Membrane behavior of exocytic vesicles: II in fate of the trichocyst membranes in paramecium after induced trichocyst discharge

A specific exocytic process, the discharge of spindle trichocysts of paramecium caudatum was examined by means of the electron microscope. This exocytosis is induced by an electric shock simultaneously in nearly all of the trichocysts (ca. 6,000-8,0000 of a single cell. Single paramecia were subjected to the shock and then fixed at defined times after the shock so that the temporal sequence of the pattern of changes of the trichocyst membranes after exocytosis could be studied. The trichocyst vacuoles fuse with the plasma membrane only for that length of time required for expulsion to take place. After exocytosis, the membrane of the vacuole does not become incorporated into the plasma membrane; rather, the collapsed vacuole is pinched off and breaks up within the cytoplasm. The membrane vesiculates into small units which can no longer be distinguished from vesicles of the same dimensions that exist normally within the cell's cytoplasm. the entire process is completed within 5-10 min. These results differ from the incorporation of mucocyst membranes into the plasma membrane as proposed for tetrahymena.     

Glycosphingolipid-binding specificity of the mannose-binding protein from human sera

Mannose-binding protein was purified from human serum to apparent homogeneity by affinity chromatography on mannose-Sepharose, followed by affinity chromatography on underivatized Sepharose. Approximately 0.4 mg protein was obtained from 1 liter serum. The glycosphingolipid-binding specificity of the purified protein was examined by chromatogram overlay and solid phase assays. It binds with high affinity to Lc-3Cer (GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide) and n-Lc5Cer (GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1ceramide). It does not bind to many other glycosphingolipids without terminal N-acetylglucosamine residues that were tested. Thus, these data suggest that N-acetylglucosamine-terminated glycosphingolipids may serve as cell-surface attachment sites for mannose-binding protein in vivo. In addition, the binding specificity of the protein can be used as a sensitive probe for determining the levels of Lc3Cer and nLc5Cer in tissues, as it exhibits half-maximal binding to about 10 pmol of these lipids in solid phase assays, and detects less than 20 pmol of Lc3Cer in chromatogram overlay assays. This technique was utilized to demonstrate that one sample of chronic myeloid leukemia cells contains both Lc3Cer and nLc5Cer.     

Taxonomic studies of the genus Hypoxis in East Africa

East African material of the genus Hypoxis L. has preliminarily been divided into the heterogenous, probably apomictic H. obtusa Burch- complex (2n = 40–50, ca. 75, 76, ca. 85, >86, ca. 92, ca. 98, ca. 108, 130–135, 160–200) and 5 rather homogenous species: H. angustifolia Lam. (2n = 14, 28), H. goetzei Harms (2n = ca. 62), H. kilimanjarica Bak., H. malosana Bak. (2n = 14) and H. macrocarpa Holt & Staubo sp. nov. H. kilimanjarica is divided into ssp. kilimanjarica and ssp. prostrata Holt & Staubo ssp. nov.     

A cationic chalcogenoxanthylium photosensitizer effective in vitro in chemosensitive and multidrug-resistant cells

Pentacyclic thio- (1) and seleno- (2) analogues of tetramethylrosamine (TMR) were prepared with a julolidyl fragment replacing the 3-dimethylamino substituent in the xanthylium core. The pentacylic structure increases the lipophilicity of 1 and 2 relative to TMR-S and TMR-Se and locks the lone-pair of electrons on the julolidyl N atom into conjugation with the xanthylium core. This conformational rigidization leads to longer wavelengths of absorption, but has little impact on other photophysical properties such as quantum yields for fluorescence and singlet-oxygen generation and fluorescence lifetimes in 1 and 2 relative to TMR-S and TMR-Se. Both 1 and 2 are effective photosensitizers against chemosensitive AUXB1 cells in vitro at 1 × 10⁻⁷ M and compound 2 is an effective photosensitizer against multidrug-resistant CR1R12 cells in vitro at 1 × 10⁻⁷ M. While the uptake TMR-S into CR1R12 cells as measured by fluorescence is significantly lower than uptake into chemosensitive AUXB1 cells, there is no significant difference in the uptake of 1 into either AUXB1 or CR1R12 cells. The addition of 2 × 10⁻⁴ M verapamil to the cells prior to treatment with 1 had no significant effect on the uptake of 1 into either AUXB1 or CR1R12 cells. Treating lipid-activated, purified Pgp with 2 and light gave complete inhibition of Pgp ATPase activity.     

Deletions of muscle mitochondrial DNA in mitochondrial myopathies: sequence analysis and possible mechanisms.

Forty per cent of patients with mitochondrial myopathies, a diverse group of multisystem diseases predominantly affecting skeletal muscle and the brain, have large deletions of a proportion of muscle mitochondrial DNA (mt DNA). These appeared to be identical in 13 of 28 cases, contained within the region 8286-13595 bp. Analysis of the deletion junction in two cases showed a 13 nucleotide sequence which occurred in the normal genome as a direct repeat flanking the region deleted in the mutant mt DNAs. Mt DNA deletions may arise from recombination or slippage between short sequence repeats during replication.     

Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113-0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.     

A Salmonella Typhimurium-Typhi genomic chimera: a model to study Vi polysaccharide capsule function in vivo

The Vi capsular polysaccharide is a virulence-associated factor expressed by Salmonella enterica serotype Typhi but absent from virtually all other Salmonella serotypes. In order to study this determinant in vivo, we characterised a Vi-positive S. Typhimurium (C5.507 Vi(+)), harbouring the Salmonella pathogenicity island (SPI)-7, which encodes the Vi locus. S. Typhimurium C5.507 Vi(+) colonised and persisted in mice at similar levels compared to the parent strain, S. Typhimurium C5. However, the innate immune response to infection with C5.507 Vi(+) and SGB1, an isogenic derivative not expressing Vi, differed markedly. Infection with C5.507 Vi(+) resulted in a significant reduction in cellular trafficking of innate immune cells, including PMN and NK cells, compared to SGB1 Vi(-) infected animals. C5.507 Vi(+) infection stimulated reduced numbers of TNF-α, MIP-2 and perforin producing cells compared to SGB1 Vi(-). The modulating effect associated with Vi was not observed in MyD88(-/-) and was reduced in TLR4(-/-) mice. The presence of the Vi capsule also correlated with induction of the anti-inflammatory cytokine IL-10 in vivo, a factor that impacted on chemotaxis and the activation of immune cells in vitro.     

Hearing sensitivity in two black bass species using the auditory brainstem response approach

Recently, several bioacoustic studies have focused on the red eye bass (Micropterus coosae). One of these studies documented sound production, while the other played back sounds produced by prey items in order to determine their attractiveness to M. coosae. Surprisingly, the hearing ability of fishes in the genus Micropterus has received very little attention. The need for audiograms describing hearing in Micropterus is apparent. This study utilized the auditory brainstem response (ABR) approach to determine hearing sensitivity in terms of both sound pressure level (SPL) and particle acceleration in two black bass species, the red eye bass (M. coosae) and the Alabama bass (M. henshalli). Audiograms produced in this study expressed in both SPL and particle acceleration showed a positive relationship between hearing threshold and frequency. Micropterus coosae was most sensitive to frequencies that overlap with the peak frequencies of their vocalizations, and the vocalizations of a prey species, Cyprinella trichroistia. Bass hearing sensitivities at lower frequencies, measured in terms of particle acceleration, were similar to several sciaenid species.