ELECTRON MICROSCOPIC OBSERVATIONS OF RAT AND MOUSE CEREBELLUM IN TISSUE CULTURE

Closely ordered stages of myelin formation in cultures of newborn rat and mouse cerebellum, selected by direct light microscopy, were studied with the electron microscope. Electron micrographs of these cultures reveal the presence of neurons, axons, neuroglia, microglia, and ependymal cells. The appearance of the neuron is identical to that previously described in vivo. The neuroglial cell has long, branching processes, and its cytoplasm is characterized by packets of long, narrow fibrils. During myelin formation, a glial cell process surrounds the axon. This process may form an internal mesaxon and may spiral for several turns around the axon. Other glial cell processes may interdigitate with or overlay the innermost process to contribute to the multilamellated structure. The glial processes flatten and the cytoplasmic surfaces of the cell membrane come into contact to form the lamellae of the myelin sheath. These adhesions may be temporarily incomplete as evidenced by sequestered islands of glial cytoplasm among the myelin lamellae. Ultimately, a compact, apparently spiral, myelin sheath is formed. These findings are discussed in relation to in vivo central myelin formation.     

Study of the in vitro bioactivation of albendazole in human liver microsomes and hepatoma cell lines

The metabolism of albendazole (ABZ), a benzimidazole anthelminthic, was studied in either microsomal preparations of human liver biopsies or cultured human hepatoma cell lines. Metabolites were analyzed by HPLC. Our data show that microsomes from human biopsies and two human cell lines, HepG2 and Hep3B, oxidize the drug to the sulfoxide very efficiently, whereas the third cell line tested, SK-HEP-1, does not. Both cytochrome P-450 dependent monooxygenases and favin-containing monooxygenases appear to be involved in human ABZ metabolism. Using the cell line displaying the highest ABZ-metabolizing activity, HepG2, the cytotoxic and the inducing effects of the parent drug ABZ and of two primary metabolites, the sulfoxide and the sulfone were studied. These three chemicals provoked a rise in mitotic index resulting from cell division blockage at the prophase or at the metaphase (ABZ metabolites) stage, and ABZ was more cytotoxic than its metabolites. With regard to enzyme-inducing effects, our data clearly demonstrate that the sulfoxide and, to a lesser degree, the sulfone are potent inducers of some drug metabolizing enzymes (i.e., cytochrome P-488 dependent monooxygenases and UDP glucuronyltransferase), whereas ABZ fails to increase and even slightly decreases these enzymatic activities. In conclusion, the HepG2 human hepatoma cell line appears to be suitable for the study of many parameters of metabolism and action of ABZ and other structurally related compounds in humans.Abbreviations ABZ albendazole - B[a]P benzo[a]pyrene - HPLC high-performance liquid chromatography - MC 3-methylcholanthrene - MFO mixed-function oxidase - UDPGT UDP-glucuronyltransferase     

Deacylated lipopolysaccharide inhibits plasminogen activator inhibitor-1, prostacyclin, and prostaglandin E2 induction by lipopolysaccharide but not by tumor necrosis factor-alpha

Bacterial LPS and TNF induce vascular endothelial cells to express a variety of response molecules. LPS that is partially deacylated (dLPS) by a human neutrophil enzyme blocks the ability of LPS, but not TNF, to augment one of these responses, the expression of endothelial cell surface molecules that promote neutrophil adherence (J. Exp. Med. 1987; 165:1393-1402). We show that dLPS can inhibit the ability of LPS, but not TNF, to elicit the expression of plasminogen activator inhibitor-1 (PAI-1), prostacyclin, and PGE2 by human umbilical vein endothelial cells. dLPS also prevented the accumulation of specific PAI-1 mRNA in response to LPS, but not to TNF. Neither the LPS- or TNF-induced expression of PAI-1 nor the dLPS inhibition of the LPS response was mediated by prostanoids. These results indicate that dLPS can specifically block a variety of endothelial cell responses to LPS and provide support for the hypotheses 1) that dLPS and LPS may interact with a common target molecule on or in endothelial cells, and 2) that dLPS, produced by enzymatic deacylation of LPS in vivo, could inhibit endothelial cell stimulation by LPS and thereby limit the host inflammatory response to invasive gram-negative bacteria.     

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.     

A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

The contrasting role of auxin in submergence-induced petiole elongation in two species from frequently flooded wetlands

The involvement of auxin in the submergence-induced petiole elongation has been investigated in Rumex palustris and Ranunculus sceleratus. Both wetland species are capable of enhanced petiole elongation upon submergence or treatment with exogenous ethylene (5μl l⁻¹). Treatment of intact Rumex palustris plants with 1-naphthalene acetic acid (NAA) at 10⁻⁴ M enhanced petiole elongation, while treatment with N -1-naphthylphthalamic acid (NPA) had no effect on petiole elongation. The elongation response after NAA or NPA treatment was comparable for plants in both submerged and drained conditions. Pre-ageing of detached petioles of Rumex palustris for 3 h in light or in dark conditions had no effect on the submergence-induced elongation. In comparison to intact plants, detached petioles of Rumex palustris , with or without lamina, did not show significant differences in responsiveness to IAA between drained or submerged conditions. This was in contrast to Ranunculus sceleratus where submergence caused a clear increase in responsiveness towards IAA. Removal of the lamina, the putative source of auxin, or treatment with NPA did not hinder the submergence-induced elongation of detached Rumex palustris petioles, but severely inhibited elongation of detached Ranunculus sceleratus petioles. This inhibition could be restored by application of NAA, suggesting the specific involvement of auxin in the submergence response of Ranunculus sceleratus. It is concluded that, in contrast to Ranunculus sceleratus , auxin is probably not involved in the submergence-induced petiole elongation of Rumex palustris.     

A telescopic method for photographing within 8×8 cm minirhizotrons

The volatile organic compounds produced during a sequence of soil incubations under controlled conditions, with either added NH₄ ⁺-N or NO₃ ^--N, were collected and identified. The nature and relative amounts of the volatile organic compounds produced by the microorganisms in the soils were remarkably reproducible and consistent.     

Suppressors of thermosensitive mutations in the DNA polymerase δ gene of Saccharomyces cerevisiae

DNA polymerases (Pol) α, δ and ε are necessary for replication of nuclear DNA. Po1δ interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Po18. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase δ gene (POL3). We have attempted to clone two recessive extragenic suppressors of such is mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdpl and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, D1S2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.