The mechanism of biliary secretion of reduced glutathione. Analysis of transport process in isolated rat-liver canalicular membrane vesicles

Transport of reduced glutathione (GSH) was studied in isolated rat liver canalicular membrane vesicles by a rapid filtration technique. The membrane vesicles exhibit uptake of [2-3H]glycine--labeled GSH into an osmotically reactive intravesicular space. Although the canalicular membrane vesicles possess gamma-glutamyltransferase and aminopeptidase M, enzymes that hydrolyze glutathione into component amino acids, inactivation of the vesicle-associated transferase by affinity labeling with L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125) had no effect on the initial rate of GSH transport. Chemical analysis revealed that intact GSH accounted for most of vesicle-associated radioactivity. The initial rate of transport followed saturation kinetics with respect to GSH concentration; an apparent Km of 0.33 mM and V of 1.47 nmol/mg protein in 20 s were calculated. These results indicate that transport of GSH across the canalicular membranes is a carrier-mediated process. Replacement of NaCl in the transport medium by KCl, LiCl or choline chloride had no effect on the transport activity of the vesicles. The rate of GSH uptake by the vesicles was enhanced by valinomycin-induced K+-diffusion potential (vesicle inside-positive) and was inhibited by probenecid, indicating that GSH transport across the canalicular membranes is electrogenic and involves the transfer of negative charge. The transport of GSH was inhibited by oxidized glutathione or S-benzyl-glutathione. This transport system in canalicular plasma membranes may function in biliary secretion of GSH and its derivatives which are synthesized in hepatocytes by oxidative processes or glutathione S-transferase.     

Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity

Summary Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.     

Effects of Legal Abortion on Gynaecology

The effect of the Abortion Act on gynaecological work in one provincial teaching hospital is that despite an increase in patient turnover of 45% the waiting list has increased by 200%. It is suggested that the Act has had little effect on the birth rate or illegitimacy and that there may have been an increase in criminal abortion.     

Comparison of the localization of nucleic acid synthesis during bud formation on leaf fragments and on intact undetached leaves ofBegonia rex Putz.

The budding capacity ofBegonia rex leaf fragments is well known; that of undetached leaves has been shown by us only recently after treating the leaves with 6γγ DMAAP. Benzyladenine is as effective as 6γγ DMAAP in stimulating budding. Lower temperatures (17°, 22–12°, 12°) are also capable of inducing bud formation but only after a small cut has been made in a main vein of the undetached leaf. Root formation can also be provoked on undetached leaves which have a cut in the main leaf vein by higher temperatures (24–22°) or by an IAA treatment. Differences in the first stages of bud formation on leaf fragments and on undetached leaves are observed using histochemical and histoautoradiographic techniques.     

Association of three highly mutable genes involving pigmentation in Anthirrhinum majus

Unstable alleles at three unlinked loci (delilah, nivea and pallida) can mutate somatically to allow the production of clones of pigmented cells (“flakes”) in particular parts of the corolla. The size of the flakes depends on the timing of the mutations during the development of the flower.     

Lipids of the Spirochaetales: Comparison of the Lipids of Several Members of the Genera Spirochaeta, Treponema, and Leptospira

The lipid compositions of 17 spirochetes belonging to the genera Spirochaeta and Treponema were investigated and compared with data previously derived from 11 strains of Leptospira. The lipid compositions and lipid metabolism of any of these genera is sufficiently different to be characteristic of that genus and to differentiate it from the other two genera. Members of the genus Leptospira are characterized by their ability to beta-oxidize long chain fatty acids as their major carbon and energy source. With few exceptions, they are incapable of synthesizing fatty acids de novo. The major phospholipid found was phosphatidyl ethanolamine. No glycolipid or phosphatidyl choline was found in these organisms. Members of the genus Treponema studied were incapable of beta-oxidation as well as de novo synthesis of fatty acids. Phosphatidyl choline is the major phospholipid of this genus. The glycolipid, monogalactosyl diglyceride, is a major component of the Treponema. Members of the Spirochaeta did synthesize fatty acids de novo. Although these spirochetes contain a monoglycosyl diglyceride, the hexose content of the glycolipid varied from species to species. Neither phosphatidyl ethanolamine nor phosphatidyl choline was found in the Spirochaeta.     

A mass-spectrometric sequence study of the enzyme ribitol dehydrogenase from Klebsiella aerogenes

The first detailed results of the application of a low-resolution mixture analysis approach to the sequence analysis of an enzyme, ribitol dehydrogenase, are given. Examples of the interpretation of the spectra of peptide mixtures derived from this protein are described. Evidence for new fragmentation patterns observed is reported, together with an explanation of the generation of ambiguous sequences by use of a low-specificity enzyme, thermolysin. The overall sequencing strategy evolved is assessed.     

Thorotrast uptake and transit in embryonic glia, heart fibroblasts and neurons in vitro

Thorotrast (colloidal ThO₂) is incorporated into coated vesicles, various agranular vesicles and sacs, and a surface-associated system of membranous channels in times as short as 1 min by single cultured glial and heart cells. Thorotrast appears in ‘C’-shaped bodies and in small, dense bodies of the lysosomal series within ca. 25 min. With longer chase periods, thorotrast ‘clears’ from all cytoplasmic organelles except the lysosomal series. The technique of applying thorotrast and using varying chase periods fails to distinguish a class of membranous organelles, located close to the cell periphery, that might serve as a source of new cell surface during locomotory activity. Similarly, thorotrast (colloidal ThO₂) is incorporated into almost all classes of membrane-bounded organelles of growth cones and axons of single nerve cells in vitro in times as short as 1 min. This includes elements of the smooth endoplasmic reticulum. No thorotrast enters the lysosomal granules in this short time. During various chase periods, the tracer disappears from the initial sites of incorporation and accumulates in dense bodies of the lysosome series within growth cones and axons. ‘C’-shaped bodies may be an intermediate in that process. No unique sites of endocytotic activity or of a complete absence of endocytosis were observed that could be correlated with growth cone function and axonal elongation, though the presence of the tracer in agranular sacs of the smooth endoplasmic reticulum in growth cones could reflect hypothesized cycling of cell surface (Bray, 1973).