Biomedical subjects

R Linnemann

Publications and source records attributed to R Linnemann.

9 recordsLinked to original sources

PubMed2000

Cardiovascular overexpression of transforming growth factor-beta(1) causes abnormal yolk sac vasculogenesis and early embryonic death.

Transforming growth factor-beta(1) (TGF-beta(1)) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-beta(1) expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-beta(1) in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-beta(1) is expressed from the SM22alpha promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22alpha-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22alpha-beta-galactosidase transgene. SM22alpha- beta-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22alpha-TGF-beta(1) transgenic mice died before E11.5. SM22alpha-TGF-beta(1) transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-beta(1) from the SM22alpha promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-beta(1) expression in adults may require a conditional transgenic approach. Moreover, because the SM22alpha promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22alpha promoter to drive expression of "vasculoactive" transgenes may be particularly likely to cause embryonic death.

Animals↗

PubMed2000

Expression of Fas ligand in arteries of hypercholesterolemic rabbits accelerates atherosclerotic lesion formation.

Fas ligand (FasL) is expressed by cells of the arterial wall and is present in human atherosclerotic lesions. However, the role of FasL in modifying the initiation and progression of atherosclerosis is unclear. To investigate the role of arterial FasL expression in the development of atherosclerosis, we first established a model of primary lesion formation in rabbit carotid arteries. In this model, infusion of adenoviral vectors into surgically isolated, nondenuded arteries of hypercholesterolemic rabbits leads to the formation of human-like early atherosclerotic lesions. Expression of FasL in arterial endothelium in this model decreased T-cell infiltration and expression of vascular cell adhesion molecule-1 but did not affect expression of intercellular adhesion molecule-1. Intimal lesions grew more rapidly in FasL-transduced arteries than in arteries transduced with a control adenovirus that did not express a transgene. Total intimal macrophage accumulation was increased in FasL-transduced arteries; however, the proportion of lesion area occupied by macrophages was not elevated. The accelerated lesion growth was primarily due to the accumulation of intimal smooth muscle cells with a synthetic proliferative phenotype. There was no significant apoptosis in FasL-transduced or control arteries and no granulocytic infiltrates. Thus, the net result of elevated FasL expression is to accelerate atherosclerotic lesion growth by increasing lesion cellularity. Vascular expression of FasL may contribute to the progression of atherosclerosis.

Adenoviridae↗

PubMed1997

Endocytosis and retrograde transport of pertussis toxin to the Golgi complex as a prerequisite for cellular intoxication.

The uptake mechanism of pertussis toxin (PT) in CHO and insulin-producing HIT-T15 cells was studied. By electron microscopy after direct labeling of the toxin with gold particles, PT was found to be taken up by receptor-mediated endocytosis. The presence of active pertussis toxin in the Golgi complex was shown by subcellular fractionation. The importance of the Golgi localization of pertussis toxin for the S1-dependent ADP-ribosylation of G-proteins was investigated employing Brefeldin A (BFA) treatment to disrupt Golgi structures. Treatment with Brefeldin A completely blocked the pertussis toxin mediated ADP-ribosylation of cellular G-proteins in CHO and HIT-T15 cells, whereas the BFA-resistant MDCK cells were not protected. A mutant CHO cell line (V24.1) exhibiting a temperature-sensitive Golgi complex could be protected when grown at restrictive conditions. These results strongly indicate that retrograde transport to the Golgi network is a necessary prerequisite for pertussis toxin mediated ADP-ribosylation of G-proteins and thus also for cellular intoxication.

Adenosine Diphosphate Ribose↗

PubMed1995

Identification of binding proteins for pertussis toxin on pancreatic beta cell-derived insulin-secreting cells.

The ability of pertussis toxin (PT) to recognize and bind to surface proteins on cells derived from pancreatic insulin-secreting beta cells and alpha cell-like glucagon-producing cells was investigated employing HIT-T15 (beta cell-derived) and In-R1-G9 (alpha cell-like) cell lines. PT recognition of membrane binding proteins on HIT-T15 and In-R1-G9 cells was first assessed with immunofluorescence microscopy in tissue culture. Both cell lines were equally well recognized by PT. N-octylglucoside extracts of whole cells and isolated membranes were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. PT, the B-oligomer, or the isolated PT dimers S2-S4 and S3-S4 recognized distinct proteins in HIT-T15 and In-R1-G9 cells of about 220 kDa. Recognition by the sialic acid specific Sambucus nigrica lectin identified these proteins as sialoglycoproteins. Incubation of the blotted membrane proteins with sialidase or pretreatment of PT with anti-PT polyclonal antibodies abolished the recognition and binding of these proteins by PT. To demonstrate that these glycoproteins are also able to transduce PT mediated effects and thus might serve as PT binding proteins, the stimulation of insulin secretion in HIT-T15 cells was assessed. As the secretion of insulin in HIT-T15 cells increased about 30% upon interaction with PT it was concluded that these glycoproteins are indeed functional as PT receptors.

Animals↗

PubMed1986