Impact of disease frequency and host density on pollination and transmission of an African anther-smut fungus

The vast majority of flowering plants rely solely on insects for pollination. A number of pathogens have evolved mechanisms to exploit these close associations and use pollinators as vectors of infective propagules. Factors that affect pollinator movements and successful pollination may in turn also influence successful transmission of fungal spores. Here we investigate the effect of host density and the frequency of diseased Oxalis lanata individuals infected by the anther-smut fungus, Thecaphora capensis, on the likelihood of receiving pollen and fungal spores. Specifically, we determined the numbers of spores and pollen grains deposited on stigmatic surfaces of selected flowers under natural and standardized conditions where host density and disease frequency varied. The effect of host flower density and diseased flower frequency on pollen and spore transfer was variable under natural conditions and these factors interacted significantly. However, an increase in host density and disease frequency significantly influenced pollen and spore deposits under standardized conditions. The effect of host density was, however, not linear and an optimal flower density for pollen and fungal spore transmission was found. Similar to other systems of vector-borne disease, the transmission of anther-smut of Oxalis lanata is more frequency-dependent than density-dependent. This study represents a first step towards understanding the disease transmission process of T. capensis on Oxalis and lays the foundation for future comparative studies between this and other systems to develop and test general hypotheses of disease dynamics in vector-borne disease transmission systems.     

Lesions of the Basal Forebrain Cholinergic System in Mice Disrupt Idiothetic Navigation

Loss of integrity of the basal forebrain cholinergic neurons is a consistent feature of Alzheimer’s disease, and measurement of basal forebrain degeneration by magnetic resonance imaging is emerging as a sensitive diagnostic marker for prodromal disease. It is also known that Alzheimer’s disease patients perform poorly on both real space and computerized cued (allothetic) or uncued (idiothetic) recall navigation tasks. Although the hippocampus is required for allothetic navigation, lesions of this region only mildly affect idiothetic navigation. Here we tested the hypothesis that the cholinergic medial septo-hippocampal circuit is important for idiothetic navigation. Basal forebrain cholinergic neurons were selectively lesioned in mice using the toxin saporin conjugated to a basal forebrain cholinergic neuronal marker, the p75 neurotrophin receptor. Control animals were able to learn and remember spatial information when tested on a modified version of the passive place avoidance test where all extramaze cues were removed, and animals had to rely on idiothetic signals. However, the exploratory behaviour of mice with cholinergic basal forebrain lesions was highly disorganized during this test. By contrast, the lesioned animals performed no differently from controls in tasks involving contextual fear conditioning and spatial working memory (Y maze), and displayed no deficits in potentially confounding behaviours such as motor performance, anxiety, or disturbed sleep/wake cycles. These data suggest that the basal forebrain cholinergic system plays a specific role in idiothetic navigation, a modality that is impaired early in Alzheimer’s disease.     

Multiple Bacteriocin Production by Leuconostoc mesenteroidesTA33a and Other Leuconostoc/Weissella Strains

Leuconostoc (Lc.) mesenteroides TA33a produced three bacteriocins with different inhibitory activity spectra. Bacteriocins were purified by adsorption/desorption from producer cells and reverse phase high-performance liquid chromatography. Leucocin C-TA33a, a novel bacteriocin with a predicted molecular mass of 4598 Da, inhibited Listeria and other lactic acid bacteria (LAB). Leucocin B-TA33a has a predicted molecular mass of 3466 Da, with activity against Leuconostoc/Weissella (W.) strains, and appears similar to mesenterocin 52B and dextranicin 24, while leucocin A-TA33a, which also inhibited Listeria and other LAB strains, is identical to leucocin A-UAL 187. A survey of other known bacteriocin-producing Leuconostoc/Weissella strains for the presence of the three different bacteriocins revealed that production of leucocin A-, B- and C-type bacteriocins was widespread. Lc. carnosum LA54a, W. paramesenteroides LA7a, and Lc. gelidum UAL 187-22 produced all three bacteriocins, whereas W. paramesenteroides OX and Lc. carnosum TA11a produced only leucocin A- and B-type bacteriocins. Received: 11 April 1997 / Accepted: 10 June 1997     

Glucocorticoid action on the immune system

Glucocorticoids have profound effects on immune function that are mediated, in part, by steroid-induced cell death. Our studies have been aimed at identifying the mechanism of this lymphocytolytic process using the rat thymocyte as a model system. Administration of glucocorticoids in vivo resulted in internucleosomal cleavage of the lymphocyte genome that was detectable within 2 h of treatment and increased with time after hormone administration. Six h after steroid treatment greater than 50% of the genome was degraded, yet cell viability remained greater than 90% indicating that this event preceded cell death. Furthermore, this process appeared to be mediated by the glucocorticoid receptor since the antagonist RU 486 blocked glucocorticoid-mediated DNA degradation. To further characterize this lymphocytolysis we have analyzed glucocorticoid-treated thymocytes for nucleases. Two families of nuclear proteins have been identified, a 30-32 kDa doublet and a series of 3-4 proteins that are 12-19 kDa, both of which are induced by glucocorticoid treatment (137 +/- 6% and 342 +/- 24%, respectively) and have prominent nuclease activity. These nucleases can also be induced in vitro indicating that glucocorticoids act directly on thymocytes to mediate this response. Moreover, this nuclease induction, like glucocorticoid-mediated DNA degradation, could be blocked by RU 486. Based on these findings we propose a working model of glucocorticoid-mediated lymphocytolysis in which these steroids, acting via a receptor mediated process, induce the expression of a lysis gene product (nuclease) which degrades the genome and results in cell death.     

Purification and characterization of the beta-adrenergic receptor kinase

The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.     

Morphine and Enkephalins Potently Inhibit [3H]Noradrenaline Release from Rat Brain Cortex Synaptosomes: Further Evidence for a Presynaptic Localization of μ-Opioid Receptors

Synaptosomes prepared from rat cerebral cortex and labeled with [3H]noradrenaline (NA) were superfused with calcium-free Krebs-Ringer-bicarbonate medium and exposed to 10 mM K+ plus 0.1 mM Ca2+ so that [3H]NA release was induced. 6,7-Dihydroxy-N,N-dimethyl-2-aminotetralin (TL-99) strongly inhibited synaptosomal K+-induced [3H]NA release (EC50 = 5-10 nM) by activating alpha 2-adrenoceptors. Release was also inhibited (maximally by 40-50%) by morphine (EC50 = 5-10 nM), [Leu5]enkephalin (EC50 = approximately 300 nM), [D-Ala2,D-Leu5]enkephalin (DADLE), and Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) (EC50 values = approximately 30 nM). In contrast to the mu-selective opioid receptor agonists morphine and DAGO, the highly delta-selective agonist [D-Pen2,D-Pen5]enkephalin (1 microM) did not affect [3H]-NA release. Furthermore, the inhibitory effect of DADLE, an agonist with affinity for both delta- and mu-opioid receptors, was antagonized by low concentrations of naloxone. The findings strongly support the view that, like alpha 2-adrenoceptors, mu-opioid receptors mediating inhibition of NA release in the rat cerebral cortex are localized on noradrenergic nerve terminals.     

Factors responsible for the differences in cultural estimates and direct microscopical counts of populations of bacterivorous nanoflagellates

Bacterivorous nanoflagellates (microflagellates) have been routinely enumerated in marine and freshwater samples using either a Most Probable Number (MPN) culture method or by a direct microscopical counting method (DC). These two techniques typically yield highly disparate estimates of the density of nanoflagellates in natural samples. We compared these methods with seawater and marine snow (macroscopic detrital aggregate) samples collected from surface waters throughout the North Atlantic and in freshwater samples collected at three stations in Lake Ontario. Densities of nanoflagellates determined by the two methods differed by as much as four orders of magnitude; the MPN estimate rarely exceeded 10% of the microscopical count, and averaged 1% of this count. The MPN estimate constituted a higher percentage of the DC value in environments with high concentrations of nanoflagellates relative to environments with low concentrations of nanoflagellates. The ratio of the culture count to the microscopical count (MPNDC) increased along an environmental gradient from oligotrophy to eutrophy, and was positively correlated with the density of bacteria in the samples. In laboratory experiments with two species of bacterivorous nanoflagellates, the MPN count constituted a much greater percentage of the DC count during the exponential growth phase of the nanoflagellate than during the stationary growth phase. Differences in the estimates of nanoflagellate density obtained with these two techniques probably can be explained by the trophic mode of these protozoa, their growth stage, and the amenability of these species to laboratory culture.     

Genetic convergence during serial in vitro passage of a polyclonal squamous cell carcinoma

A cell line was established from an in situ squamous cell carcinoma of the skin (Bowen's disease), and its in vitro karyotypic evolution was cytogenetically analyzed. Initially, considerable genetic heterogeneity was evident. Nine cytogenetically abnormal clones, eight of which were apparently unrelated, were found among the 83 metaphases analyzed from the primary culture and the first passage. With increasing time in culture this complexity was reduced, so that a single clone dominated passages 7-11. The clone that emerged from this genetic convergence had a t(12;17)(p13;q21) as the sole abnormality. Our findings indicate that the cytogenetic multiclonality that has been repeatedly detected in short-term cultures of squamous cell carcinomas is not caused by the in vitro conditions. Instead, the principles of Darwinian selection apply: the altered, but stable, selection pressure facing a newly established and initially multiclonal cell line will lead to a reduction of genetic heterogeneity until the one clone that now has the proliferative advantage outgrows the other subpopulations.     

Reconstituted skin in culture:a simple method with optimal differentiation

Human skin is a unique organ, which can be reconstituted in vitro and represents an interesting system for studying cell proliferation and differentiation. A simple technique for producing reconstituted skin with optimal epidermal differentiation is described and characterized. A 4-mm punch biopsy of normal human skin is deposited on the epidermal side of mortified de-epidermized human dermis maintained at the air-liquid interface with a metallic support. The culture medium contains insulin, epidermal growth factor (EGF), cholera toxin, hydrocortisone, penicillin/streptomycin and fungizone. A well-differentiated epidermis develops within 15 days. Morphological and ultrastructural studies show a neoepidermis resembling normal skin. Differentiation markers such as involucrin, filaggrin, and various cytokeratins detected with pancytokeratin antibody are present and confirm this resemblance. The keratin profile is comparable to that observed in other skin culture models. A basement-membrane-like structure is reconstituted with hemidesmosomes and anchoring-filament formation. Bullous pemphigoid (BP) antigen is observed at the dermo-epidermal junction after 21 days of culture. Moreover, both dermal substrates and punch biopsies can be kept frozen for long-term storage, with little or no loss of epidermal growth kinetics and morphology. This skin culture technique is rapid, simple, economical and reproducible. Characterization has here shown high-quality epidermal differentiation. Scientists interested in epidermal in vitro studies should take interest in all these advantages.