Neoglycolipid analogues of ganglioside GM1 as functional receptors of cholera toxin.

We synthesized several lipid analogues of ganglioside GM1 by attaching its oligosaccharide moiety (GM1OS) to aminophospholipids, aliphatic amines, and cholesteryl hemisuccinate. We incubated GM1-deficient rat glioma C6 cells with each of the derivatives as well as native GM1 and assayed the cells for their ability to bind and respond to cholera toxin. On the basis of the observed increase in binding of 125I-labeled cholera toxin, it was apparent that the cells took up and initially incorporated most of the derivatives into the plasma membrane. In the case of the aliphatic amine derivatives, the ability to generate new toxin binding sites was dependent on chain length; whereas the C10 derivative was ineffective, C12 and higher analogues were effective. Increased binding was dependent on both the concentration of the neoglycolipid in the medium and the time of exposure. Cells pretreated with the various derivatives accumulated cyclic AMP in response to cholera toxin, but there were differences in their effectiveness. The cholesterol and long-chain aliphatic amine derivatives were more effective than native GM1, whereas the phospholipid derivatives were less effective. The distance between GM1OS and the phospholipid also appeared to influence its functional activity. The neoglycolipid formed by cross-linking the amine of GM1OS to phosphatidylethanolamine (PE) with disuccinimidyl suberate was less effective than the neoglycolipid formed by directly attaching GM1OS to PE by reductive amination. Furthermore, insertion of a C8 spacer in the former neoglycolipid rendered it even less effective.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid fluorometric method for the estimation of DNA in cultured cells

We present here a procedure for the rapid measurement of both DNA and protein from the same aliquot of cell lysate. DNA estimates obtained by this method were compared to replicate determinations using the method of Kissane and Robins (1). The optimal range for the estimation of DNA (1 to 20 μg) is well suited for use with portions of extracts from individual cell cultures; the remainder of the extract remains available for enzyme assays or other parallel determinations.     

Discrimination and consistency of five myosin ATPase stains in human normal and duchenne dystrophic muscle

Summary Limitations in the ability of the human visual system to assess accurately the relative staining densities of individual fibers in muscle tissue stained for myosin ATPase can complicate the objective evaluation of fiber type populations. In this study a novel approach is employed which utilizes human visual capabilities to provide accurate fiber classification. Using this approach, the ability of five ATPase staining techniques to discriminate fiber type categories in single samples of human normal and Duchenne dystrophic skeletal muscle is evaluated, as is the consistency of the fiber type classifications between stains. While no major discrepancies in fiber typing were observed in the sample of normal muscle, significant differences in classification, along with a decrease in the ability to discriminate fiber types were noted in the sample of Duchenne muscular dystrophy. For the most part, these discrepancies were resolved by a re-interpretation of the staining characteristics of fibers in one stain.This work was supported in part by NIH grant NS 15584, and by a grant from the Muscular Dystrophy Association     

Phages I alpha and I2-2: IncI plasmid-dependent bacteriophages

Phage I alpha was isolated from sewage from Windhoek, South West Africa. It formed relatively clear plaques about 2 mm in diameter, on sensitive strains of Escherichia coli K12 and Salmonella typhimurium LT2. The phage had an hexagonal outline with a diameter of about 24 nm, contained RNA and was resistant to chloroform. Phage I alpha formed plaques or propagated only on organisms carrying I1 plasmids or the I gamma plasmid R621a. The efficiency of plating was higher on E. coli than on S. typhimurium hosts. The phage adsorbed along the length of shafts of I1 pili. Phage I2-2 was isolated from Pretoria sewage. It was a filamentous virus and individual virions varied considerably in length. Phage I2-2 formed turbid plaques which varied from pin point to about 1 mm in diameter on all hosts. It was resistant to RNAase and sensitive to chloroform. Phage I2-2 had a spectrum of activity limited to strains harbouring I2 plasmids but the adsorption site could not be demonstrated. The phage was not related serologically to phages Ifl or PR64FS.     

Differentiation of normal human mammary epithelial cells in culture: an ultrastructural study.

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a larger number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days were prominent in the cells of the stratified layer. After 28 days in the culture, the cells began to round up and slough off the culture plate.     

Biology of the Galapagos shark,Carcharhinus galapagensis, in Hawai'i

Synopsis Catch records from the Hawai'i Cooperative Shark Research and Control Program, which operated in Hawai'i from 1967–1969, were examined and data on the Galapagos shark,Carcharhinus galapagensis were analyzed. A total of 304 Galapagos sharks was caught, predominantly with longlines. More female sharks were caught than males, and the catch was skewed geographically. On the island of O'ahu the highest catch rates occurred along the north and south coasts. High catch rates also occurred near points of land, where longshore currents converge. Average depth of capture was greater for juveniles (45.1 m) and mature males (60.2 m), than for subadults (38.8 m) and mature female sharks (34.2 m). Males appear to reach maturity between 205 and 239 cm total length, and females between 215 and 245 cm. Litter size ranged from 4 to 16 pups, with an average of 8.7. In Hawaiian waters Galapagos sharks are born at just over 80 cm total length. Mating and parturition apparently occur early in the year, and gestation is estimated to be about 12 months. Stomach contents consisted mainly of teleosts and benthic prey, and ontogenetic changes in diet occurred as sharks increased in size. Sharks consumed a smaller proportion of teleosts and more elasmobranchs with increasing size. Dietary diversity also increased with increasing size of shark.     

Differential effect of three-repeat and four-repeat tau on mitochondrial axonal transport

Tau protein is present in six different splice forms in the human brain and interacts with microtubules via either 3 or 4 microtubule binding repeats. An increased ratio of 3 repeat to 4 repeat isoforms is associated with neurodegeneration in inherited forms of frontotemporal dementia. Tau over-expression diminishes axonal transport in several systems, but differential effects of 3 repeat and 4 repeat isoforms have not been studied. We examined the effects of tau on mitochondrial transport and found that both 3 repeat and 4 repeat tau change normal mitochondrial distribution within the cell body and reduce mitochondrial localization to axons; 4 repeat tau has a greater effect than 3 repeat tau. Further, we observed that the 3 repeat and 4 repeat tau cause different alterations in retrograde and anterograde transport dynamics with 3 repeat tau having a slightly stronger effect on axon transport dynamics. Our results indicate that tau-induced changes in axonal transport may be an underlying theme in neurodegenerative diseases associated with isoform specific changes in tau's interaction with microtubules.     

Characteristics and function of thick and thin conjugative pili determined by transfer-derepressed plasmids of incompatibility groups I1, I2, I5, B, K and Z

Eleven transfer-derepressed plasmids from incompatibility groups I1, I5, B, K and Z were constructed using the dnaG3 mutant Escherichia coli strain BW86. All were found to determine thin flexible and thick rigid pili constitutively. Immune electron microscopy was used to relate thick and thin pilus serotypes with incompatibility grouping. Mutant plasmids that determined only thick pili constitutively transferred efficiently on an agar surface but not in a liquid, whereas plasmids with both kinds of pili transferred equally well in both environments. A mutant of the IncI2 plasmid R721 determined thin pili constitutively, and thick pili at a repressed level, as indicated by electron microscopy. Experiments with this indicated that thin pili were apparently not involved directly in conjugation but were only used to stabilize mating aggregates.     

P2 pyridine N-oxide thrombin inhibitors: a novel peptidomimetic scaffold

In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).