyrosine phosphorylated paxillin, instead than to FAK, and interacts with EGF receptor (EGFR). This complex, when activated by EGF and FN, signals for the rescue of EDS cells from anoikis. Therefore, EDS cells, through the alphavbeta3 integrin-EGFR complexes, engage a paxillin- but not FAK-mediated pathway of cell survival.