Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

Porphyrin content of the cysticercus of Taenia solium

The strong red fluorescence of the cysticercus of Taenia solium depends on the presence of several porphyrins in the vesicular fluid of the parasite: probably protoporphyrin IX, coproporphyin I or III, and 2 decarboxylated porphyrins intermediate between uroporphyrin and coproporphyrin. Cyst porphyrins associated to form conglomerates of high molecular weight that dissociated in acid solutions and were not antigenic themselves nor associated with antigenic molecules. An appreciable fraction of the porphyrins was capable of undergoing oxidation and reduction, indicating that some of the porphyrins were complexed with metal ions. The metabolic basis for the accumulation of porphyrins is unknown. Preliminary results suggest that conditions deleterious to the cysticercus cause release of porphyrins so that the appearance of porphyrins in the cerebrospinal fluid of neurocysticercotic patients may prove useful in monitoring therapeutic attacks on the parasite.     

Phosphorylation of nonmuscle myosin heavy chain IIA on Ser1917 is mediated by protein kinase C beta II and coincides with the onset of stimulated degranulation of RBL-2H3 mast cells

Dynamic remodeling of the actinomyosin cytoskeleton is integral to many biological processes. It is regulated, in part, by myosin phosphorylation. Nonmuscle myosin H chain IIA is phosphorylated by protein kinase C (PKC) on Ser(1917). Our aim was to determine the PKC isoform specificity of this phosphorylation event and to evaluate its potential role in regulated secretion. Using an Ab against the phosphorylated form of Ser(1917), we show that this site is not phosphorylated in unstimulated RBL-2H3 mast cells. The physiological stimulus, Ag, or the pharmacological activators, PMA plus A23187, induced Ser(1917) phosphorylation with a time course coincident with the onset of granule mediator secretion. Dephosphorylation at this site occurred as Ag-stimulated secretion declined from its peak, but dephosphorylation was delayed in cells activated with PMA plus A23187. Phosphate incorporation was also enhanced by PMA alone and by inhibition of protein phosphatase 2A. G?6976, an inhibitor of conventional PKC isoforms, abolished secretion and Ser(1917) phosphorylation with similar dose dependencies consistent with involvement of either PKCalpha or PKCbeta. Phorbol ester-stimulated Ser(1917) phosphorylation was reconstituted in HEK-293 cells (which lack endogenous PKCbeta) by overexpression of both wild-type and constitutively active PKCbetaII but not the corresponding PKCbetaI or PKCalpha constructs. A similar selectivity for PKCbetaII overexpression was also observed in MIN6 insulinoma cells infected with recombinant PKC wild-type adenoviruses. Our results implicate PKC-dependent phosphorylation of myosin H chain IIA in the regulation of secretion in mast cells and suggest that Ser(1917) phosphorylation might be a marker of PKCbetaII activation in diverse cell types.     

Fatal pulmonary acariasis in an aged indoor rhesus macaque (Macaca mulatta)

Pulmonary acariasis is a sporadic, incidental finding in colony‐raised rhesus macaques (Macaca mulatta). Prophylactic treatment in indoor‐raised and indoor‐housed macaques is not routine due to low prevalence, lack of clinical significance, and potential risk of toxicosis. This case is an unusually severe infestation of Pneumonyssus simicola in an indoor‐housed rhesus macaque, which ultimately resulted in this animal's death.     

Temporal dynamics and relationship between climate,limnological variables and zooplankton composition in climate-sensitive Lake Bosumtwi,Ghana

The community composition and the factors affecting seasonal and interannual dynamics of zooplankton in Lake Bosumtwi were studied biweekly at a central index station during 2005 and 2006. The lake zooplankton community was species poor. Mesocyclops bosumtwii was numerically superior seasonally and interannually and was endemic to the lake. Minor constituents included Moina micrura, six rotifer species (except for Hexarthra intermedia) and Chaoborus ceratopogones larvae. Low variance of cyanobacteria-dominated phytoplankton biomass underlined stable zooplankton community structure. Emergence of rare species of rotifers occurred seasonally. The climatic signature on the lake’s stratification and mixing regime was strongly influenced by atmospheric temperature, but weakly by wind strength, because of sheltering of the lake by high crater walls. Increasing mixing depth entrained high TP concentrations from below the thermocline seasonally, but reflected poorly in the phytoplankton biomass behaviour. Total zooplankton abundance did not differ seasonally, but varied markedly from year to year in its timing and magnitude. Herbivores were squeezed between food limitation and high predation pressure from Chaoborus all year round. The low fish planktivory (high fishing pressure) on Chaoborus may create a trophic bottleneck restricting energy transfer efficiency from zooplankton to fish.     

Implications phylogénétiques de I'ultrastructure de la spermatogenèse, du spermatozoïde et de I'ovogenèse du TurbellariéUrastoma cyprinae ("Prolecithophora", Urastomidae)

Spermiogenesis in Urastoma cyprinae (Graff. 1882) involvcs a progressive lengthening of the spermatid. Free flagella are only transitory. The mature spermatozoon is fusibrm. 45μm in length and 2.5 μm in width. It contains two incorporated axonermes of the flatworm 9+"1" pattern, two elongated mitochondria. an elongated nucleus and a row or cortical longitudinal microtubules. Observations on oogenesis concerncd only the immuturc ovary. lmmature oocytes contain few dense granules and accssory cells were not ohserved. Phylogenetic implications of a biflagellate spermatozoon in a Prolecithophora are important. The presence of two 9 +"1" axonemes confirms that Urastoma (and the Prolecithophora) belongs to the taxon Trepaxonemata Ehlers. Previous electron microscope studies on spermitozoa of Prolecithophora (four genera) only dealt with aflagellate spermatozoa. On this basis, Ehlers (1985) proposed two autapomorphics for the taxon Prolecithophora: aflagellate spermatozoon and spermatozoal mitochondrial derivatives with abundant membranes. The present observations on Urastoma contradict these two autapomorphics. The taxon Prolecithophora cannot be defined by autapomorphies of the spermatozoon.     

Stoichiometric relationships in vernal pond plankton communities

1. The light-nutrient hypothesis (LNH) predicts that changes in light supply can alter the balance of nutrient and energy limitation in primary producers. We tested this prediction by examining temporal changes in vernal forest ponds, which are highly dynamic systems with respect to seasonal change in light and nutrient supply. In three vernal ponds that differ in productivity, we measured changes in light, total and seston nitrogen and phosphorus, and seston carbon and chlorophyll during the spring, before and after tree leaf-out. We also quantified changes in the population dynamics of the major zooplankton grazers in these systems.
2. In each pond, nutrient levels increased and light levels declined, creating a temporal shift in light-nutrient supply to the plankton. Results generally supported predictions of stoichiometric theory and the LNH, but there were notable exceptions.
3. Seston C : N : P ratios rapidly changed in response to dramatic increases in N and P supply rates. However, seston N : P was typically lower than values for total N : P in the water. Furthermore, as predicted, we observed a decline in seston C : P as the light : nutrient ratio declined, but seston C : N simultaneously increased. These results suggest an unexpected shift towards potential nitrogen limitation. Alternatively, this change in nutrient ratios may be driven by a seasonal change in phytoplankton composition or nutritional mode.
4. Seston carbon concentrations remained stable despite seasonal changes in grazing intensity associated with the phenology of large-bodied Daphnia grazers. However, chlorophyll concentrations declined dramatically as the season progressed, resulting in a simultaneous decline in the C : Chlorophyll ratio of seston. Both pond shading and increased grazing probably contributed to the decline in chlorophyll.     

Transmembrane signaling in P815 mastocytoma cells by transfected IgE receptors

In order to delineate structural-functional relationships of the mast cell receptor for IgE (Fc epsilon RI) by molecular-genetic analysis, a transfectable cell must be identified which resembles mast cells except for being deficient in receptors. We have found that the well known murine mastocytoma P815 is suitable. These cells express no Fc epsilon RI, lack mRNA for the alpha and beta subunits of the receptor, but contain some mRNA for gamma chains. After transfection with the cDNA for each of the subunits, stable clones could be isolated which expressed several hundred thousand normal Fc epsilon RI and synthesized large amounts of mRNA for alpha, beta, and gamma, the last at 3-fold higher levels than in the untransfected cells. Aggregation of the transfected receptors led to opening of presumptive calcium channels and to activation of phospholipase C, phospholipase A2, and protein kinase C. The kinetics and other characteristics of the signals were similar to those observed after stimulation of the rat tumor mast cells from which the receptor genetic material had been derived but were smaller in magnitude. These weaker signals most likely result from an overall reduced reactivity exhibited by the P815 cells since stimulation by other ligands led to weaker or even no responses. The cells failed to degranulate after either receptor aggregation or reaction with ionophores with or without phorbol ester. Both the transfected and untransfected P815 cells express Fc receptors for IgG (Fc gamma RII) which, interestingly, independently triggered similar responses despite their apparently simpler subunit structure.     

Comparative cytotoxicity of fumonisin B1 in two cell lines derived from normal human bronchial epithelial cells using four distinct bioassay techniques

This study focuses on the cytotoxic effects of fumonisin B₁ (FB₁) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two related cell lines, the clonogenic assay, measuring cell survival and proliferation, indicated that FB₁ had a more toxic effect on the nontransfected cells. This kind ofin vitro approach using cells which retain many characteristics of normal cell growth and differentiation can go some way to developing evaluation models for food safety in the case of mycotoxin contamination without resorting totally to whole animal testing. Nevertheless, one or two cytotoxicity tests may be inadequate for a complete appraisal of toxic potential: rather, as wide a range of methodologies as feasible should be employed initially before meaningful conclusions may be drawn.