Interferon-gamma increases monocyte HLA-DR expression without effects on glucose and fat metabolism in postoperative patients.

Tissue injury is associated with decreased cellular immunity and enhanced metabolism. Immunodepression is thought to be counteracted by interferon (IFN)-gamma, which increases human leukocyte antigen (HLA)-DR expression. Hypermetabolism could be enhanced by IFN-gamma because cytokines induce a hypermetabolic response to stress. In healthy humans, IFN-gamma enhanced HLA-DR expression without effects on glucose and fat metabolism. In the present study, we evaluated whether IFN-gamma lacks potential harmful side effects on metabolic and endocrine pathways while maintaining its beneficial effects on the immune system under conditions in which the inflammatory response system is activated. In 13 patients scheduled for major surgery, we studied HLA-DR expression on peripheral blood monocytes before surgery and postoperatively randomized the patients into an intervention and a placebo group. Subsequently, we evaluated the effects of a single dose of IFN-gamma vs. saline on short-term monocyte activation, glucose and lipid metabolism, and glucose and lipid regulatory hormones. HLA-DR expression on monocytes was restored from postoperative levels of 54% (42-60%; median and interquartiles) to 92% (91-96%) 24 h after IFN-gamma administration but stayed low in the placebo-treated patients. IFN-gamma did not affect glucose metabolism (plasma glucose, rate of appearance and disappearance of glucose) and lipid metabolism (plasma glycerol, plasma free fatty acids, and rates of appearance and disappearance of glycerol). IFN-gamma had no effect on plasma cortisol, adrenocorticotropic hormone, growth hormone, insulin, C-peptide, glucagon, epinephrine, and norepinephrine concentrations. We conclude that IFN-gamma exerts a favorable effect on cell-mediated immunity in patients after major surgery without effects on glucose and lipid metabolism.     

Stem-like cells in human hepatoblastoma.

Hepatoblastoma is a pediatric liver tumor with epithelial components resembling embryonal and fetal liver cells. The existence of teratoid hepatoblastoma suggests the presence of stem cells in hepatoblastoma. The aim of this study was to analyze the expression of stem cell markers in hepatoblastomas. We studied specimens from 10 hepatoblastomas. Five of the hepatoblastomas were of epithelial and five of mixed type. Immunohistochemistry (IHC) for the stem cell markers CD34, Thy1, c-kit, and the hepatic or biliary lineage markers CK-18, OCH, CK-7, and CD56 was performed. Double IHC for stem cell and lineage markers was used to identify putative liver stem cells. The different markers showed distinct distributions on the tumor cells. Cells in atypical ducts were found to express simultaneously stem cell markers and hepatocytic or biliary lineage markers. Other cells in connective tissue showed c-kit expression, but not hepatic or biliary marker expression. The data show the presence of different cell populations bearing stem cell markers in human hepatoblastoma. Ductal cells co-expressing stem cell markers and hepatic lineage markers phenotypically resemble hepatic stem-like cells. These findings support the thesis that stem cells play a role in the histogenesis of hepatoblastoma.     

Detection of mutant protein in complex biological samples: glucocerebrosidase mutations in Gaucher's disease

We report a sensitive method to detect point mutations in proteins from complex samples. The method is based on surface-enhanced laser desorption/ionization time-of-flight (SELDI-ToF) MS but can be extended to other MS platforms. The target protein in this study is the lysosomal enzyme glucocerebrosidase (GC), the key enzyme in Gaucher's disease. Deficiency of GC activity results in accumulation of glucosylceramide in macrophages. The relationship between GC genotypes and Gaucher's patient phenotypes is not strict. The possibility to measure protein levels of GC in clinical samples may provide deeper insight into the phenomenology of Gaucher's disease. For this purpose, GC was isolated in a single enrichment step through interaction with an immobilized monoclonal antibody, 8E4. After on-chip digestion of the antibody-antigen complex with trypsin, a total of 25 GC peptides were identified (sequence coverage approximately 60%), including several peptides containing mutated amino acid residues. The described methodology allows mutational analysis on the protein level, directly measured on complex biological samples without the necessity of elaborate purification procedures.     

The role of calcitonin and alpha-calcitonin gene-related peptide in bone formation

The Calca gene encodes two polypeptides, calcitonin (CT) and α-calcitonin gene-related peptide (α-CGRP), generated through alternative splicing. While CT, a hormone mainly produced by thyroidal C cells, has been described as a major regulator of bone resorption, α-CGRP, a neuropeptide expressed in the cells of the central and peripheral nervous system, is mostly known as a regulator of vascular tone. Surprisingly, the generation and skeletal analyses of two mouse deficiency models has recently uncovered a physiological function for both peptides in the regulation of bone formation. In the first model, where the replacement of exons 2-5 of the Calca gene resulted in the combined deficiency of CT and α-CGRP, an increased bone formation rate (BFR) was observed, whereas decreased BFR was found in the second model, where the introduction of a translational termination codon into exon 5 of the Calca gene resulted in the specific absence of α-CGRP.     

Mechanism-based inhibition of enzyme I of the Escherichia coli phosphotransferase system. Cysteine 502 is an essential residue.

Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system. The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) of 40 and 4 min(-1), compared with 2,200 min(-1), for PEP. The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations. Z-Cl-PEP was a competitive inhibitor of PEP with a K(I) of 0.4 mm. E-Cl-PEP was not an inhibitor. Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI. Z-Cl-PEP is a suicide inhibitor. About 10-50 turnovers sufficed to inactivate EI completely. Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations. Inactivation was saturatable in Z-Cl-PEP, with an apparent K(m)(inact) of 0.2-0.4 mm. The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI. E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP. This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species. Cys-502 of EI was identified by mass spectrometry as the reacting residue. The C502A EI mutant showed less than 0.06% wild-type activity. Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.     

The IKK-2/Ikappa Balpha /NF-kappa B pathway plays a key role in the regulation of CCR3 and eotaxin-1 in fibroblasts. A critical link to dermatitis in Ikappa Balpha -deficient mice.

Tumor necrosis factor (TNF)-alpha-induced phosphorylation of the IkappaB proteins by the IkappaB kinase (IKK) complex containing IKK-2 and subsequent degradation of the IkappaB proteins are prerequisites for NF-kappaB activation, resulting in the stimulation of a variety of pro-inflammatory target genes. The C-C chemokine eotaxin-1 is a potent chemoattractant for eosinophils and Th2 lymphocytes, may play an important role in the pathogenesis of atopic dermatitis, and acts via binding to its receptor CCR3. To investigate the role of NF-kappaB signaling in the regulation of these genes, we stably expressed a transdominant mutant of IkappaBalpha and a constitutively active mutant of IKK-2 in mouse NIH3T3 fibroblasts. The transdominant IkappaBalpha mutant completely inhibited TNF-alpha-mediated induction of both eotaxin-1 and CCR3, whereas expression of constitutively active IKK-2 was sufficient to drive almost full expression of these two genes in the absence of TNF-alpha. Moreover, we observed elevated expression levels of CCR3 and eotaxin-1 protein levels in the skin of IkappaBalpha-deficient mice characterized by a widespread dermatitis. Finally, using dermal fibroblasts derived from IkappaBalpha-deficient mice, we observed elevated basal expression, enhanced inducibility by TNF-alpha, and attenuated down-regulation upon TNF-alpha withdrawal of both CCR3 and eotaxin-1 mRNA levels. These results demonstrate that the IKK-2/IkappaBalpha/NF-kappaB pathway plays a critical role for CCR3 and eotaxin-1 expression in fibroblasts and suggests a critical link to the pathogenesis of atopic dermatitis.     

A Golgi-associated PDZ domain protein modulates cystic fibrosis transmembrane regulator plasma membrane expression.

We identified a novel cystic fibrosis transmembrane conductance regulator (CFTR)-associating, PDZ domain-containing protein, CAL (CFTR associated ligand) containing two predicted coiled-coiled domains and one PDZ domain. The PDZ domain of CAL binds to the C terminus of CFTR. Although CAL does not have any predicted transmembrane domains, CAL is associated with membranes mediated by a region containing the coiled-coil domains. CAL is located primarily at the Golgi apparatus, co-localizing with trans-Golgi markers and is sensitive to Brefeldin A treatment. Immunoprecipitation experiments suggest that CAL exists as a multimer. Overexpression of CAL reduces CFTR chloride currents in mammalian cells and decreases expression, rate of insertion and half-life of CFTR in the plasma membrane. The Na(+)/H(+) exchanger regulatory factor, NHE-RF, a subplasma membrane PDZ domain protein, restores cell surface expression of CFTR and chloride currents. In addition, NHE-RF inhibits the binding of CAL to CFTR. CAL modulates the surface expression of CFTR. CAL favors retention of CFTR within the cell, whereas NHE-RF favors surface expression by competing with CAL for the binding of CFTR. Thus, the regulation of CFTR in the plasma membrane involves the dynamic interaction between at least two PDZ domain proteins.