Intracellular transport of endocytosed chylomicron [3H]retinyl ester in rat liver parenchymal cells. Evidence for translocation of a [3H]retinoid from endosomes to endoplasmic reticulum

The intracellular transport of chylomicron remnants labeled with [3H]retinyl ester was studied in rat liver parenchymal cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. The data presented indicate that endocytosed chylomicron remnant [3H]retinyl ester initially is located in low density endosomes. Radioactivity is subsequently transferred to a denser vesicle. Equilibrium as well as rate zonal centrifugation suggest that this denser [3H] retinoid-containing vesicle may represent endoplasmic reticulum. We have compared the intracellular transport of chylomicron remnant [3H]retinyl ester and 125I-asialofetuin. The receptor-mediated endocytosis of asialoglycoproteins in rat liver parenchymal cells is a thoroughly studied system. Our results suggest that the [3H] retinoid and 125I-asialofetuin follow the same path initially to the endosomes. After transit in endosomes, the intracellular transport differs. While asialofetuin is transported to the lysosomes, the retinoid is probably transferred to the endoplasmic reticulum.     

Murine Cytomegalovirus m02 Gene Family Protects against Natural Killer Cell-Mediated Immune Surveillance

The murine cytomegalovirus m02 gene family encodes putative type I membrane glycoproteins named m02 through m16. A subset of these genes were fused to an epitope tag and cloned into an expression vector. In transfected and murine cytomegalovirus-infected cells, m02, m04, m05, m06, m07, m09, m10, and m12 localized to cytoplasmic structures near the nucleus, whereas m08 and m13 localized to a filamentous structure surrounding the nucleus. Substitution mutants lacking the m02 gene (SMsubm02) or the entire m02 gene family (SMsubm02-16) grew like their wild-type parent in cultured cells. However, whereas SMsubm02 was as pathogenic as the wild-type virus, SMsubm02-16 was markedly less virulent. SMsubm02-16 produced less infectious virus in most organs compared to wild-type virus in BALB/c and C57BL/6J mice, but it replicated to wild-type levels in the organs of immunodeficient gamma(c)/Rag2 mice, lacking multiple cell types including natural killer cells, and in C57BL/6J mice depleted of natural killer cells. These results argue that one or more members of the m02 gene family antagonize natural killer cell-mediated immune surveillance.     

Morphological and Gene Expression Changes in Cattle Embryos from Hatched Blastocyst to Early Gastrulation Stages after Transfer of In Vitro Produced Embryos

A detailed morphological staging system for cattle embryos at stages following blastocyst hatching and preceding gastrulation is presented here together with spatiotemporal mapping of gene expression for BMP4, BRACHYURY, CERBERUS1 (CER1), CRIPTO, EOMESODERMIN, FURIN and NODAL. Five stages are defined based on distinct developmental events. The first of these is the differentiation of the visceral hypoblast underlying the epiblast, from the parietal hypoblast underlying the mural trophoblast. The second concerns the formation of an asymmetrically positioned, morphologically recognisable region within the visceral hypoblast that is marked by the presence of CER1 and absence of BMP4 expression. We have termed this the anterior visceral hypoblast or AVH. Intra-epiblast cavity formation and the disappearance of the polar trophoblast overlying the epiblast (Rauber’s layer) have been mapped in relation to AVH formation. The third chronological event involves the transition of the epiblast into the embryonic ectoderm with concomitant onset of posterior NODAL, EOMES and BRACHYURY expression. Lastly, gastrulation commences as the posterior medial embryonic ectoderm layer thickens to form the primitive streak and cells ingress between the embryonic ectoderm and hypoblast. At this stage a novel domain of CER1 expression is seen whereas the AVH disappears. Comparison with the mouse reveals that while gene expression patterns at the onset of gastrulation are well conserved, asymmetry establishment, which relies on extraembryonic tissues such as the hypoblast and trophoblast, has diverged in terms of both gene expression and morphology.     

Steroids excreted in urine by neonates with 21-hydroxylase deficiency: Characterization, using GC-MS and GC-MS/MS, of the D-ring and side chain structure of pregnanes and pregnenes

Steroid metabolites in urine from neonates with 21-hydroxylase deficiency are predominantly polyhydroxylated 17-hydroxyprogesterone and androgen metabolites, and most have incompletely defined structure. This study forms part of a comprehensive project to characterize and identify these in order to enhance diagnosis and to further elucidate neonatal types of steroid metabolism.Steroids were analyzed, after extraction and enzymatic conjugate hydrolysis, as methyloxime-trimethylsilyl ether derivatives on gas-chromatographs coupled to quadrupole and ion-trap mass-spectrometers. GC-MS and GC-MS/MS spectra, obtained with constant excitation conditions, were used together to determine the structure of the D-ring and the side chain of 20-oxo and 20-hydroxy pregnane(ene)s without oxo groups on the A-, B-, and C-ring.All possible combinations of D-ring and side chain configuration were considered. Most fragmentations could be interpreted as partial or complete D-ring cleavages with loss of the side chain, aided by comparison with spectra of deuterated derivatives and of borohydride reduced metabolites. Possible rearrangement ions are also discussed. More than 140 endogenous metabolites were characterized.GC-MS/MS was especially beneficial for characterization of compounds with 16,17-dihydroxy-20-oxo structure, interpreted as markers of intra-uterine enzyme induction. It also assisted the differentiation of 16-hydroxy-20-oxo metabolites, present in urine of non-affected neonates, from the diagnostic 17-hydroxy-20-oxosteroids and enabled the detection of 15,17-dihydroxy-20-oxo compounds in low concentrations. The presence of 17,21-dihydroxylated pregnane(ene)s despite the deficit in CYP21A2 is discussed.We conclude that GC-MS combined with GC-MS/MS allows reliable identification of the structure of the D-ring and side chain of pregnane(ene)s without prior isolation, even when in low concentrations in urine.     

Cloning of genes that suppress an Escherichia coli K-12 alanine auxotroph when present in multicopy plasmids.

To facilitate molecular analyses of a previously uncharacterized gene involved in alanine synthesis, attempts were made to clone the wild-type allele of this gene, alaA, with a mini-Mu plasmid element used for in vivo cloning. Seventy-six independent Ala+ plasmids were isolated and characterized. Physiological, enzymological, and restriction endonuclease analyses indicated that three different genes, none of them alaA, were cloned. These genes were avtA+, which encodes the alanine-valine transaminase (transaminase C); tyrB+, which encodes the tyrosine-repressible transaminase (transaminase D); and a previously undescribed gene, called alaB, which encodes an alanine-glutamate transaminase.     

Directional flow of sensillum liquor in blowfly (Phormia regina) labellar chemoreceptors.

Utilizing horseradish peroxidase as a tracer protein, it is shown that trichogen and tormogen cells have a secretory function. Protein tracer from the haemolymph enters these cells by endocytosis and is transported to the sensillum liquor cavity by transport vacuoles and multivesicular bodies. It is also suggested that closely associated pigment cells may be involved in macromolecular transport.     

Novel α-L-Fucosidases from a Soil Metagenome for Production of Fucosylated Human Milk Oligosaccharides

This paper describes the discovery of novel α-L-fucosidases and evaluation of their potential to catalyse the transglycosylation reaction leading to production of fucosylated human milk oligosaccharides. Seven novel α-L-fucosidase-encoding genes were identified by functional screening of a soil-derived metagenome library and expressed in E. coli as recombinant 6xHis-tagged proteins. All seven fucosidases belong to glycosyl hydrolase family 29 (GH 29). Six of the seven α-L-fucosidases were substrate-inhibited, moderately thermostable and most hydrolytically active in the pH range 6–7, when tested with para-nitrophenyl-α-L-fucopyranoside (pNP-Fuc) as the substrate. In contrast, one fucosidase (Mfuc6) exhibited a high pH optimum and an unusual sigmoidal kinetics towards pNP-Fuc substrate. When tested for trans-fucosylation activity using pNP-Fuc as donor, most of the enzymes were able to transfer fucose to pNP-Fuc (self-condensation) or to lactose. With the α-L-fucosidase from Thermotoga maritima and the metagenome-derived Mfuc5, different fucosyllactose variants including the principal fucosylated HMO 2’-fucosyllactose were synthesised in yields of up to ~6.4%. Mfuc5 was able to release fucose from xyloglucan and could also use it as a fucosyl-donor for synthesis of fucosyllactose. This is the first study describing the use of glycosyl hydrolases for the synthesis of genuine fucosylated human milk oligosaccharides.