Glycosyl transferases in mouse and human milk fat globule membranes

Summary Membranes isolated from mouse and human milk fat globules were found to contain the enzymes responsible for the synthesis of dolichol monophosphate mannose and dolichol monophosphate glucose as well as those involved in the transference of the glycosyl residues from the two dolichol derivatives to dolichol diphosphate oligosaccharides. The levels of most of the enzymes were comparable to those found in mouse mammary gland microsomes. The presence of enzymes involved in protein glycosylation via dolichol derivatives in the milk fat globule membrane provides evidence in favor of an outward flow of membrane components from the rough endoplasmic reticulum, where these enzymes are active in vivo, towards the cell surface.     

Synthesis of dolichol derivatives in human erythrocyte membranes

Summary Human erythrocyte membranes contain the enzymes responsible for the synthesis of dolichol-P-glucose, dolichol-P-mannose, dolichol-PP-N-acetylglucosamine, dolichol-PP-NN diacetylchitobiose and of dolichol-PP-oligosaccharides containing NN diacetylchitobiose and mannose or the same sugar residues plus glucose. The transfer of the oligosaccharide moieties from the dolichol-PP-oligosaccharides to endogenous proteins could not be detected. These enzymes appeared to be integral membrane proteins.Abbreviation Dol dolichol Dedicated to ProfessorLuis f. Leloir on the occasion of his 70th birthday.     

Procedure for testing kinetic models of the photocycle of bacteriorhodopsin.

Given some simple kinetic models of the photocycle of bacteriorhodopsin (bR) and data taken at many wavelengths and under conditions that avoid photoselection and steady-state cycling complications, it is shown how to extract the apparent rate constants and the spectra of the intermediates. Special consideration was given to establishing the range of error of these results. There are many criteria, which we explicitly discuss, that the spectra should satisfy in order that the kinetic model be acceptable. New data for the photocycle of purple membrane fragments in dilute buffer at pH 7.0 has been obtained at 15 measuring wavelengths and four temperatures. The procedure, which can be generalized to more complex models, has been applied to these data to test two kinds of kinetic models: the unidirectional unbranched model and the undirectional model with simple branching straight back to bR from any intermediate. In these models the spectrum of the O intermediate is highly temperature sensitive, even with branching, and/or has two broad maxima. Moreover, the spectrum of the M intermediate has a secondary maximum and two M-like states appear to be required. Thus, neither model satisfies the physical criteria.     

Studies on DNA damage: Discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate,obtained for several compounds

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically, is more strictly correlated with the initiation of DNA unwinding.     

Muscarinic Acetylcholine Receptor Enhances Phosphatidylcholine Hydrolysis via Phospholipase D in Bovine Chromaffin Cells in Culture

Although it is well-established that inositol-containing lipids serve as precursors of intracellular second messenger molecules in chromaffin cells, we describe some findings that show the formation of diacylglycerol from phosphatidylcholine in response to agonist-mediated stimulation. Stimulation of chromaffin cells by acetylcholine produced a high turnover of phosphatidylcholine, as suggested by the release of [3H]choline derived from [3H]-phosphatidylcholine in experiments performed with [3H]choline chloride-prelabeled cells. An enhanced breakdown of phosphatidylcholine was also inferred from the finding of an increased formation of [3H]diacylglycerol in chromaffin cells prelabeled with [3H]glycerol. The diacylglycerol mass that accumulated after stimulation showed a distinct temporal course and seemed to exceed the mass that has been reported to be derived from phosphatidylinositol. In keeping with the purported origin from phosphatidylcholine, diacylglycerol showed a high content in [3H]oleate molecular species. Phospholipase D activity measurements and experiments performed in the presence of propranolol (an inhibitor of phosphatidic acid:phosphohydrolase) suggested that phosphatidylcholine is hydrolyzed by a phospholipase D activity, producing phosphatidic acid, which is subsequently degraded to diacylglycerol, rather than by a phospholipase C. Incubation of chromaffin cells in the presence of atropine before addition of acetylcholine showed complete inhibition of the increased formation of [3H]-diacylglycerol, whereas d-tubocurarine failed to do so. Taken together, these results suggest that acetylcholine activates phosphatidylcholine breakdown and diacylglycerol formation in chromaffin cells via a muscarinic-type receptor.     

The UDP-Glc:glycoprotein glucosyltransferase is a soluble protein of the endoplasmic reticulum.

N-linked oligosaccharides devoid of glucose residues are transiently glucosylated directly from UDP-Glc in the endoplasmic reticulum. The reaction products have been identified, depending on the organisms, as protein-linked Glc1Man5-9GlcNAc2. Incubation of right side-sealed vesicles from rat liver with UDP-[14C]Glc, Ca2+ ions and denatured thyroglobulin led to the glucosylation of the macromolecule only when the vesicles had been disrupted previously by sonication or by the addition of detergents to the glucosylation mixture. Similarly, maximal glucosylation of denatured thyroglobulin required disruption of microsomal vesicles isolated from the protozoan Crithidia fasciculata. Treatment of the rat liver vesicles with trypsin led to the inactivation of the UDP-Glc:glycoprotein glucosyltransferase only when proteolysis was performed in the presence of detergents. The glycoprotein glucosylating activity could be solubilized upon sonication of right side-sealed vesicles in an isotonic medium, upon passage of them through a French press or by suspending the vesicles in an hypotonic medium. Moreover, the enzyme appeared in the aqueous phase when the vesicles were submitted to a Triton X-114/water partition. Solubilization was not due to proteolysis of a membrane-bound enzyme. The enzyme could also be solubilized from C. fasciculata microsomal vesicles by procedures not involving membrane disassembly. About 30% of endogenous glycoproteins glucosylated upon incubation of intact rat liver microsomal vesicles with UDP-[14C]GLc could be solubilized by sonication or by suspending the vesicles in 0.1 M Na2CO3. These and previous results show that the UDP-Glc:glycoprotein glucosyltransferase is a soluble protein present in the lumen of the endoplasmic reticulum. In addition, both soluble and membrane-bound glycoproteins may be glucosylated by the glycoprotein glucosylating activity.     

Myelin Lipids in the Developing Cerebrum, Cerebellum, and Brain Stem of Normal and Undernourished Children

Abstract: The developmental lipid profiles in the human cerebrum, cerebellum and brain stem are presented, with special reference to galactolipids as myelin markers to trace myelination in the three main parts of the human CNS. A group of undernourished children were also studied to test the vulnerability of myelinogenesis in the different regions of the human brain. Myelination was well advanced in the brain stem with regard to the other brain regions, a fact reflected in the much higher concentration of myelin lipids in the brain stem of the human foetus of 26 weeks of gestational age. The cerebrum, on the other hand, had the lowest galactolipid concentration during the prenatal period, galactolipid levels in the cerebellum being four times higher. From just before the end of gestation the accretion of galactolipids accelerated enormously in the cerebrum, whereas it slowed down considerably in the cerebellum. Consequently, in relation to prenatal levels galactolipids increased most rapidly in the cerebrum, followed by the cerebellum and finally by the brain stem. These regional differences were in clear contrast to data from the rat, as was the finding that only the cerebrum of undernourished children had a galactolipid concentration significantly decreased with respect to normal values. A relationship between the different myelination patterns in the human and the rat and the distinct vulnerability of myelinogenesis in the two species is suggested.     

Ant community composition and functional traits in new grassland strips within agricultural landscapes

1. Ongoing intensification and fragmentation of European agricultural landscapes dramatically reduce biodiversity and associated functions. Enhancing perennial noncrop areas holds great potential to support ecosystem services such as ant‐mediated pest control.
2. To study the potential of newly established grassland strips to enhance ant diversity and associated functions, we used hand collection data and predation experiments to investigate differences in (a) ant community composition and (b) biocontrol‐related functional traits, and (c) natural pest control across habitats in cereal fields, old grasslands, and new grassland transects of three years of age.
3. Ant species diversity was similar between new and old grasslands, but significantly higher in new grasslands than in surrounding cereal fields. Contrary, ant community composition of new grasslands was more similar to cereal fields and distinct from the species pool of old grasslands. The functional trait space covered by the ant communities showed the same distribution between old and new grasslands. Pest control did not differ significantly between habitat types and therefore could not be linked to the prevalence of functional ant traits related to biocontrol services in new grasslands.
4. Our findings not only show trends of convergence between old and new grasslands, but also indicate that enhancing ant diversity through new grasslands takes longer than three years to provide comparable biodiversity and functionality.
5. Synthesis and applications: Newly established grasslands can increase ant species richness and abundance and provide a consistent amount of biocontrol services in agroecosystems. However, three years after their establishment, new grasslands were still dominated by common agrobiont ant species and lacked habitat specialists present in old grasslands, which require a constant supply of food resources and long colony establishment times. New grasslands represent a promising measure for enhancing agricultural landscapes but must be preserved in the longer term to promote biodiversity and resilience of associated ecosystem services.