A small-scale method for the preparation of plant N-linked glycans from soluble proteins for analysis by MALDI-TOF mass spectrometry

The use of plants as production hosts for recombinant glycoproteins, which is rapidly developing, requires methods for fast and reliable analysis of plant N-linked glycans. This study describes a simple small-scale method for the preparation of N-linked glycans from soluble plant protein and analysis thereof by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Concentration and protease digestion of plant protein as well as deglycosylation is carried out in a single concentrator unit without the need for intermittent purification to minimize adsorptive loss and to facilitate handling. Plant protein is concentrated in a unit with a 5 kDa cutoff, and after buffer exchange, pepsin (EC 3.4.23.1) digestion is carried out in the concentrator overnight to obtain peptides as substrates for deglycosylation. Deglycosylation is carried out with peptide-N-glycosidase A (PNGase A; EC 3.5.1.52) for 24 h. Released N-glycans are purified using reverse-phase and cation exchange chromatography micro-columns for removal of peptides and desalting. N-Glycans are directly analyzed by MALDI-TOF MS without derivatization. The method for isolation of N-glycans is compatible with secreted proteins from cell culture supernatant as well as with soluble protein extracts from leaf tissue. As little as 5 μg of plant glycoprotein is sufficient for N-glycan preparation for MALDI-TOF MS analysis using this method.     

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multipotent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.     

Lichen-like association of Chlamydomonas reinhardtii and Aspergillus nidulans protects algal cells from bacteria

Organismal interactions within microbial consortia and their responses to harmful intruders remain largely understudied. An important step toward the goal of understanding functional ecological interactions and their evolutionary selection is the study of increasingly complex microbial interaction systems. Here, we discovered a tripartite biosystem consisting of the fungus Aspergillus nidulans, the unicellular green alga Chlamydomonas reinhardtii, and the algicidal bacterium Streptomyces iranensis. Genetic analyses and MALDI-IMS demonstrate that the bacterium secretes the algicidal compound azalomycin F upon contact with C. reinhardtii. In co-culture, A. nidulans attracts the motile alga C. reinhardtii, which becomes embedded and surrounded by fungal mycelium and is shielded from the algicide. The filamentous fungus Sordaria macrospora was susceptible to azalomycin F and failed to protect C. reinhardtii despite chemotactically attracting the alga. Because S. macrospora was susceptible to azalomycin F, this data imply that for protection the fungus needs to be resistant. Formation of the lichen-like association between C. reinhardtii and A. nidulans increased algal growth. The protection depends on the increased amounts of membrane lipids provided by resistant fungi, thereby generating a protective shelter against the bacterial toxin. Our findings reveal a strategy whereby algae survive lethal environmental algicides through cooperation with fungi.Subject terms: Microbial ecology, Microbiome, Microbial ecology, Antibiotics, Fungal ecology     

Ixodes scapularis: effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice.

Schoeler, G. B., Manweiler, S. A., and Wikel, S. K. 1999. Ixodes scapularis: Effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice. Experimental Parasitology 92, 239-248. Ixodes scapularis is the principal vector in the United States of Borrelia burgdorferi, the causative agent of Lyme borreliosis, the human granulocytic ehrichiosis agent, and Babesia microti. Infestation with I. scapularis nymphs has previously been shown to modulate host T lymphocyte cytokine production. Tick-induced host immunomodulation is increasingly recognized as a contributing factor in successful transmission and/or establishment of tick-borne pathogens. This study was conducted to determine the effects of repeated infestations with pathogen-free I. scapularis nymphs on the production of the macrophage cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha and the T lymphocyte cytokines IL-2, IL-4, IL-10, and interferon-gamma in both BALB/c and C3H/HeN mice. The pattern of T lymphocyte cytokine production was evaluated to determine if repeated tick infestation polarizes the immune response toward a Th-1 or Th-2 cytokine profile. Female BALB/c and C3H/HeN mice were infested one to four times with pathogen-free I. scapularis nymphs, with a 14-day tick-free period between each exposure. After each infestation, tick biology parameters were measured and macrophage and T lymphocyte cytokine production was assessed. Elaboration of T lymphocyte and macrophage cytokines was quantitated by antigen capture enzyme-linked immunosorbent assay. Acquired resistance to I. scapularis feeding was not developed by either mouse strain. Significant differences in cytokine production were observed between infested and noninfested mice, as well as between the two mouse strains, following tick infestation. Infestation of both strains with pathogen-free I. scapularis results in a polarization of the host immune response toward a Th-2, anti-inflammatory pattern, with a corresponding suppression of Th-1 responses.     

Identification of NF2 germ-line mutations and comparison with neurofibromatosis 2 phenotypes

Neurofibromatosis 2 (NF2) is an autosomal inherited disorder that predisposes carriers to nervous system tumors. To examine genotype-phenotype correlations in NF2, we performed mutation analyses and gadolinium-enhanced magnetic resonance imaging of the head and full spine in 59 unrelated NF2 patients. In patients with vestibular schwannomas (VSs) or identified NF2 mutations, the mild phenotype was defined as <2 other intracranial tumors and ≤ 4 spinal tumors, and the severe phenotype as either ≥ 2 other intracranial tumors or > 4 spinal tumors. Nineteen mutations were found in 20 (34%) of the patients and were distributed in 12 of the 17 exons of the NF2 gene, including intron-exon boundaries. Seven mutations were frameshift, six were nonsense, four were splice site, two were missense, and one was a 3-bp in frame deletion. The nonsense mutations included one codon 57 and two codon 262 C→T transitions in CpG dinucleotides. The frameshift and nonsense NF2 mutations occurred primarily in patients with severe phenotypes. The two missense mutations occurred in patients with mild phenotypes, and three of the four splice site mutations occurred in families with both mild and severe phenotypes. Truncating NF2 mutations are usually associated with severe phenotypes, but the association of some mutations with mild and severe phenotypes indicates that NF2 expression is influenced by stochastic, epigenetic, or environmental factors. Received: 4 July 1996