Biomedical subjects

R Van Antwerpen

Publications and source records attributed to R Van Antwerpen.

10 recordsLinked to original sources

PubMed1998

Evidence for a diazepam-binding inhibitor (DBI) benzodiazepine receptor-like mechanism in ecdysteroidogenesis by the insect prothoracic gland.

The diazepam-binding inhibitor (DBI) is a 10-kDa highly evolutionarily conserved multifunctional protein. In mammals, one of DBI's functions is in the activation of steroid hormone biosynthesis via binding to a specific outer mitochondrial membrane receptor (benzodiazepine receptor, BZD) and promoting cholesterol transport to the inner membrane. In this work, a multitiered approach was utilized to study the role of this receptor-like activity in ecdysteroidogenesis by larval insect prothoracic glands (PGs). First, both DBI protein and messenger RNA (mRNA) levels were correlated with peak PG ecdysteroid production. In vitro ecdysteroid production was stimulated by the diazepam analogue FGIN 1-27 and inhibited anti-DBI antibodies. The DBI protein was found distributed throughout PG cells, including regions of dense mitochondria, supposed subcellular sites of ecdysteroid synthesis. Finally, a potential mitochondrial BZD receptor in PG cells was demonstrated by photoaffinity labeling. These results suggest an important role for the insect DBI in the stimulation of steroidogenesis by prothoracic glands and indicate that a pathway for cholesterol mobilization leading to the production of steroid hormones appears to be conserved between arthropods and mammals.

Animals↗

PubMed1996

A new type of highly polymerized yolk protein from the cochineal insect Dactylopius confusus.

A female specific protein was isolated from eggs and female hemolymph of cochineal insects, using density gradient ultracentrifugation, ammonium sulfate precipitation, and size exclusion column chromatography. The protein was found to consist of four different subunits with apparent molecular weights (Mr) 45,000, 49,000, 53,000, and 56,000, respectively. All four subunits were found to be glycosylated; no association of lipids was detected. Size exclusion column chromatography and non-denaturing polyacrylamide gel electrophoresis demonstrated that the native yolk protein exists as large polymers. Electron microscopy showed that these molecules are long, helical ribbons of variable size which are found in both hemolymph and eggs. Using cryo-electron microscopy, it was shown that the ribbons were 14.6 +/- 1.5 nm wide; the helix they form has a repeat distance of 104.9 +/- 11.3 nm and a diameter of 42.1 +/- 5 nm. A clear substructure of the ribbons was recognized. The newly identified protein is the major yolk protein of Dactylopius confusus and no other proteins resembling the more familiar vitellins of other insect species were detected. Moreover, the D. confusus yolk protein appears to be unique both in its subunit structure and in its polymerizing qualities. Thus, the cochineal yolk protein (CYP) is suggested to represent a new type of insect yolk protein.

Amino Acid Sequence↗

PubMed1994

Cryo-electron microscopy reveals human low density lipoprotein substructure.

In the present study, we have examined the structure of human low density lipoprotein (LDL) using cryo-electron microscopy. Human LDL particles were analyzed in a vitrified frozen-hydrated condition, without chemical fixation or any form of staining. Hence, the lipoproteins were visualized close to their native state. Contrary to current spherical models, the overall shape of human LDL is indicated to be discoidal. The observed LDL disks have a diameter of 21.4 +/- 1.3 nm and a height of 12.1 +/- 1.1 nm (mean +/- standard deviation). The average volume of LDL particles in cryo-electron microscopic preparations is estimated to be 4352 nm3. This value corresponds well with the LDL volume that has been determined by sedimentation equilibrium studies [4130-4803 nm3; Kahlon et al., 1982. Lipids. 17: 323-330]. Details of LDL ultrastructure, visible as a result of local differences in mass density, are indicated to reflect the distribution of protein within the lipoprotein particle. Thus, apolipoprotein B-100 (apoB) appears to form two ring-shaped structures that are organized around the perimeter of the LDL disk.

Freezing↗

PubMed1993

Protein and lipoprotein uptake by developing oocytes of the hawkmoth Manduca sexta. An ultrastructural and immunocytochemical study.

The ultrastructure of developing Manduca sexta oocytes is described with respect to the endocytic pathway for protein incorporation. Three major (lipo) protein components of mature M. sexta eggs, lipophorin, vitellogenin and microvitellogenin, were localized along this pathway by immuno-fluorescence and immuno-gold labeling techniques. Labeling of the antigens was observed in the extracellular spaces of the follicle. In those cases where fixation and en bloc staining procedures did not destroy antigenicity, antigens were detected in coated pits and coated vesicles near the plasma membrane of the oocyte. All three antigens were demonstrated to be present in endosomes in the cortex of the oocyte. Both the morphology and the labeling pattern of the endosomes indicate that this organelle is a compartment of uncoupling of receptor and ligand. Tubular elements at the surface of the endosome, interpreted to be involved in the recycling of receptors and membrane to the oocyte surface, were not labeled. Strong labeling of lipophorin, vitellogenin and microvitellogenin was observed in the developing yolk bodies, the main protein storage compartment of the oocyte. The uptake and storage of hemolymph proteins and lipoproteins by M. sexta oocytes is discussed in comparison with other insect and vertebrate endocytic systems.

Animals↗

PubMed1993

Immunocytochemical localization of a follicle specific protein of the hawkmoth Manduca sexta.

A follicle specific protein (FSP-I) from the hawkmoth Manduca sexta, has been localized in developing follicles by immuno-fluorescence and immuno-gold labeling techniques. At the light microscopical level, the protein was demonstrated to be present in both the basolateral and apical parts of follicular epithelial cells, as well as in clearly defined, spherical compartments in the cortex of the developing oocyte. Immuno-gold labeling at the electron microscopical level revealed the localization of FSP-I in endoplasmic compartments of the follicular epithelial cells, in the extracellular matrix of the follicle and in endocytic compartments of the oocyte. Our results indicate that M. sexta FSP-I is synthetized and secreted by the follicular epithelial cells, after which it is taken up by the developing oocyte through endocytic routes.

Animals↗

PubMed1992

Lipophorin lipase from the yolk of Manduca sexta eggs: identification and partial characterization.

In the hawkmoth Manduca sexta high density lipophorin from adult insects (HDLp-A) delivers lipids to developing oocytes. During this lipid delivery HDLp-A is taken up by the oocyte and converted to a very high density lipophorin (VHDLp), which is stored in protein storage granules (yolk bodies). A membrane-free lysate of isolated M. sexta yolk bodies was demonstrated to contain lipoprotein lipase activity that hydrolyses the diacylglycerol of HDLp-A. With HDLp-A as a substrate yolk body lipophorin lipase (YBLpL) activity was shown to be maximal between pH 9 and pH 9.5. NaCl concentration was optimal between 0.7 M and 1 M. YBLpL activity required neither bovine serum albumin nor calcium ions but appeared to be stimulated by 5 mM EDTA. Diisopropyl fluorophosphate effectively inhibited YBLpL activity, indicating the presence of a serine in the active site of the enzyme. The identified lipase activity co-eluted with lipophorins and vitellins from the yolk in the void volume of a Sephadex G-75 gel filtration column. This observation suggests that the lipase has a Mr of more than 80,000, or that the enzyme is associated with the lipoproteins. Incubation of HDLp-A with yolk body lysate converted HDLp-A to two classes of higher density lipophorins. The highest density lipophorins produced during this incubation approached the density of VHDLp as it is isolated from mature eggs. The possible role of YBLpL activity in the delivery of lipids to developing oocytes is discussed.

Animals↗

PubMed1990

Manduca sexta lipid transfer particle acts upon a lipoprotein to catalyze lipid and apoprotein disproportionation.

A novel reaction, catalyzed by Manduca sexta lipid transfer particle (LTP), transforms low density lipophorin (LDLp) into two distinct lipoprotein species. A population of LDLp particles serves as lipid donor or acceptor in LTP-catalyzed production of a very low density lipophorin (VLDLp) and a high density lipophorin (HDLp) product. The products result from facilitated net transfer of lipid mass from donor LDLp particles to acceptor LDLp particles. Transfer of apolipophorin III (apoLp-III) from donor to acceptor lipoprotein occurs during the reaction to produce a lipid- and apoLp-III-enriched VLDLp species and lipid- and apoLp-III-depleted HDLp species. The VLDLp produced in this in vitro reaction contains more lipid and apoLp-III than any previous lipophorin species reported and further demonstrates the scope of the lipid binding capacity of lipophorin. Lipid analysis and radiolabeling studies confirmed that unidirectional net transfer of lipid mass and apoLp-III from donor to acceptor occurs. When 3H-lipid-LDLp was used as substrate in the LTP-catalyzed disproportionation reaction the density distribution of radioactivity and protein provided evidence of vectorial transfer of diacylglycerol, phospholipid, and free fatty acids. Electron micrographs of the original LDLp population and of the LTP-induced product lipoprotein population provided further support for the interpretation derived from biochemical studies. This LTP-catalyzed disproportionation was observed only with apoLp-III-rich LDLp suggesting that the presence of increased amounts of this apoprotein dramatically affects the properties of the particle and appears to be directly related to the capacity of the lipoprotein to bind lipid.

Animals↗

PubMed1990

Interaction of lipophorin with the plasma membrane of locust flight muscles.

Binding of high-density lipophorin (HDLp) to a plasma membrane preparation of locust flight muscle tissue was studied using a radiolabelled ligand binding assay and ligand blotting techniques. Analysis at 33 degrees C of the concentration-dependent total binding of tritium-labelled HDLp ([3H]HDLp) to the membrane preparation revealed the presence of a single specific binding site with an equilibrium dissociation constant of Kd = 9 (+/- 2) X 10(-7) M and a maximal binding capacity of 84 (+/- 10) ng X (micrograms protein)-1. Unlabelled HDLp as well as unlabelled low-density lipophorin (LDLp) competed with [3H]HDLp for binding to the identified binding site. In addition, ligand blotting demonstrated that both HDLp and LDLp bind specifically to a 30-kDa protein in the plasma membrane preparation, suggesting the involvement of this protein in the binding of lipophorins to the isolated membranes. A possible relationship between the identified binding of lipophorins and the observed co-purification of lipophorin lipase activity with the plasma membranes is discussed.

Animals↗

PubMed1988

Immunocytochemical localization of lipophorins in the flight muscles of the migratory locust (Locusta migratoria) at rest and during flight.

Locust lipoproteins (lipophorins) were localized by indirect immunofluorescence- and immunogold labelling in cryosections of dorsolongitudinal flight muscles. Immunolabelling was performed with monoclonal antibodies against apolipoproteins epitopes that are exposed at the surfaces of the lipophorin particles. Both at rest and during flight, lipophorins were located only in the wider spaces of the extracellular matrix, in the basement membranes of the individual muscle fibers and in the extracellular spaces that surround interfibrillar tracheoles. No internalization of lipophorins by the flight muscle cells was observed. Our results indicate that the unloading of lipophorins at the flight muscles is an extracellular event. Similarities with the vertebrate system of chylomicron and very-low-density lipoprotein degradation are discussed.

Animals↗

PubMed1984

A major human histone gene cluster on the long arm of chromosome 1.

A human histone gene cluster was assigned to chromosome 1 by Southern blot analysis of DNA's from a series of mouse-human somatic cell hybrids with 32P-labeled cloned human H4 and H3 histone DNA as probes. Localization of this histone gene cluster on the long arm of chromosome 1 was confirmed by in situ hybridization of this DNA probe to metaphase chromosomes.

Animals↗