Identification of Armillaria field isolates using isozymes and mycelial growth characteristics

This research was conducted to develop procedures based on mycelial growth characteristics and patterns of esterase (EST) and polyphenol oxidase (PPO) production by diffuse mycelia for identification of Armillaria field isolates from Quercus-Carya-Pinus forests in the Ozark Mountains (central USA). The 285 isolates collected were first identified by standard diploid-haploid pairing tests as A. gallica, A. mellea, or A. tabescens. A strong PPO band was diagnostic for A. gallica. All A. mellea isolates tested and 91% of the A. tabescens isolates tested were distinguished based on production of EST bands in three standardized R f ranges. A procedure based on mycelial growth and morphology on tannic acid medium (TA) at 24 °C and on malt extract medium (ME) at 33 °C correctly identified 98% of A. gallica isolates and all A. mellea and A. tabescens isolates. On TA, A. gallica grew slowest. On ME, A. mellea grew slowest: mycelial morphology differed among species; A. gallica typically stained the agar and produced an appressed/submerged growth pattern with concentric bands of decreasing hyphal density, A. mellea typically did not stain the agar and produced round mycelia with smooth margins and abundant aerial hyphae, A. tabescens typically stained the agar and grew appressed/submerged with very irregular margins and patchy hyphal density. These are the first published systems evaluating the potential for identifying Armillaria field isolates based on their mycelial growth characteristics and EST and PPO complements. This revised version was published online in June 2006 with corrections to the Cover Date.     

Alkaloids and ethnobotany of Mexican peyote cacti and related species

Some Mexican cacti of the genera Ariocarpus, Mammillaria, Obregonia, Pelecyphora, Solisia and Turbinicarpus have been studied with respect to their alkaloids and ethnobotany. Seven previously known cactus alkaloids were identified. N,N-Dimethyl-4-hydroxy-3-methoxyphenethylamine was found to occur in Ariocarpus agavoides. In Pelecyphora aselliformis N,N-dimethyl-3-hydroxy-4, 5-dimethoxyphenethylamine was identified as the major alkaloid. The possible relationship between the vernacular names of these plants and their alkaloid content is discusse.     

The apparent temperature response of leaf respiration depends on the timescale of measurements: a study of two cold climate species

Productivity and climate models often use a constant Q10 for plant respiration, assuming tight control of respiration by temperature. We studied the temperature response of leaf respiration of two cold climate species (the Australian tree Eucalyptus pauciflora and the subantarctic megaherb Pringlea antiscorbutica, both measured in a field setting) on a short timescale (minutes) during different times within a diel course, and on a longer timescale, using diel variations in ambient temperature. There were great variations in Q10 depending on measuring day, measuring time and measuring method. When Q10 was calculated from short-term (15 min) manipulations of leaf temperature, the resulting values were usually markedly smaller than when Q10 was calculated from measurements at ambient leaf temperatures spread over a day. While for E. pauciflora, Q10 estimates decreased with rising temperature (corroborating the concept of a temperature-dependent Q10), the opposite was the case for P. antiscorbutica. Clearly, factors other than temperature co-regulate both leaf respiration rates and temperature sensitivity and contribute to diel and seasonal variation of respiration.     

Antitumoral and antileishmanial dioncoquinones and ancistroquinones from cell cultures of Triphyophyllum peltatum (Dioncophyllaceae) and Ancistrocladus abbreviatus (Ancistrocladaceae)

From the methanolic extracts of solid callus cultures from two species of the closely related palaeotropical plant families Dioncophyllaceae and Ancistrocladaceae seven new natural naphthoquinones were isolated, dioncoquinones A (4) and B (5) from Triphyophyllum peltatum, and ancistroquinones B (6), C (7), D (9), E (10), and F (12) from Ancistrocladus abbreviatus. Their structures were elucidated by spectroscopic, chemical, and computational methods. Furthermore, the already known naphthoquinones plumbagin (2), droserone (3), malvone A (8), and nepenthone A (11) were found in the extract of A. abbreviatus. Dioncoquinones A (4) and B (5) showed good - and specific - activity against Leishmania major, while they were not active against other protozoic parasites. Moreover, treatment with 4 and 5 strongly induced apoptosis in human tumor cells derived from two different B cell malignancies, B cell lymphoma and multiple myeloma, without any significant toxicity towards normal peripheral mononuclear blood cells.     

Latent manganese deficiency increases transpiration in barley (Hordeum vulgare)

To investigate if latent manganese (Mn) deficiency leads to increased transpiration, barley plants were grown for 10 weeks in hydroponics with daily additions of Mn in the low n M range. The Mn-starved plants did not exhibit visual leaf symptoms of Mn deficiency, but Chl a fluorescence measurements revealed that the quantum yield efficiency of PSII (F_v/F_m) was reduced from 0.83 in Mn-sufficient control plants to below 0.5 in Mn-starved plants. Leaf Mn concentrations declined from 30 to 7 μg Mn g⁻¹ dry weight in control and Mn-starved plants, respectively. Mn-starved plants had up to four-fold higher transpiration than control plants. Stomatal closure and opening upon light/dark transitions took place at the same rate in both Mn treatments, but the nocturnal leaf conductance for water vapour was still twice as high in Mn-starved plants compared with the control. The observed increase in transpiration was substantiated by ¹³C-isotope discrimination analysis and gravimetric measurement of the water consumption, showing significantly lower water use efficiency in Mn-starved plants. The extractable wax content of leaves of Mn-starved plants was approximately 40% lower than that in control plants, and it is concluded that the increased leaf conductance and higher transpirational water loss are correlated with a reduction in the epicuticular wax layer under Mn deficiency.     

Forest farming of shiitake mushrooms: An integrated evaluation of management practices

Two outdoor shiitake (Lentinula edodes) cultivation experiments, established in Missouri USA in 1999 and 2000, produced mushrooms in 2000–2005. We examined shiitake production in response to substrate species, inoculum form, inoculum strain, and inoculation timing, using total mushroom weight per log as the primary response variable with log characteristics as covariates. The significantly greater mushroom weight produced by sugar maple logs compared with white or northern red oak was attributable to the higher proportion of undiscolored wood volume in the maple logs, rather than to bark thickness or log diameter. The “wide temperature range” shiitake strain produced significantly greater yield compared with the “warm” or “cold” weather strains. Both the wide-range and warm-weather strains were stimulated to fruit by significant rain events, while the cold-weather strain was responsive to temperature. Inoculation with sawdust spawn gave significantly greater yield than colonized wooden dowels or pre-packaged “thimble” plug inoculum. The second and third full years following inoculation were the most productive.     

Antibacterial Activity of Marine Culturable Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs of Marine Organisms

The purpose of the present study was to isolate marine culturable bacteria with antibacterial activity and hence a potential biotechnological use. Seawater samples (244) and 309 swab samples from biotic or abiotic surfaces were collected on a global Danish marine research expedition (Galathea 3). Total cell counts at the seawater surface were 5 × 10⁵ to 10⁶ cells/ml, of which 0.1–0.2% were culturable on dilute marine agar (20°C). Three percent of the colonies cultured from seawater inhibited Vibrio anguillarum, whereas a significantly higher proportion (13%) of colonies from inert or biotic surfaces was inhibitory. It was not possible to relate a specific kind of eukaryotic surface or a specific geographic location to a general high occurrence of antagonistic bacteria. Five hundred and nineteen strains representing all samples and geographic locations were identified on the basis of partial 16S rRNA gene sequence homology and belonged to three major groups: Vibrionaceae (309 strains), Pseudoalteromonas spp. (128 strains), and the Roseobacter clade (29 strains). Of the latter, 25 strains were identified as Ruegeria mobilis or pelagia. When re-testing against V. anguillarum, only 409 (79%) retained some level of inhibitory activity. Many strains, especially Pseudoalteromonas spp. and Ruegeria spp., also inhibited Staphylococcus aureus. The most pronounced antibacterial strains were pigmented Pseudoalteromonas strains and Ruegeria spp. The inhibitory, pigmented Pseudoalteromonas were predominantly isolated in warmer waters from swabs of live or inert surfaces. Ruegeria strains were isolated from all ocean areas except for Arctic and Antarctic waters and inhibitory activity caused by production of tropodithietic acid.     

Second messenger function of nicotinic acid adenine dinucleotide phosphate revealed by an improved enzymatic cycling assay

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent activator of Ca2+ release from intracellular stores known today. Although recent reports have suggested an important function of NAADP in human T lymphocytes, direct evidence for receptor-induced formation of NAADP is yet missing in these cells. Thus, we developed a highly sensitive and specific enzyme assay capable of quantifying low fmol amounts of NAADP. In unstimulated T cells, the NAADP concentration amounted to 4.4 +/- 1.6 nm (0.055 +/- 0.028 pmol/mg of protein). Stimulation of the cells via the T cell receptor/CD3 complex resulted in biphasic elevation kinetics of cellular NAADP levels and was characterized by a bell-shaped concentration-response curve for NAADP. In contrast, the NAADP concentration was elevated neither upon activation of the ADP-ribose/TRPM2 channel Ca2+ signaling system nor by an increase of the intracellular Ca2+ concentration upon thapsigargin stimulation. T cell receptor/CD3 complex-mediated NAADP formation was dependent on the activity of tyrosine kinases because genistein completely blocked NAADP elevation. Thus, we propose a regulated formation of NAADP upon specific stimulation of the T cell receptor/CD3 complex, suggesting a function of NAADP as a Ca2+-mobilizing second messenger during T cell activation.     

Allo-Reactivity of Mesenchymal Stem Cells in Rhesus Macaques Is Dose and Haplotype Dependent and Limits Durable Cell Engraftment In Vivo

The emerging paradigm that MSCs are immune privileged has fostered the use of “off-the-shelf” allogeneic MSC-based therapies in human clinical trials. However, this approach ignores studies in experimental animals wherein transplantation of MSCs across MHC boundaries elicits measurable allo-immune responses. To determine if MSCs are hypo-immunogeneic, we characterized the immune response in rhesus macaques following intracranial administration of allogeneic vs. autologous MSCs. This analysis revealed unambiguous evidence of productive allo-recognition based on expansion of NK, B and T cell subsets in peripheral blood and detection of allo-specific antibodies in animals administered allogeneic but not autologous MSCs. Moreover, the degree of MHC class I and II mismatch between the MSC donor and recipient significantly influenced the magnitude and nature of the allo-immune response. Consistent with these findings, real-time PCR analysis of brain tissue from female recipients administered varying doses of male, allogeneic MSCs revealed a significant inverse correlation between MSC engraftment levels and cell dose. Changes in post-transplant neutrophil and lymphocyte counts also correlated with dose and were predictive of overall MSC engraftment levels. However, secondary antigen challenge failed to elicit a measurable immune response in allogeneic recipients. Finally, extensive behavior testing of animals revealed no main effect of cell dose on motor skills, social development, or temperament. Collectively, these data indicate that allogeneic MSCs are weakly immunogenic when transplanted across MHC boundaries in rhesus macaques and this negatively impacts durable engraftment levels. Therefore the use of unrelated donor MSCs should be carefully evaluated in human patients.