An improved procedure for root surface disinfection suitable for observations of proliferation of intraradical hyphae of arbuscular mycorrhizal fungusGlomus fistulosum

A disinfection procedure for mycorrhizal root segment surface was improved using neomycin and polymyxin B besides sodium hypochlorite, streptomycin and penicillin G, to obtain clean material for observation of proliferation of hyphae of arbuscular mycorrhizal (AM) fungi. The procedure of surface disinfection is more efficient, in terms of incidence of contamination, compared to the original procedure involving only sodium hypochlorite, streptomycin and penicillin G. A further decrease of visually detectable contamination can be achieved using bacteriostatic concentrations of rolitetracycline which cause a decrease of hyphal growth of the AM fungusGlomus fistulosum. The technique was used in experiment studying the uptake ofU-¹⁴C-glucose by proliferating hyphae. An active uptake of glucose occurred even in the presence of all four antibiotics at concentrations of 500 mg/L.     

Oxidative stress increases levels of endogenous amyloid-beta peptides secreted from primary chick brain neurons

Oxidative damage is associated with Alzheimer's disease and mild cognitive impairment, but its relationship to the development of neuropathological lesions involving accumulation of amyloid-beta (Abeta) peptides and hyperphosphorylated tau protein remains poorly understood. We show that inducing oxidative stress in primary chick brain neurons by exposure to sublethal doses of H(2)O(2 )increases levels of total secreted endogenous Abeta by 2.4-fold after 20 h. This occurs in the absence of changes to intracellular amyloid precursor protein or tau protein levels, while heat-shock protein 90 is elevated 2.5-fold. These results are consistent with the hypothesis that aging-associated oxidative stress contributes to increasing Abeta generation and up-regulation of molecular chaperones in Alzheimer's disease.     

Genetic diversity and its implications in the conservation of endangered<Emphasis Type="Italic">Zostera japonica</Emphasis> in Korea

As part of the on-going effort to conserve endangeredZostera japonica Ascher. & Graebn. in Korea, we have used RAPD band patterns to analyze its genetic structure and diversity. Out of 50 primers tested, 45 formed amplified bands with its genome, including 814 polymorphic and 28 monomorphic bands. The highest number (120) was found in the population of Geoje-do; the smallest (58), in Anmyeon-do. An examination of its genetic structure with AMOVA revealed that about 50% of all variations could equally be assigned to within and between populations. The statistical value G_st (index of genetic differences) was 0.49, and the average number of individuals exchanged between populations per generation (N _e m) was calculated as 0.26. Although the habitats ofZ. japonica in Korea are disappearing at an alarming rate, significant levels of genetic variation still exist, especially in the Geoje-do population. Therefore, any recovery strategy for this endangered species should be planned on the basis of this genetic diversity among populations.     

Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates

A multicenter study was conducted with the objective to evaluate a reverse line blot (RLB) assay to detect resistance to rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB) in clinical isolates of Mycobacterium tuberculosis. Oligonucleotides specific for wild type and mutant (drug resistance linked) alleles of the selected codons in the genes rpoB, inhA, ahpC, rpsL, rrs, embB, were immobilized on a nylon membrane. The RLB assay conditions were optimized following analysis of DNA samples with known sequences of the targeted genes. For validation of the method at different geographical locations, the membranes were sent to seven laboratories in six countries representing the regions with high burdens of multudrug-resistant tuberculosis. The reproducibility of the assay for detection of rpoB genotypes was initially evaluated on a blinded set of twenty reference DNA samples with known allele types and overall concordant results were obtained. Further mutation analysis was performed by each laboratory on the local strains. Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306. Mutations in the inhA and ahpC promoter regions, conferring resistance to INH, were successfully identified in respectively 16.9% and 13.2% of INH-resistant strains. Likewise, mutations in rrs513 and rpsL88 that confer resistance to STR were identified in respectively 15.1% and 10.7% of STR-resistant strains. It should be mentioned that mutation analysis of the above targets usually requires rather costly DNA sequencing to which the proposed RLB assay presents rapid and inexpensive alternative. Furthermore, the proposed method requires the same simple equipment as that used for spoligotyping and permits simultaneous analysis of up to 40 samples. This technique is a first attempt to combine different targets in a single assay for prediction of antituberculosis drugs resistance. It is open to further development as it allows easy incorporation of new probes for detection of mutations in other genes associated with resistance to second-line (e.g., fluoroquinolones) and new antituberculosis compounds.     

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.     

Hierarchical recruitment by AMPA but not staurosporine of pro-apoptotic mitochondrial signaling in cultured cortical neurons: evidence for caspase-dependent/independent cross-talk

Excitotoxicity mediated via the ( S )-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for l -glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up- and downstream of mitochondrial activation. AMPA-mediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (ΔΨ_m), early release of cytochrome c (cyt c ), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of ΔΨ_m, and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke ΔΨ_m as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c , SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis.     

Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.     

Direct analysis of the extracellular proteome from two strains of Helicobacter pylori

Helicobacter pylori extracellular proteins are of interest because of possible roles in pathogenesis, host recognition, and vaccine development. We utilized a unique approach by growing two strains (including one nonsequenced strain) in a defined serum-free medium and directly analyzing the proteins present in the culture supernatants by LC-MS/MS. Over 125 proteins were identified in the extracellular proteomes of two H. pylori strains. Forty-five of these proteins were enriched in the extracellular fraction when compared to soluble cell-associated protein samples. Our analysis confirmed and expanded on the previously reported H. pylori extracellular proteome. Extracellular proteins of interest identified here included cag pathogenicity island protein Cag24 (CagD); proteases HP0657 and HP1012; a polysaccharide deacetylase, HP0310, possibly involved in the hydrolysis of acetyl groups from host N-acetylglucosamine residues or from residues on the cell surface; and HP0953, an uncharacterized protein that appears to be restricted to Helicobacter species that colonize the gastric mucosa. In addition, our analysis found eight previously unidentified outer membrane proteins and two lipoproteins that could be important cell surface proteins.     

Rabs and other small GTPases in ciliary transport

The non-motile primary cilium is a single, microtubule-based hair-like projection that emanates from most, if not all, non-dividing mammalian cells. Enriched in a variety of signalling receptors and accessories, the cilium mediates crucial sensory and regulatory functions during development and postnatal tissue homoeostasis. Maintenance of ciliary morphology and function requires continuous IFT (intraflagellar transport), and recent findings have shed light on some molecular details of how ciliogenesis is dependent on targeted exocytic membrane trafficking from the Golgi. The ARL [Arf (ADP ribosylation factor)-related] small GTPase Arf4 functions in TGN (trans-Golgi network) sorting of cilia-targeted rhodopsin into carrier vesicles, while Arl6 (Arf-like 6) and Arl13b regulate aspects of ciliary transport and IFT. Ciliogenesis and ciliary functions are also regulated by small Rabs. Rab8a, in conjunction with Rab11a, and via its interaction with a multitude of proteins associated with the ciliary basal body and axoneme/membrane, appears to be critical for ciliogenesis. Rab8's close homologue Rab10 may also play a ciliogenic role in some cells. Rab23, the depletion or inactivation of which affects cilia formation, may regulate specific ciliary protein targeting and turnover, particularly those involved in Shh (Sonic hedgehog) signalling. Recent findings have also implicated Ran, a small GTPase better known for nuclear import, in ciliary targeting of the KIF17 motor protein. We highlight and discuss recent findings on how Rabs and other small GTPases mediate ciliogenesis and ciliary traffic.     

Diversity and decay ability of basidiomycetes isolated from lodgepole pines killed by the mountain pine beetle

When lodgepole pines (Pinus contorta Douglas ex Louden var. latifolia Engelm. ex S. Watson) that are killed by the mountain pine beetle (Dendroctonus ponderosae) and its fungal associates are not harvested, fungal decay can affect wood and fibre properties. Ophiostomatoids stain sapwood but do not affect the structural properties of wood. In contrast, white or brown decay basidiomycetes degrade wood. We isolated both staining and decay fungi from 300 lodgepole pine trees killed by mountain pine beetle at green, red, and grey stages at 10 sites across British Columbia. We retained 224 basidiomycete isolates that we classified into 34 species using morphological and physiological characteristics and rDNA large subunit sequences. The number of basidiomycete species varied from 4 to 14 species per site. We assessed the ability of these fungi to degrade both pine sapwood and heartwood using the soil jar decay test. The highest wood mass losses for both sapwood and heartwood were measured for the brown rot species Fomitopsis pinicola and the white rot Metulodontia and Ganoderma species. The sap rot species Trichaptum abietinum was more damaging for sapwood than for heartwood. A number of species caused more than 50% wood mass losses after 12 weeks at room temperature, suggesting that beetle-killed trees can rapidly lose market value due to degradation of wood structural components.