Manfred Hüttinger

Heterogeneous lipoprotein (a) size isoforms differ by their interaction with the low density lipoprotein receptor and the low density lipoprotein receptor-related protein/α2-macroglobulin receptor

Lipoprotein (a) (Lp(a)) is a complex of low density lipoprotein (LDL) with apolipoprotein (apo) (a). To examine the size distribution of Lp(a), plasma was separated by fast flow gel filtration and Lp(a): B complexes were determined in the eluate by enzyme immunoassays, in which detection was performed with monoclonal antibodies specific for apoB Lp(a): B particles displayed apparent molecular masses (Mr ) of 2 × 106 to at least 10 × 106. Lp(a) size isoforms differed by the expression of apoB epitopes and their interaction with cultured human skin fibroblasts. LDL was more effective in inhibiting binding, uptake, and degradation of low Mr , Lp(a) than of high Mr Lp(a). In contrast, Glu‐plasmmogen, α2‐macroglobulin and tissue‐type plasminogen activator were more effective in competing for the cellular degradation of high Mr Lp(a) than of low Mr Lp(a). Ligand blotting revealed that Lp(a) bound to the low density lipoprotein receptor, the low density lipoprotein receptor‐related protein/α2‐macroglobulin receptor (LRP) and to two other endosomal membrane proteins. We propose that the LDL receptor preferentially internalizes low Mr Lp(a), whereas LRP may have a role in the clearance of high Mr Lp(a).

The low density lipoprotein receptor gene family. Differential expression of two alpha2-macroglobulin receptors in the brain

LR7/8B is a member of the low density lipoprotein receptor gene family that is specifically synthesized in the brain. Here we have functionally expressed in 293 cells the splice variant harboring eight ligand binding repeats (LR8B). As assessed by confocal microscopy, the expressed receptor is localized to the plasma membrane. Importantly, in cell binding experiments, we demonstrate that this protein is a receptor for activated α2-macroglobulin. Because to date low density lipoprotein receptor-related protein (LRP) has been shown to be the only α2-macroglobulin receptor in brain, we became interested in the expression pattern of both proteins at the cellular level in the brain. LR7/8B is expressed in large neurons and Purkinje cells of the cerebellum and in cells constituting brain barrier systems such as the epithelial cells of the choroid plexus, the arachnoidea, and the endothelium of penetrating blood vessels. Anti-LR7/8B antibody stains the plasma membrane, dendrites, and vesicular structures close to the cell membrane of neurons, especially of Purkinje cells. In contrast, LRP is present in patchy regions around large neurons and most prominently in the glomeruli of the stratum granulare of the cerebellum. This suggests that, contrary to LR7/8B, LRP is expressed in synaptic regions of the neurons; furthermore, there is a striking difference in the expression patterns of LR7/8B and LRP in the choroid plexus. Whereas LRP shows baso-lateral and apical localization in the epithelial cells, LR7/8B is restricted to the apical cell aspect facing the cerebrospinal fluid. Finally, these studies were extended to cultured primary rat neurons, where double immunofluorescence labeling with anti-LR7/8B and anti-microtubuli-associated protein 2 (MAP2) confirmed the somatodendritic expression of the receptor. Based upon these data, we propose that LR7/8B is involved in the clearance of α2-macroglobulin·proteinase complexes and/or of other substrates bound to α2-macroglobulin from the cerebrospinal fluid and from the surface of neurons.

Effect of clofibrate application on morphology and enzyme content of liver peroxisomes.

SummaryMale albino rats (Sprague Dawley) were fed for 2–6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase.Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to “normal” sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes.

Effects of apolipoprotein E (apoE) isoforms, β-amyloid (Aβ) and apoE/Aβ complexes on Protein Kinase C-α (PKC-α) translocation and amyloid precursor protein (APP) processing in human SH-SY5Y neuroblastoma cells and fibroblasts

We investigated the effects of different apolipoprotein E (apoE) isoforms, Abeta (1-42), and apoE/Abeta complexes on PKC-alpha translocation and APP processing in human SH-SY5Y neuroblastoma cells and fibroblasts. Treatment of cells with either 10 nM apoE3 or apoE4, 10 microM Abeta (1-42), or apoE/Abeta complexes induced significant translocation of PKC-alpha in both cell types. Effects were seen using both human recombinant apoE and apoE loaded into beta-very low density lipoprotein (beta-VLDL) particles. Time course (5-24 h) studies of APP processing revealed that some conditions induced transient or moderate increases in the secretion of proteins detected by 22C11. In contrast, the secretion of alpha-secretase cleaved APP was either not modified or transiently decreased, as determined by immunoblotting with the antibody 6E10. These results suggest that apoE, Abeta (1-42) and apoE/Abeta complexes can modulate PKC activity but do not have major consequences for APP processing. These effects could contribute to the reported PKC alterations seen in AD. However, it is unlikely that the contribution of different apoE isoforms to AD pathology occurs via effects on APP processing.

β-oxidation in peroxisomes of brown adipose tissue

Peroxisomes and mitochondria from brown adipose tissue of the rat were separated by differential pelleting and isopycnic gradient centrifugation. Both fractions oxidized palmitoyl-CoA with comparable specific activities. Unlike the mitochondrial beta-oxidation the peroxisomal activity was not influenced by carbon monoxide. Peroxisomal beta-oxidation together with carnitine acetyl-transferase, which is also located in peroxisomes, might be involved in chemical thermogenesis by delivering acetyl groups to the mitochondria.

Catalase-positive particles (“microperoxisomes”) from rat preputial gland and bovine adrenal cortex☆

Abstract Catalase activity was detected histochemically within membrane-bound cell organelles in epithelial cells of rat preputial gland and bovine adrenal cortex. These particles are oval to worm-like in rat preputial gland, 0.08 – 0.15 μm thick and up to 1.0 μm long. In bovine adrenal cortex the shape of catalase-positive particles is rather spherical (diameter 0.1 to 0.3 μm). Particles of both organs lack crystalline or dense cores. Biochemical examination of cell fractions prepared from tissue homogenates by differential centrifugation revealed the presence of two typical peroxisomal oxidases, viz. α-hydroxy acid and d -amino acid oxidase, with maximal relative specific activities in the ‘microsomal’ fraction (preputial gland) and in the ‘lysosomal’ fraction (adrenal cortex), respectively. Urate oxidase is absent in both tissues. The concomitant occurrence of catalase and hydrogen peroxide producing oxidases in the particles described characterizes them as true peroxisomal systems (‘microperoxisomes’).

Enzymic and morphological studies on catalase positive particles from brown fat of cold adapted rats

SummaryBrown adipose tissue of normal and cold-adapted adult rats has been investigated morphologically and cytochemically. In thin sections catalase-positive particles appear as circular, oval or elongated profiles lying either as single particles or forming groups. Biochemical studies on peroxisomal enzymes show an increase of catalase activity to the tenfold amount after cold adaptation. The tissue is devoid of D-aminoacid oxidase and glycolate oxidase, while low activities of middle-chain α-hydroxyacid oxidases could be detected. The catalase-positive particles were purified by differential and isopycnic gradient centrifugation. The density of the particles (1.20 g/cm3) is lower than that of the liver peroxisomes. Enzymic investigations of the fractions render it probable that particles contain carnitine acetyltransferase, whereas they are lacking NAD-dependent glycerophosphate dehydrogenase. The pellets derived from the gradient centrifugation have been checked morphologically for purity. After performing DAB-cytochemistry for identification of the peroxidatic activity of catalase, most of the particles were shown to be structurally intact and homogeneously filled with reaction product.

β-VLDL protects against Aβ(1-42) and apoE toxicity in human SH-SY5Y neuroblastoma cells

The toxic effects of β-amyloid (Aβ) (1-42), apolipoprotein E (apoE) isoforms, and apoE/Aβ complexes were studied in human SH-SY5Y neuroblastoma cells and fibroblasts using MTT reduction. In SH-SY5Y cells, Aβ(1-42) gave time-dependent toxicity over 2–48 h, which was reduced by co-incubation with rabbit β-very low density lipoproteins (β-VLDL). Human recombinant apoE3 and E4 isoforms were also toxic by themselves and also potentiated Aβ effects when used alone, but not when associated with β-VLDL. None of the treatments were toxic to human fibroblasts. These results suggest that β-VLDL has a protective role on Aβ-induced neurotoxicity and that the status of apoE or the conformation of lipoprotein containing apoE particles may be important for determining the contribution of apoE to neurodegeneration.

Preparation and characterization of catalase-positive particles ('microperoxisomes') from Harder's gland of the rat.

Abstract Catalase-positive particles (diameter 0.1–0.3 μm) from Harders gland of the rat were prepared by differential centrifugation. It was demonstrated that these particles do not contain the oxidases thought to be characteristic of peroxisomal systems (i.e. urate oxidase, d -amino acid oxidase, and α-hydroxy acid oxidase). Cytochemical DAB reaction was employed to demonstrate the organelles in the gland tissue and in subcellular fractions by electron microscopy.

Catalase positive particles from pig lung

SummaryIn pig lung tissue catalase positive particles (CPs) are abundant especially in type II pneumocytes and in Clara cells.In both cell types they occur circular, oval or elongated membrane profiles surrounding a moderately electron dense matrix lacking a crystalline core. In Clara cells and in part of type II pneumocytes they are located as individual particles without any evident morphological relation to other cell organelles. In part, of type II pneumocytes 5–8 particles are forming a group and their close relation to agranular endoplasmic reticulum cisterns is evident. The particles can be purified from lung homogenates by fractionated pelleting and subsequent rate sedimentation in a sucrose gradient using a zonal rotor. The catalase rich fraction bands in the middle of the gradient whereas cytochrome oxidase and part of the acid phosphatase sediments at its heavy end. A second part of acid phosphatase stays at the light end of the gradient and — according to morphological control — seems to correspond to lamellar bodies of the type II pneumocytes. The purified catalase positive particles do not contain hydroxyacid and d-aminoacid oxidases thought to be characteristic H2O2 producing enzymes of peroxisomal systems. The buoyant density of the particles (d=1.195 g/cm3) is lower than that of liver peroxisomes.Cytochemical controls of the peroxisomal pellets exhibit the particles partly uniformly filled with reaction product, partly irregularly stained.