Correlation of bacterial viability with uptake of [14C] acetate into phenolic glycolipid-1 ofMycobacterium leprae within Schwannoma cells

The viability ofMycobacterium leprae, maintained within 33B Schwannoma cells, was estimated in terms of incorporation of [¹⁴C] acetate into its specific phenolic glycolipid-1. This measure of viability was correlated with two other assays,viz., fluorescein diacetate/ethidium bromide staining and mouse footpad growth. Observation of a 2-fold increase in the number of intracellularMycobacterium leprae over an experimental period of 12 days also corroborated this contention. Furthermore, on addition of anti-leprosy drugs to these intracellularMycobacterium leprae there was significant decrease in phenolic glycolipid-1 synthesis indicative of loss of viability of the organisms. This study also established the importance of the host cell for active bacillary metabolism, asMycobacterium leprae maintained in cell-free conditions showed no incorporation into phenolic glycolipid-1. Moreover, compromising the host’s protein synthesis capacity with cycloheximide, also led to reduction in bacillary metabolism. As this system measures the metabolic synthesis of a uniqueMycobacterium leprae component, it would be useful for development and screening of compounds acting against specific bacillary targets.     

Spontaneous abortion and confined chromosomal mosaicism

Summary Confined placental chorionic mosaicism is reported in 2% of viable pregnancies cytogenetically analyzed on chorionic villi samplings (CVS) at 9–12 weeks of gestation. In follow-up studies this mosaicism has been shown to be associated with increased frequency of second and third trimester pregnancy loss or intrauterine fetal growth retardation. We have studied 54 spontaneous abortions (SA) for the detection of confined placental mosaicism and found 11 of them to be mosaic. All mosaic cases were identified among first trimester spontaneous abortions, and the mosaicism was confined to specific placental or embryonic/fetal cell lineages. These results indicate that the previously reported mosaicism in SA represents both confined and generalized types of mosaicism and that its accepted frequency of 5%–10% in SA will likely be higher. Over the whole gestational period, the confined placental mosaicism is more common than the reported rate of 1%–2% seen in viable pregnancies at CVS, and a higher proportion of pregnancy complications than previously suspected may be associated with confined placental mosaicism.     

Some cytochemical studies on the cell surface ofAmphidinium carterae (Dinophyceae)

Summary The surface coat of the dinoflagellateAmphidinium carterae Hulburt was examined by fluorescence and transmission electron microscopy, using various fluorochromes and cationic dyes. The overall results showed cell-surface reactions typical of acid mucopolysaccharides. The cationic dye staining revealed an outer fine fibrillar layer (15–70 nm thick) overlying a dense anionic coat (40–60 nm thick) which appeared to thicken progressively with age. In general, the structure of the amphiesmal vesicles was similar to that previously described by other investigators. However, an acidic mucopolysaccharide layer was observed on the inner surface of these vesicles. Each of these structures is traversed by 1–3 pores and at least 2 types of extrusomes are formed, the spindle trichocysts and the mucocysts. Cell to cell adhesion through the surface coat was frequently observed. Evidence was also obtained for internalization of all the surface-coat markers used.This investigation forms part of a doctoral thesis submitted by the first author to the University of British Columbia, Vancouver, B.C.     

Some observations on the urea-degrading enzyme of the diatom Cyclotella cryptica and the role of nickel in its production

The urea-degrading enzyme of Cyclotella cryptica was testedin crude cell-free extracts for effects from chemical reagentsknown to distinguish between urease and ATP:urea amidolyase.Inhibition of the enzyme by hydroxyurea and its indifferenceto added ATP, Mg²⁺ or K⁺ avidin or biotin clearly characterizedthe enzyme as urease (EC 3.5.1.5 [EC] ). The Cyclotella urease wasunaffected by thiourea addition, as was also the growth of thediatom in the presence of this substrate analogue. Indirectevidence was obtained from growth studies of the diatom andcorresponding urease production showing that the enzyme: (i)contains Ni²⁺ tightly bound to an apoprotein; (ii) is producedconstitutively even from growth on nitrate and does not requireextracellular urea for its synthesis, although quantitativelythe activity is greatest from growth on urea. It is concludedthat Cyclotella urease is a Ni²⁺ constitutive enzyme similarin many respects to those previously reported from Phaeodactylumtricornutwn and Tetraselmis maculata.     

Mechanism of killing by virus-induced cytotoxic T lymphocytes elicited in vivo.

The mechanism of lysis by in vivo-induced cytotoxic T lymphocytes (CTL) was examined with virus-specific CTL from mice infected with lymphocytic choriomeningitis virus (LCMV). LCMV-induced T cells were shown to have greater than 10 times the serine esterase activity of T cells from normal mice, and high levels of serine esterase were located in the LCMV-induced CD8+ cell population. Serine esterase was also induced in purified T-cell preparations isolated from mice infected with other viruses (mouse hepatitis, Pichinde, and vaccinia). In contrast, the interferon inducer poly(I.C) only marginally enhanced serine esterase in T cells. Serine esterase activity was released from the LCMV-induced T cells upon incubation with syngeneic but not allogeneic LCMV-infected target cells. Both cytotoxicity and the release of serine esterase were calcium dependent. Serine esterase released from disrupted LCMV-induced T cells was in the form of the fast-sedimenting particles, suggesting its inclusion in granules. Competitive substrates for serine esterase blocked killing by LCMV-specific CTL, but serine esterase-containing granules isolated from LCMV-induced CTL, in contrast to granules isolated from a rat natural killer cell tumor line, did not display detectable hemolytic activity. Fragmentation of target cell DNA was observed during the lytic process mediated by LCMV-specific CTL, and the release of the DNA label [125I]iododeoxyuridine from target cells and the accompanying fragmentation of DNA also were calcium dependent. These data support the hypothesis that the mechanism of killing by in vivo-induced T cells involves a calcium-dependent secretion of serine esterase-containing granules and a target cell death by a process involving nuclear degradation and DNA fragmentation.     

The use of fluorochromes in the cytochemical characterization of some phytoflagellates

Summary Sixteen fluorochromes were tested for the cytochemical characterization of two dinoflagellates (Amphidinuim carterae, Prorocentrum micans) and one chlorophycean flagellate (Dunaliella tertiolecta). Depending on the fluorochrome used, various cellular components (incuding the plasma membrane, thecal plates, pusule, trichocysts, nucleus, lipid bodies and vacuoles) were revealed. The different colours obtained from single or double fluorochrome staining enabled the differentiation and identification of most cellular components. Protoplasmic staining with Fluorescein diacetate suggested the occurrence of esterases in the three phytoflagellates. Rhodamine B, Neutral Red, FluoroBora P and Nile Blue revealed extensive occurrence of lipoid bodies inA. carterae, but Nile Blue showed considerable difference from the other stains in the inclusion size and intracellular location of these bodies. Chlortetracycline binding, and its inhibition by the Ca²⁺ionophore A23187, indicated that the plasma membrane, pusule system and trichocysts contain sites of Ca²⁺ binding. Calcofluor White ST proved superior to Congo Red and Lucifer Yellow in elucidating structural details of the thecal plates ofP. micans. While Acridine Orange revealed the presence of surface-coat acidic polysaccharides, the fluoresceinated lectins established their glycoconjugate nature in all the three flagellates. Possible mechanisms of fluorochrome uptake are discussed.     

Revisiting estimates of CTL killing rates in vivo

Recent experimental advances have allowed the estimation of the in vivo rates of killing of infected target cells by cytotoxic T lymphocytes (CTL). We present several refinements to a method applied previously to quantify killing of targets in the spleen using a dynamical model. We reanalyse data previously used to estimate killing rates of CTL specific for two epitopes of lymphocytic choriomeningitis virus (LCMV) in mice and show that, contrary to previous estimates the "killing rate" of effector CTL is approximately twice that of memory CTL. Further, our method allows the fits to be visualized, and reveals one potentially interesting discrepancy between fits and data. We discuss extensions to the basic CTL killing model to explain this discrepancy and propose experimental tests to distinguish between them.     

Neuraminidase inhibitor resistance in influenza: assessing the danger of its generation and spread

Neuraminidase Inhibitors (NI) are currently the most effective drugs against influenza. Recent cases of NI resistance are a cause for concern. To assess the danger of NI resistance, a number of studies have reported the fraction of treated patients from which resistant strains could be isolated. Unfortunately, those results strongly depend on the details of the experimental protocol. Additionally, knowing the fraction of patients harboring resistance is not too useful by itself. Instead, we want to know how likely it is that an infected patient can generate a resistant infection in a secondary host, and how likely it is that the resistant strain subsequently spreads. While estimates for these parameters can often be obtained from epidemiological data, such data is lacking for NI resistance in influenza. Here, we use an approach that does not rely on epidemiological data. Instead, we combine data from influenza infections of human volunteers with a mathematical framework that allows estimation of the parameters that govern the initial generation and subsequent spread of resistance. We show how these parameters are influenced by changes in drug efficacy, timing of treatment, fitness of the resistant strain, and details of virus and immune system dynamics. Our study provides estimates for parameters that can be directly used in mathematical and computational models to study how NI usage might lead to the emergence and spread of resistance in the population. We find that the initial generation of resistant cases is most likely lower than the fraction of resistant cases reported. However, we also show that the results depend strongly on the details of the within-host dynamics of influenza infections, and most importantly, the role the immune system plays. Better knowledge of the quantitative dynamics of the immune response during influenza infections will be crucial to further improve the results.