Biomedical subjects

Friedrich Spener

Publications and source records attributed to Friedrich Spener.

28 records · Page 2Linked to original sources

PubMed2003

Specific localization of epidermal-type fatty acid binding protein in dendritic cells of splenic white pulp.

Dendritic cells in the splenic white pulp of mice were intensely immunoreactive for epidermal-type fatty acid binding protein (E-FABP). This specific immunostaining revealed a clear difference in morphology between the dendritic cells in the periarterial lymphoid sheath (PALS) and follicular dendritic cells in the follicles in terms of cell sizes and process branching. No immunoreactivity was detected in dendritic cells in the marginal zones and the red pulp, although endothelial cells of almost all capillaries in the red pulp were immunoreactive for E-FABP. After peritoneal injection of lipopolysaccharide, the immunoreactive cells in PALS progressively enlarged and became rounded in shape with a peak in size at 24 h postinjection and they eventually resumed the dendritic form at 48 h postinjection. Within each of the enlarged immunoreactive cell perikarya were included small immunonegative apoptotic cells, presumptive lymphocytes. Taken together, E-FABP is useful as a marker for dendritic cells in the splenic white pulp, and may be involved through combination with fatty acids in antigen presentation and retention as well as in cytokine production.

Animals↗

PubMed2003

Vitamin E deficiency reduces surfactant lipid biosynthesis in alveolar type II cells.

Reactive oxygen species play an important role in development of lung injury. Neonates exhibit a high risk of developing acute and/or chronic lung disorder, often associated with surfactant deficiency, and in parallel they show low vitamin E concentration. We investigated whether the vitamin E status of adult rats affects the content of phospholipids (PL) in bronchoalveolar lavage and alveolar type II cells. Phosphatidylcholine (PtdCho) is the dominant and functional most important PL in lung surfactant. Therefore, we determined its formation via de novo synthesis and reacylation of lyso-PtdCho in type II cells. Vitamin E depletion caused a decrease of PL content in bronchoalveolar lavage and type II cells and decreased glycerol-3-phosphate O-acyltransferase (G3P-AT) activity, de novo synthesis of PtdCho, and reacylation of lyso-PtdCho in type II cells. Preincubation of type II cell homogenates with dithiothreitol restored the activity of G3P-AT and de novo synthesis but inhibited reacylation. Reacylation was strongly reduced by chelerythrine-mediated inhibition of protein kinase C. We conclude that antioxidant and PKC-modulating properties of vitamin E regulate de novo synthesis of PtdCho and reacylation of lyso-PtdCho in alveolar type II cells. Vitamin E depletion reduced the two pathways of PL synthesis and caused a decrease of PL content in alveolar surfactant of rats.

Acylation↗

PubMed2003

Induction of epidermal fatty acid binding protein in intravascular monocytes of renal allografts.

During acute rejection of rat renal allografts, numerous activated monocytes accumulate in the vasculature of the graft. These monocytes seem to be involved in allograft destruction. Proinflammatory and effector functions of monocytes and macrophages can be down-regulated by peroxisome proliferators, which are probably transported in the cytoplasm by fatty acid binding proteins (FABPs). We performed renal transplantation in rats in the Dark Agouti-to-Lewis strain combination. Intravascular graft leukocytes were harvested 4 days posttransplantation. Epidermal (E)-FABP mRNA and protein expression were investigated by reverse-transcriptase polymerase chain reaction and immunoblotting, respectively. E-FABP-expressing cells were identified by immunofluorescence. After allogeneic transplantation, intravascular graft leukocytes expressed E-FABP mRNA and protein. In isografts, significantly lower expression levels were observed. E-FABP protein was detected in monocytes expressing ED1 and in alphabeta-T-cell receptor positive T lymphocytes. E-FABP might regulate monocyte activation and may represent a promising target for a therapeutic intervention in allograft rejection.

Animals↗

PubMed2002

Branched chain fatty acids induce nitric oxide-dependent apoptosis in vascular smooth muscle cells.

Clinical observations in patients with peroxisomal disorders and studies employing corresponding mouse models have shown that supraphysiological concentrations of dietary branched chain fatty acids (BCFAs) are associated with a high level of toxicity, which is poorly understood at present. Here we show that phytanic and pristanic acid, two BCFAs that are metabolized in peroxisomes, promote apoptosis in cultured vascular smooth muscle cells of human, rat, and porcine origin. Under the conditions used, the apoptosis-promoting effect of BCFAs was neither shared by saturated or unsaturated straight chain fatty acids nor by artificial peroxisome proliferators, which, like phytanic and pristanic acid, have been shown to activate the peroxisome proliferator-activated receptor alpha (PPARalpha). We could demonstrate, however, that BCFA induced tumor necrosis factor alpha (TNFalpha) activation and secretion, which is an obligatory step required for induction of apoptosis by BCFAs. Furthermore, incubation of VSMCs with BCFA increased inducible nitric-oxide synthase (iNOS) mRNA and protein concentrations markedly within 2 h of treatment. Correspondingly, apoptosis was significantly reduced when the cells were co-treated with the competitive NOS inhibitors monomethyl-L-arginine monoacetate and aminoguanidine. Moreover, co-incubation with TGFbeta1, previously shown to destabilize iNOS mRNA, also abolished apoptosis. These results establish a new signaling cascade in which natural BCFA induced NO-dependent apoptosis, which is apparently triggered by autocrine secretion of TNFalpha in cultured VSMCs.

Animals↗

PubMed2002

Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds.

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2002

Solution structure and backbone dynamics of human epidermal-type fatty acid-binding protein (E-FABP).

Human epidermal-type fatty acid-binding protein (E-FABP) belongs to a family of intracellular 14-15 kDa lipid-binding proteins, whose functions have been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to understanding the structure-function relationship, we report in the present study features of its solution structure and backbone dynamics determined by NMR spectroscopy. Applying multi-dimensional high-resolution NMR techniques on unlabelled and 15N-enriched recombinant human E-FABP, the 1H and 15N resonance assignments were completed. On the basis of 2008 distance restraints, the three-dimensional solution structure of human E-FABP was subsequently obtained (backbone atom root-mean-square deviation of 0.92+/-0.11 A; where 1 A=0.1 nm), consisting mainly of 10 anti-parallel beta-strands that form a beta-barrel structure. 15N relaxation experiments (T1, T2 and heteronuclear nuclear Overhauser effects) at 500, 600 and 800 MHz provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S(2)>0.8, with an average value of 0.88+/-0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the beta-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated in the present study differ markedly from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid-binding protein, implying a strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family.

Amino Acid Sequence↗

PubMed2002

Heterologous expression and characterization of recombinant purple acid phosphatase from red kidney bean.

Purple acid phosphatases (PAPs) are dinuclear metallohydrolases of widespread occurrence. In a first step to understand structure-function relationship of PAP from red kidney bean (kbPAP), we cloned its cDNA and functionally expressed the enzyme in insect cells. kbPAP cDNA encodes a protein of 459 amino acids with 99% identity to the published primary structure (T. Klabunde et al., Eur. J. Biochem. 226 (1994) 369-375). N-terminally the cDNA encodes 27 amino acids with characteristics for a signal directing the nascent protein to the endoplasmic reticulum. A baculovirus vector was constructed containing cDNAs of kbPAP and green fluorescent protein, the latter to serve as transfection and infection marker. Heterologous expression in High Five insect cells afforded a dimeric, disulfide-linked phosphatase of 110 kDa, identical to the mass of native kbPAP. Purification in three steps yielded 1.5 mg recombinant protein per liter of culture medium with a specific activity of 266 units/mg, slightly exceeding that of native kbPAP. The recombinant protein was functionally indistinguishable from native kbPAP, despite differences in glycosylation and sensitivity to redox reagents.

Acid Phosphatase↗

PubMed2002

Insights into binding of fatty acids by fatty acid binding proteins.

Members of the phylogenetically related intracellular lipid binding protein (iLBP) are characterized by a highly conserved tertiary structure, but reveal distinct binding preferences with regard to ligand structure and conformation, when binding is assessed by the Lipidex method (removal of unbound ligand by hydrophobic polymer) or by isothermal titration calorimetry, a true equilibrium method. Subfamily proteins bind retinoids, subfamily II proteins bind bulky ligands, examples are intestinal bile acid binding protein (I-BABP) and liver fatty acid binding protein (L-FABP) which binds 2 ligand molecules, preferably monounsaturated and n-3 fatty acids. Subfamily III intestinal fatty acid binding protein (I-FABP) binds fatty acid in a bent conformation. The fatty acid bound by subfamily IV FABPs has a U-shaped conformation; here heart (H-) FABP preferably binds n-6, brain (B-) FABP n-3 fatty acids. The ADIFAB-method is a fluorescent test for fatty acid in equilibrium with iLBP and reveals some correlation of binding affinity to fatty acid solubility in the aqueous phase; these data are often at variance with those obtained by the other methods. Thus, in this review published binding data are critically discussed, taking into account on the one hand binding increments calculated for fatty acid double bonds on the basis of the 'solubility' hypothesis, on the other hand the interpretation of calorimetric data on the basis of crystallographic and solution structures of iLBPs.

Animals↗

PubMed2002

Plasma concentration of intestinal- and liver-FABP in neonates suffering from necrotizing enterocolitis and in healthy preterm neonates.

Both early diagnostic and prognostic assessment of the acute abdomen in preterm infants are hampered by the lack of a sensitive and specific parameter for intestinal injury. In this prospective clinical study we wanted to estimate the value of intestinal (I-) and liver (L-) fatty acid binding protein (FABP) in diagnosing necrotizing enterocolitis (NEC). Using highly sensitive and specific sandwich ELISAs which employ recombinant human I- and L-FABP as standard proteins (limit of detection 0.1 ng/ml plasma), the L-FABP concentration (median 7.6 ng/ml) was determined to be about 3 fold that of I-FABP (median 2.52 ng/ml) in plasma of healthy preterm infants. I- and L-FABP concentrations significantly increased with birth weight (1.6 and 5.0 ng/ ml per kg, respectively). At onset of symptoms, I-FABP concentration was significantly higher in infants who later developed severe NEC compared to healthy infants and those, whose illness remained confined to stage I or II. L-FABP was significantly elevated compared to the control group at onset of symptoms regardless of the further course of NEC. In conclusion, I-FABP appears to be a specific parameter for early detection of intestinal injury leading to severe NEC stage III. L-FABP, however, is a promising sensitive marker even for stage I of NEC.

Animals↗

PubMed2001

Localization of epidermal-type fatty acid binding protein in the thymic epithelial cells of mice.

The immunoreactivity for epidermal-type fatty acid binding protein of epidermis type (E-FABP) was selectively localized in the epithelial cells of both cortex and medulla of mouse thymus. The cortical epithelial cytoreticulum was clearly visible with the intense immunoreactivity and the immunoreactive cytoreticulum extended intricately throughout the thymic cortex to enclose thymocytes. In the thymic medulla, the immunoreactivity was variable in intensity among the epithelial cells and there was a tendency that epithelial cells containing more numerous tonofilament bundles were less immunoreactive. Considering the possibility that FABPs function as intracellular carriers for unsaturated long chain fatty acids, the present finding suggests that E-FABP in the thymic epithelial cells, especially the cortical ones because of their extensive location, are intimately involved in the metabolic processes of fatty acids including production of bioactive substances, such as prostaglandin and leukotriene, which are known to exert some regulation of thymic immune responses.

Animals↗