BBA - Molecular Basis of Disease (v.1500, #1)
Enhancement of macrophage migration inhibitory factor (MIF) expression in injured epidermis and cultured fibroblasts by Riichiro Abe; Tadamichi Shimizu; Akira Ohkawara; Jun Nishihira (1-9).
After the cDNA of human macrophage migration inhibitory factor (MIF) was cloned in 1989, this protein has been re-evaluated as a pro-inflammatory cytokine, pituitary hormone and glucocorticoid-induced immunoregulatory protein. We previously reported the expression of MIF in the basal cell layers of the epidermis, but its pathophysiological function in the skin has not been well understood. In this study, we examined the expression of MIF during the wound healing of rat skin injured by excision. Reverse transcription-polymerase chain reaction in combination with Southern blot analysis revealed that the increase of MIF mRNA expression was biphasic. The maximum peaks were observed at 3 and 24 h after the injury. Similarly, maximal increases of the serum MIF level were observed at 3 and 24 h after the injury. Immunohistochemical analysis at 12 h after injury demonstrated enhanced expression of MIF protein in the whole epidermal lesion of the wound tissue. By the Boyden chamber assay, we demonstrated that MIF had a chemotactic effect on freshly prepared keratinocytes from rat skin. Additionally, cultured fibroblasts from the skin wound lesion secreted a higher amount of MIF in response to lipopolysaccharide compared to those of the normal skin. Furthermore, administration of anti-MIF antibodies induced a delay of wound healing in vivo. Taken together, these results suggest that MIF contributes to the wound healing process of skin tissue.
Keywords: Fibroblast; Macrophage migration inhibitory factor; Skin; Wound;
Increased activities of antioxidant enzymes and decreased ATP concentration in cultured myoblasts with the 3243A→G mutation in mitochondrial DNA by Harri Rusanen; Kari Majamaa; Ilmo E. Hassinen (10-16).
The MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is most commonly caused by the 3243A→G mutation in mitochondrial DNA, resulting in impaired mitochondrial protein synthesis and decreased activities of the respiratory chain complexes. These defects may cause a reduced capacity for ATP synthesis and an increased rate of production of reactive oxygen species. Myoblasts cultured from controls and patients carrying the 3243A→G mutation were used to measure ATP, ADP, catalase and superoxide dismutase, which was also measured from blood samples. ATP and ADP concentrations were decreased in myoblasts with the 3243A→G mutation, but the ATP/ADP ratio remained constant, suggesting a decrease in the adenylate pool. The superoxide dismutase and catalase activities were higher than in control cells, and superoxide dismutase activity was slightly, but not significantly higher in the blood of patients with the mutation than in controls. We conclude that impairment of mitochondrial ATP production in myoblasts carrying the 3243A→G mutation results in adenylate catabolism, causing a decrease in the total adenylate pool. The increase in superoxide dismutase and catalase activities could be an adaptive response to increased production of reactive oxygen species due to dysfunction of the mitochondrial respiratory chain.
Keywords: Mitochondrial disease; MELAS syndrome; Muscle; ATP production; Superoxide dismutase; Catalase;
Large-scale analysis of differential gene expression in the hindlimb muscles and diaphragm of mdx mouse by Andrei V Tkatchenko; Ginette Le Cam; Jean J Léger; Claude A Dechesne (17-30).
The mdx mouse is an animal model for Duchenne muscular dystrophy (DMD), which is caused by the absence of dystrophin. Mdx limb muscles substantially compensate for the lack of dystrophin while the diaphragm is affected like DMD skeletal muscles. To understand better the complex cascade of molecular events leading to muscle degeneration and compensatory processes in mdx muscles, we analyzed alterations of gene expression in mdx hindlimb and diaphragm muscles as compared to their normal counterparts. The strategy was based on suppression subtractive hybridization followed by reverse Northern quantitative hybridization. Four subtracted/normalized libraries, containing cDNA clones up- or downregulated in mdx hindlimb muscles or diaphragm, were constructed and a total of 1536 cDNA clones were analyzed. Ninety-three cDNAs were found to be differentially expressed in mdx hindlimb muscles and/or diaphragm. They corresponded to 54 known genes and 39 novel cDNAs. The potential role of the known genes is discussed in the context of the mdx phenotype.
Keywords: Mdx mouse; Muscle regeneration; Differential gene expression; Suppression subtractive hybridization; Reverse Northern;
Molecular assembly of endogenous and synthetic big atrial natriuretic peptide (ANP) and its amyloidogenic implications by Emanuela Maioli; Claudia Torricelli; Annalisa Santucci; Adriana Pacini (31-40).
The aggregation process of α-hANP has been investigated in vitro at physiological concentrations by gel chromatographic procedures using a radiolabeled tracer incubated in PBS and in plasma. In PBS big forms of ANP are organized as a peak eluting from both Sephacryl S-100 and S-300 HR in the void volume of the columns; in plasma, besides this major peak, a second radioactive peak is evident, eluting from Sephacryl S-100 HR around the HSA region. After gel chromatography on Sephacryl S-300 HR the major peak appears to consist of three components of different molecular size. Some information about the nature of these peak materials comes from the result of parallel incubations of partially aggregated (seed or nucleus) and aggregate depleted tracer. The comparison between the two time courses of big ANP formation indicates that: (a) ANP aggregation is a nucleation-dependent process, with a lag time longer than 8 days, at picogram peptide levels and (b) the aggregated forms of peptide are those eluting in the void volume, the other plasma peaks being probably expression of a binding, neither saturable or reversible, to some plasma components. The principle of seeded polymerization, used to detect ANP aggregates present in the plasma, indicates that: (a) the endogenous big ANP cannot act as a nucleus for polymerization and it likely consists of non-fibrillar ANP aggregates and/or bound ANP, and (b) this experimental approach can be suitable to evidence ANP binding plasma factors for further characterization studies.
Keywords: Amyloidogenic peptide; Atrial natriuretic peptide; Aggregation; Big form; Disulfide bond;
Expression of monocyte chemotactic protein-3 mRNA in rat vascular smooth muscle cells and in carotid artery after balloon angioplasty by Xinkang Wang; Xiang Li; Tian-Li Yue; Eliot H Ohlstein (41-48).
Monocyte chemotactic protein-3 (MCP-3) is a CC chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells and dendritic cells. The activation of the target cells by MCP-3 is via specific chemokine receptors CCR2 and CCR3, of which CCR2 is shared with MCP-1. MCP-1 and CCR2 have been implicated in vascular diseases including atherosclerosis and restenosis, that are known to be involved in inflammation (accumulation of T lymphocytes and monocytes) and smooth muscle cell (SMC) activation (proliferation, migration and matrix deposition). To investigate a potential role of MCP-3 in vascular injury, the present work examined its mRNA expression in rat aortic SMCs stimulated with various inflammatory stimuli including LPS, TNF-α, IL-1β, IFN-γ and TGF-β. A time- and concentration-dependant induction of MCP-3 mRNA in SMCs was observed by means of Northern analysis. A strikingly similar expression profile was observed for MCP-3 and MCP-1 mRNA in SMCs. Furthermore, MCP-3 mRNA expression was induced in rat carotid artery after balloon angioplasty. A significant induction in MCP-3 mRNA was observed in the carotid artery at 6 h (41-fold increase over control, P<0.001), 1 day (13-fold increase, P<0.001) and 3 days (6-fold increase, P<0.01) after balloon angioplasty as quantitated by reverse transcription and polymerase chain reaction. These data provide evidence for the cytokine-induced expression of MCP-3 in SMCs and in carotid artery after balloon angioplasty, suggesting a potential role of MCP-3 in the pathogenesis of restenosis and atherosclerosis.
Keywords: Chemokine; Inflammation; Monocyte chemotactic protein-3; Restenosis; Smooth muscle cell;
α-Crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde by Elena Ganea; John J Harding (49-58).
The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether α-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether α-crystallin could renature G6PD denatured by MDA. α-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without α-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. α-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by α-crystallin, but it was stabilised and protected against further denaturation. Complex formation between α-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that α-crystallin is able to protect the enzyme against this damage.
Keywords: Chaperone; α-Crystallin; Glucose 6-phosphate dehydrogenase; Lens; Malondialdehyde;
Expression and intracellular processing of chimeric and mutant CFTR molecules 1 1 This work is dedicated to the memory of the late pediatrician R. Van Geffel who pioneered the treatment of CF children in Belgium. by J.-F. Pollet; J. Van Geffel; E. Van Stevens; R. Van Geffel; R. Beauwens; A. Bollen; P. Jacobs (59-69).
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride channel comprising two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs) and a unique regulatory (R) domain. The most frequent cystic fibrosis (CF) mutation, a deletion of Phe508 in NBD1, results in the retention of the ΔF508 CFTR in the endoplasmic reticulum, as do many other natural or constructed mutations located within the first NBD. In order to further define the role of NBD1 in CFTR folding and to determine whether the higher frequency of mutations in NBD1 with respect to NBD2 results from its position in the molecule or is related to its primary sequence, we constructed and expressed chimeric CFTRs wherein NBD domains were either exchanged or deleted. Synthesis, maturation and activity of the chimeras were assessed by Western blotting and iodide efflux assay after transient or stable expression in COS-1 or CHO cells respectively. The data showed that deletion of NBD1 prevented transport of CFTR to the cytoplasmic membrane whereas deletion of NBD2 did not impair this process but resulted in an inactive chloride channel. On the other hand, substituting or inverting NBDs in the CFTR molecule impaired its processing. In addition, while the NBD1 R555K mutation is known to partially correct the processing of CFTR ΔF508 and to increase activity of both wild-type and ΔF508 individual channels, it showed no positive effect when introduced into the double NBD1 chimera. Taken together, these observations suggest that the proper folding process of CFTR results from complex interactions between NBDs and their surrounding domains (MSDs and/or R domain).
Keywords: Cystic fibrosis transmembrane conductance regulator; Chloride channel; Nucleotide binding domain; Chimeric recombinant protein;
Migration of CD18-deficient neutrophils in vitro: evidence for a CD18-independent pathway induced by IL-8 by Clare M Morland; Bruce J Morland; Phil J Darbyshire; Robert A Stockley (70-76).
Neutrophils isolated from a child with severe leukocyte adhesion deficiency 1 (LAD1) had a complete absence of expression of the CD11/CD18 β2 integrin family of adhesion molecules, and were shown to be deficient in the in vitro adhesion and migration properties. However, we found that interleukin-8 (IL8), a potent chemoattractant for neutrophils, and sputum sol phase induced these LAD1 neutrophils to migrate through an endothelial cell layer in vitro, and confirmed that this migration was CD18-independent. These findings add to evidence of CD18-independent mechanisms of neutrophil recruitment, in particular neutrophil infiltration into the lungs, where IL8 may be an important recruitment factor.
Keywords: Neutrophil; Chemotaxis; Adhesion;
Molecular characterization of a Leishmania donovanii cDNA clone with similarity to human 20S proteasome a-type subunit 1 The sequence data reported in this paper have been submitted to EMBL/GenBank and DDJB data libraries under accession No. AF088882. 1 by Claus B.V. Christensen; Louise Jørgensen; Anja T.R. Jensen; Soha Gasim; Ming Chen; Arsalan Kharazmi; Thor G. Theander; Keld Andresen (77-87).
Using plasma from patients infected or previously infected with Leishmania donovanii, we isolated a L. donovanii cDNA clone with similarity to the proteasome a-type subunit from humans and other eukaryotes. The cDNA clone, designated LePa, was DNA sequenced and Northern blot analysis of L. donovanii poly(A+)mRNA indicated the isolation of a full length cDNA clone with a transcript size of 1.9 kb. The expressed recombinant LePa fusion protein induced proliferation of peripheral blood mononuclear cells in one out of seven patients who had suffered from visceral leishmaniasis. Plasma from 16 out of 25 patients with visceral leishmaniasis and four out of 18 patients with cutaneous leishmaniasis contained IgG antibodies which reacted with the purified LePa fusion protein as evaluated in an ELISA. The LePa DNA sequence was inserted into an eukaryotic expression vector and Balb/c mice were vaccinated. DNA vaccination of Balb/c mice with LePa generated an initial significant reduction in lesion size after challenge.
Keywords: Leishmania; cDNA library; Proteasome a-subunit; DNA vaccination;
Differences in mRNA expression of the proteins secreted by the adipocytes in human subcutaneous and visceral adipose tissues by Eric Dusserre; Philippe Moulin; Hubert Vidal (88-96).
We have investigated the difference in gene expression of six proteins secreted by adipocytes in paired biopsies from visceral and abdominal subcutaneous adipose tissue in nine individuals with various degrees of obesity. The mRNAs levels of leptin, TNFα, angiotensinogen, acylation stimulating protein (ASP), cholesterol ester transfer protein (CETP) and phospholipid transfer protein (PLTP) were quantified by RT-competitive PCR. ASP and angiotensinogen mRNA levels were higher in the visceral fat, whereas the mRNA levels of leptin and CETP were higher in the subcutaneous depot. TNFα mRNA expression was similar in the two sites. For angiotensinogen, the difference was more pronounced in the subjects with body mass index (BMI) lower than 30 kg/m2 whereas for ASP, CETP and leptin, the difference was observed regardless the BMI of the subjects. PLTP mRNA levels in subcutaneous, but not in the visceral, adipose tissue were positively related to the BMI of the subjects. These results strongly suggest that visceral and subcutaneous adipocytes may have different properties in the production of bioactive molecules.
Keywords: Angiotensinogen; Acylation stimulating protein; Cholesterol ester transfer protein; Phospholipid transfer protein; TNFα; Leptin;
Molecular cloning of canine bullous pemphigoid antigen 2 cDNA and immunomapping of NC16A domain by canine bullous pemphigoid autoantibodies 1 This paper was presented in part, as an abstract, at the Annual Meeting of the Society for Investigative Dermatology, April 23–27, 1997, Washington, DC. 1 by Luting Xu; Edel A. O’Toole; Thierry Olivry; Claudia Hernandez; Jun Peng; Mei Chen; Lawrence S. Chan (97-107).
The autoantibody-mediated subepidermal blistering skin disease bullous pemphigoid affects both humans and dogs. We previously demonstrated that canine bullous pemphigoid patient’s autoantibodies targeted skin basement membrane component and a 180-kDa keratinocyte protein. We extend our works to partially isolate the cDNA encoding canine bullous pemphigoid antigen 2 (BPAg2, BP180). Total RNA extracted from a papillomavirus-immortalized canine keratinocyte cell line and a cultured canine squamous carcinoma cell line SCC 2/88 were used to isolate fragments of cDNA encoding BPAg2 by reverse transcription-PCR and 5′-rapid amplification of cDNA end. The isolated sequence included the 5′-untranslated region, the entire intracellular, transmembranous, and extracellular NC16A autoantigenic domains, plus a small segment of the collagenous domain. Sequence analyses of the isolated cDNA showed 87 and 85% identities between canine and human at the nucleotide sequence and at the deduced amino acid sequence levels, respectively. The canine BPAg2 sequence was confirmed by a rabbit antibody raised against a 18-amino acid peptide deduced from the canine NC16A nucleotide sequence. Autoantibodies from canine bullous pemphigoid patients’ sera recognized epitopes within the human NC16A domain. The cloning of the cDNA encoding this disease-associated protein may allow us to develop a canine model in dissecting the immunopathologic mechanism underlying bullous pemphigoid.
Keywords: Autoimmunity; Veterinary immunology; ELISA; Peptide; Cloning; Canine;
Phenotypic changes in neutrophils related to anti-inflammatory therapy by A.E. Barton; D.L. Bayley; M. Mikami; C.G. Llewellyn-Jones; R.A. Stockley (108-118).
Previous work from the group has shown that non-steroidal anti-inflammatory agents given to volunteers and patients inhibit PMN function possibly by affecting the developing neutrophil during the differentiation process. In this study indomethacin treatment in vivo reduced neutrophil chemotaxis and proteolytic degradation of fibronectin, with a maximal effect after 14 days. Stimulated neutrophil adherence to fibronectin was also reduced but this was not due to quantitative changes in β2 integrin expression or function. L-Selectin expression on resting and stimulated neutrophils was increased after 14 days and there was a small decrease in plasma levels of soluble L-selectin. These effects, however, could not be reproduced by treatment of neutrophils with indomethacin in vitro, suggesting they are due to effects on differentiating/maturing PMNs. In an attempt to interpret these changes, studies were performed with dexamethasone, which is known to alter neutrophil function and kinetics. Dexamethasone treatment reduced chemotaxis and increased superoxide generation after 1 day and was associated with increased expression of activated β2 integrins and reduced L-selectin expression on resting neutrophils. This suggests the appearance of mainly ‘activated’ cells as a result of demargination and indicates that the effects of indomethacin are distinctive and not related to changes in compartmentalisation.
Keywords: Non-steroidal anti-inflammatory drug; Indomethacin; Dexamethasone; Chemotaxis; L-selectin; Adhesion;
Human placental indoleamine 2,3-dioxygenase: cellular localization and characterization of an enzyme preventing fetal rejection by Yoshiki Kudo; C.A.R Boyd (119-124).
In order to test the hypothesis (Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, Mellor, Science 281 (1998) 1191–1193) that localized placental tryptophan catabolism prevents immune rejection of the mammalian fetus, the cellular localization and characteristics of human placental indoleamine 2,3-dioxygenase (EC 184.108.40.206) were studied. The localization of indoleamine 2,3-dioxygenase activity was determined quantitatively using cell fractionation by differential and discontinuous sucrose gradient centrifugation. Enzyme activity was looked for in isolated brush border microvillous plasma membranes of placental syncytiotrophoblast. We found that this membrane preparation (which showed a 32.4-fold purification from the starting homogenate with reference to the activity of a membrane marker enzyme, alkaline phosphatase (EC 220.127.116.11)) was strongly negatively enriched with indoleamine 2,3-dioxygenase (which showed a one twenty-fifth decrease in its specific activity). Placental indoleamine 2,3-dioxygenase is thus not expressed in the maternal facing brush border membrane of syncytiotrophoblast. 1-Methyl-dl-tryptophan which was used by Munn et al. as a key experimental tool for inhibiting indoleamine 2,3-dioxygenase in the murine model showed a competitive inhibition of human placental indoleamine 2,3-dioxygenase with l-tryptophan. The hypothesis, based on experiments performed in mouse, may therefore be applicable to avoidance of immune rejection of the fetus in human pregnancy.
Keywords: Human placenta; Indoleamine 2,3-dioxygenase; l-Tryptophan; Pregnancy;
Bacterial lipopolysaccharide induces cytoskeletal rearrangement in small intestinal lamina propria fibroblasts: actin assembly is essential for lipopolysaccharide signaling by Dipshikha Chakravortty; K.S Nanda Kumar (125-136).
Cytoskeletal proteins are major components of the cell backbone and regulate cell shape and function. The purpose of this study was to investigate the effect of lipopolysaccharide (LPS) on the dynamics and organization of the cytoskeletal proteins, actin, vimentin, tubulin and vinculin in human small intestinal lamina propria fibroblasts (HSILPF). A noticeable change in the actin architecture was observed after 30 min incubation with LPS with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 2 h. Reorganization of the vimentin network into vimentin bundling was conspicuous at 2 h. With further increase in the time period of LPS exposure, diffused staining of vimentin along with vimentin bundling was observed. Vinculin plaques distributed in the cell body and cell periphery in the control cells rearrange to cell periphery in LPS-treated cells by 30 min of LPS exposure. However, there was no change in the tubulin architecture in HSILPF in response to LPS. LPS increased the F-actin pool in HSILPF in a concentration-dependent manner with no difference in the level of G-actin. A time-dependent study depicted an increase in the G-actin pool at 10 and 20 min of LPS exposure followed by a decrease at further time intervals. The F-actin pool in LPS-treated cells was lower than the control levels at 10 and 20 min of LPS exposure followed by a sharp increase until 120 min and finally returning to the basal level at 140 and 160 min. Further 35S-methionine incorporation studies suggested a new pool of actin synthesis, whereas the synthesis of other cytoskeletal filaments was not altered. Cytochalasin B, an actin-disrupting agent, severely affected the LPS induced increased percentage of ‘S’ phase cells and IL-6 synthesis in HSILPF. We conclude that dynamic and orchestrated organization of the cytoskeletal filaments and actin assembly in response to LPS may be a prime requirement for the LPS induced increase in percentage of ‘S’ phase cells and IL-6 synthesis
Keywords: Lamina propria fibroblast; Lipopolysaccharide; Actin; Vimentin; Vinculin; Tubulin;
Assembly and functional expression of murine glutathione reductase cDNA: a sequence missing in expressed sequence tag libraries by Rimma Iozef; Katja Becker; Catharina C. Boehme; R.Heiner Schirmer; Dieter Werner (137-141).
Glutathione reductase (GR) is a chemotherapeutic target. Murine GRcDNA, which contains 85% GC in the 38 codons following the start codon, was assembled from the PCR-amplified exon 1 and a downstream cDNA prior to expression in Escherichia coli as a His6-tagged protein. Recombinant GR, an FAD-containing homodimer, corresponds in its enzymic and spectral properties to GR isolated from murine Ehrlich ascites tumor cells. Another cDNA, representing GR with a mitochondrial targeting sequence, yielded two distinct enzymically active expression products.
Keywords: Mouse glutathione reductase; GC rich sequence; Mitochondrial targeting sequence; Flavin spectra; Brain tumor; Murine malaria;
Molecular cloning and tissue-specific expression of a new member of the regenerating protein family, islet neogenesis-associated protein-related protein 1 The sequence data reported in this paper have been deposited to DDBJ/EMBL/Genbank databases under the accession no. AB028625. 1 by Kenji Sasahara; Takashi Yamaoka; Maki Moritani; Katsuhiko Yoshimoto; Yasuhiro Kuroda; Mitsuo Itakura (142-146).
Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.
Keywords: Islet neogenesis-associated protein-related protein; Islet neogenesis-associated protein; Regenerating protein; Pancreatitis-associated protein;
Inositol phosphoryl transferases from human pathogenic fungi by Steven A. Heidler; Jeffrey A. Radding (147-152).
The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.
Keywords: Inositolphosphoryl transferase; AUR1/IPC1; Fungal infection; Antifungal treatment; (Human);