Heinz Hohenberg

Brown adipose tissue activity controls triglyceride clearance

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.

High-pressure freezing of cell suspensions in cellulose capillary tubes.

A procedure for efficient cryoimmobilization of large volumes of cell suspensions or micro‐organisms by high‐pressure freezing is described. This procedure uses transparent, porous cellulose capillary tubes with an inner diameter of 200 μm, into which the suspensions are drawn by capillary action. The tubes are processed by high‐pressure freezing and freeze‐substitution as if they were tissue samples. Centrifugation of suspensions at low temperatures is no longer necessary and cryopreparation is greatly facilitated.

A conformational switch controlling HIV‐1 morphogenesis

Assembly of infectious human immunodeficiency virus type 1 (HIV‐1) proceeds in two steps. Initially, an immature virus with a spherical capsid shell consisting of uncleaved Gag polyproteins is formed. Extracellular proteolytic maturation causes rearrangement of the inner virion structure, leading to the conical capsid of the infectious virus. Using an in vitro assembly system, we show that the same HIV‐1 Gag‐derived protein can form spherical particles, virtually indistinguishable from immature HIV‐1 capsids, as well as tubular or conical particles, resembling the mature core. The assembly phenotype could be correlated with differential binding of the protein to monoclonal antibodies recognizing epitopes in the HIV‐1 capsid protein (CA), suggesting distinct conformations of this domain. Only tubular and conical particles were observed when the protein lacked spacer peptide SP1 at the C‐terminus of CA, indicating that SP1 may act as a molecular switch, whose presence determines spherical capsid formation, while its cleavage leads to maturation.

Real-time magnetic resonance imaging and quantification of lipoprotein metabolism in vivo using nanocrystals

Semiconductor quantum dots and superparamagnetic iron oxide nanocrystals have physical properties that are well suited for biomedical imaging. Previously, we have shown that iron oxide nanocrystals embedded within the lipid core of micelles show optimized characteristics for quantitative imaging. Here, we embed quantum dots and superparamagnetic iron oxide nanocrystals in the core of lipoproteins--micelles that transport lipids and other hydrophobic substances in the blood--and show that it is possible to image and quantify the kinetics of lipoprotein metabolism in vivo using fluorescence and dynamic magnetic resonance imaging. The lipoproteins were taken up by liver cells in wild-type mice and displayed defective clearance in knock-out mice lacking a lipoprotein receptor or its ligand, indicating that the nanocrystals did not influence the specificity of the metabolic process. Using this strategy it is possible to study the clearance of lipoproteins in metabolic disorders and to improve the contrast in clinical imaging.

Self-organization of dissolved organic matter to micelle-like microparticles in river water.

In aquatic systems, the concept of the ‘microbial loop’ is invoked to describe the conversion of dissolved organic matter to particulate organic matter by bacteria. This process mediates the transfer of energy and matter from dissolved organic matter to higher trophic levels, and therefore controls (together with primary production) the productivity of aquatic systems. Here we report experiments on laboratory incubations of sterile filtered river water in which we find that up to 25% of the dissolved organic carbon (DOC) aggregates abiotically to particles of diameter 0.4–0.8 micrometres, at rates similar to bacterial growth. Diffusion drives aggregation of low- to high-molecular-mass DOC and further to larger micelle-like microparticles. The chemical composition of these microparticles suggests their potential use as food by planktonic bacterivores. This pathway is apparent from differences in the stable carbon isotope compositions of picoplankton and the microparticles. A large fraction of dissolved organic matter might therefore be channelled through microparticles directly to higher trophic levels—bypassing the microbial loop—suggesting that current concepts of carbon conversion in aquatic systems require revision.

Involvement of actin filaments in budding of measles virus: Studies on cytoskeletons of infected cells

Cytoskeletons were prepared from measles virus infected HeLa cells to investigate the involvement of cytoskeletal filaments in virus budding at the plasma membrane. The cytoskeletons retained nearly 80% of measles virus hemagglutinin, the major viral polypeptides, including P, NP, and M, and 2 to 12% of the total cell bound infectivity. As demonstrated with platinum- and carbon-shadowed cytoskeletons, all stages of budding, i.e., virus specific strands, stub-like protrusions, and completely rounded virus particles, are associated with actin filaments composing the outer part of the cytoskeletal network. As shown with ultrathin sections of flat embedded extracted cells, actin filaments identified with heavy meromyosin almost exclusively protrude into virus particles with their barbed ends and are in close association with viral nucleocapsids. The data support previous suggestions that actin is involved in virus budding and show that budding itself is possibly the result of a vectorial growth of actin filaments.

Cytoplasmic retention of mutant tsp53 is dependent on an intermediate filament protein (Vimentin) scaffold

The temperature-sensitive mutant tsp53val135 accumulates in the cytoplasm of cells kept at the non-permissive temperature (39°C), but is rapidly transported into the cell nucleus at the permissive temperature (30°C). tsp53 thus may serve as a model for analysing cellular parameters influencing the subcellular location of p53. Here we provide evidence that retention of tsp53 in the cytoplasm at the non-permissive temperature is due to cytoskeletal anchorage of the p53 protein. Two sublines of C6 rat glioma cells differing in their expression of the intermediate filament protein vimentin (vimentin expressing or vimentin negative cells) were stably transfected with a vector encoding tsp53. Whereas cells of vimentin expressing C6 subclones retained tsp53 in the cytoplasm at the non-permissive temperature, cells of vimentin negative subclones exclusively harbored the tsp53 within their nuclei. Intermediate filament deficient cells that had been reconstituted with a full length vimentin protein again showed a cytoplasmic localization of tsp53, whereas in cells expressing a C-terminally truncated (tail-less) vimentin tsp53 localized to the nucleus. We conclude that cytoplasmic sequestration of tsp53 requires an intact intermediate filament system.

Protein-A gold particles as markers in replica immunocytochemistry: high resolution electron microscope investigations of plasma membrane surfaces.

Due to their high atomic number contrast in transmission electron microscopy, gold particles are ideal markers in surface replicas of cultured cells. The suitability of protein‐A‐coated gold particles in replica immunocytochemistry for labelling surface antigens is demonstrated using measles virus‐infected cells as a model system. Labelled areas can easily be distinguished from unlabelled areas, and even markers positioned in the evaporation shadow of large structures can be accurately identified, which is a prerequisite for an exact quantification and mapping of antigen. In addition, the ultrastructure of labelled areas can still be visualized because of the small size of the marker.

Inhibition of measles virus budding by phenothiazines

HeLa cells infected with measles virus show an accumulation of virus-specific strands at the plasma membrane after addition of the anticalmodulin drugs trifluoperazine (TFP) and chlorpromazine (CPZ), whereas spherical virus particles are almost completely absent. At low drug concentrations (10-15 microM TFP; 30-40 microM CPZ) the inhibitory effect is dependent on the presence of extracellular calcium. The strands complete the budding process after removal of the drugs. Restoration of virus budding is not sensitive to cycloheximide and immunoprecipitation experiments give evidence that the viral protein synthesis is not qualitatively altered in the presence of TFP. The data indicate that both drugs arrest the budding process at an intermediate stage at the plasma membrane. The inability of the strands to comigrate with cytochalasin B-induced actin patches suggests that the inhibition of budding is probably the result of an impaired interaction of viral structures with the cytoskeleton.

High‐pressure freezing and freeze‐substitution of native rat brain: Suitability for preservation and immunoelectron microscopic localization of myelin glycolipids

Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral cortex tissue, obtained by fine‐needle biopsy, was cryoimmobilized by high‐pressure freezing and dehydrated by freeze‐substitution before embedding in Epon. This procedure resulted in an excellent preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well‐defined monoclonal antibodies (mAbs) O1, O4, and R‐mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R‐mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this “postembedding glycolipid‐labeling technique” holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level. J. Neurosci. Res. 53:465–474, 1998.