Patient-derived fibroblasts indicate oxidative stress status and may justify antioxidant therapy in OXPHOS disorders.

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.

Autosomal dominant Alport syndrome caused by a COL4A3 splice site mutation.

BACKGROUND: Alport syndrome (AS) is a clinically and genetically heterogeneous renal disorder, predominantly affecting the type IV collagen alpha 3/alpha 4/alpha 5 network of the glomerular basement membrane (GBM). AS can be caused by mutations in any of the three genes encoding these type IV collagen chains. The majority of AS families (85%) are X-linked (XL-AS) involving mutations in the COL4A5 gene. Mutations in the COL4A3 and COL4A4 genes cause autosomal recessive AS (AR-AS), accounting for approximately 14% of the cases. Recently, autosomal dominant AS (AD-AS) was linked to the COL4A3/COL4A4 locus in a large family. METHODS: COL4A3 and COL4A4 cDNAs were generated by nested reverse transcription-polymerase chain reaction and were analyzed by DNA sequence analysis. Denaturating high-performance liquid chromatography (DHPLC) was used for mutation and segregation analysis at the genomic DNA level. RESULTS: In the AD-AS family, a splice site mutation resulting in skipping of exon 21 of the COL4A3 gene was detected. The mutation does not alter the reading frame and is predicted to result in a COL4A3 chain with an internal deletion. CONCLUSION: As the NC domain is intact, this chain may be incorporated and distort the collagen triple helix, thereby causing the dominant effect of the mutation. The finding of a specific COL4A3 mutation in AD-AS completes the spectrum of type IV collagen mutations in all genetic forms of AS.

Identification of COL4A5 defects in Alport's syndrome by immunohistochemistry of skin.

BACKGROUND: The COL4A3-COL4A4-COL4A5 network in the glomerular basement membrane is affected in the inherited renal disorder Alport's syndrome (AS). Approximately 85% of the AS patients are expected to carry a mutation in the X-chromosomal COL4A5 gene and 15% in the autosomal COL4A3 and COL4A4 genes. The COL4A5 chain is also present in the epidermal basement membrane (EBM). It is predicted that approximately 70% of the COL4A5 mutations prevent incorporation of this chain in basement membranes. METHODS: We investigated whether or not COL4A5 defects could be detected by immunohistochemical analysis of the EBM. Punch skin biopsies were obtained from 22 patients out of 17 families and two biopsy specimens from healthy males were used as controls. RESULTS: In four cases with the COL4A5 frameshift or missense mutations, the COL4A5 chain was either lacking from the EBM (male) or showed a focally negative pattern (female). In three other patients with a COL4A5 missense mutation, a COL4A3 and a COL4A4 mutation, respectively, the COL4A5 staining was normal. A (focally) negative EBM-COL4A5 staining was found in three patients of six families with a diagnosis of AS and in one family of a group of four families with possible AS. CONCLUSIONS: The (focal) absence of COL4A5 in the EBM of skin biopsy specimens can be used for fast identification of COL4A5 defects. Combined with polymorphic COL4A5 markers, both postnatal and prenatal DNA diagnosis are possible in the family of the patient.

A comparison of NSP-reticulons with conventional neuroendocrine markers in immunophenotyping of lung cancers.

Neuroendocrine-specific protein (NSP)-reticulons are endoplasmic reticulum-associated protein complexes, which have been identified as markers for neuroendocrine differentiation. In this study, the expression of two members of the family of NSP-reticulons, NSP-A and NSP-C, have been investigated in different types of lung cancer and compared with the expression patterns of five conventional neuroendocrine markers, the neural cell adhesion molecule (NCAM), synaptophysin, chromogranin A, Leu-7, and neurofilament proteins. NSP-A and NSP-C antibodies were reactive with most carcinoid tumour and small cell lung carcinoma (SCLC) cases, while atypical carcinoid tumours showed a variable expression. In the total group of neuroendocrine tumours, a high concordance of expression was found between NSP-A and NSP-C, while their expression correlated well with NCAM and synaptophysin positivity. Chromogranin A, Leu-7, and neurofilament proteins were shown to be expressed to a limited extent in these neuroendocrine tumours. In a selected group of non-SCLCs known to exhibit neuroendocrine features, NSP-A expression was detected at much higher frequency than NSP-C. In virtually all NSP-A positive cases, this expression was associated with one or more of the other neuroendocrine markers. NSP-A expression showed a stronger correlation with conventional neuroendocrine markers than NCAM. In detecting neuroendocrine differentiation in non-SCLC, NSP-A is more sensitive than synaptophysin, chromogranin A, Leu-7, and neurofilament proteins. It is concluded that NSP-reticulons are valuable markers in the diagnosis of neuroendocrine differentiation in non-SCLC and should be used in conjunction with NCAM.

Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant.

Recently we described a protein, smoothelin, that has been exclusively found in smooth muscle cells (SMC). The human cDNA has been cloned from a colon cDNA library and the putative protein sequence was deduced. Smoothelin does not belong to a known protein family but shows a partial homology with members of the spectrin family. Transfection studies revealed that smoothelin has an affinity for actin and is either capable of forming filamentous structures or colocalizes with such structures. The protein is expressed in visceral as well as vascular tissues of all vertebrate classes. A study on the distribution of smoothelin in the vascular and placental system showed that smoothelin expression was largely restricted to the muscular pulsating blood vessels. Therefore, we hypothesized that smoothelin is expressed in contractile SMC only (36, 37). No expression of smoothelin was observed in established cell lines of SMC. In tissue explants smoothelin mRNA concentration decreases to undetectable levels within 12 hours after dissection as was in general the case in primary cell cultures. Here we report on continued smoothelin expression for several passages observed in a human prostate primary cell culture system. Smoothelin was demonstrated to colocalize with actin stress fibers but not with desmin filaments. This culture system offers opportunities to study the cytological localization of smoothelin, interactions with other proteins and should provide a system to test the promoter of the smoothelin gene. On immunoblots the molecular weight of smoothelin differed between visceral and vascular smooth muscle tissue with apparent molecular weights of respectively 59 kDa and 94 kDa. There is no evidence for the existence of another gene coding for the 94 kDa smoothelin. Thus, posttranslational modification, alternative splicing and dual promoter control are the alternatives for the expression of two isoforms of smoothelin.

Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells.

The characterization of a novel 59-kD cytoskeletal protein is described. It is exclusively observed in smooth muscle cells by Northern blotting and immunohistochemical analysis and therefore designated "smoothelin." A human smooth muscle cDNA library was screened with the monoclonal antibody R4A, and a full-size cDNA of the protein was selected. The cDNA was sequenced and appeared to contain a 1,113-bp open reading frame. Based on the cDNA sequence, the calculated molecular weight of the polypeptide was 40 kD and it was demonstrated to contain two N-glycosylation sites. Computer assisted analysis at the protein level revealed a 56-amino acid domain with homologies of approximately 40% with a sequence bordering the actin-binding domains of dystrophin, utrophin, beta-spectrin and alpha-actinin. In situ hybridization demonstrated that human smoothelin is encoded by a single copy gene which is located on chromosome 22. Immunohistochemistry and Western blotting revealed synthesis of smoothelin in smooth muscle of species evolutionarily as far apart as human and teleost. Northern blotting indicated that sequence as well as size of the mRNA (approximately 1,500 bases) are conserved among vertebrates. Cell fractionation studies and differential centrifugation showed that the protein cannot be extracted with Triton X-100, which indicates that it is a part of the cytoskeleton. Transfection of the human cDNA into smooth muscle cells and COS7 cells produced a protein of 59 kD, which assembled into a filamentous network. However, in rat heart-derived myoblasts association with stress fibers was most prominent. Smoothelin was not detected in primary or long term smooth muscle cell cultures. Also, transcription of smoothelin mRNA was almost instantly halted in smooth muscle tissue explants. We conclude that smoothelin is a new cytoskeletal protein that is only found in contractile smooth muscle cells and does not belong to one of the classes of structural proteins presently known.

Neuroendocrine-specific protein C (NSP-C): subcellular localization and differential expression in relation to NSP-A.

A mouse monoclonal antibody RNL-4, as well as rabbit polyclonal antiserum POL-8 were raised against a synthetic peptide, encompassing the first twenty unique amino-terminal amino acid residues of NSP-C. The specificity of both immunoreagents was established in an ELISA assay using the synthetic peptide and by their immunoreactivity to NSP-C fusion proteins. Immunofluorescence analysis of COS-1 cells, transfected with NSP-C cDNA, showed staining of the endoplasmic reticulum with RNL-4 and POL-8. No cross-reactivity of these reagents with NSP-A or NSP-B was seen. Immunohistochemical studies in normal human tissues showed expression of NSP-C in tissues of neural and neuroendocrine origin, i.e. neurons of the central and peripheral nervous system, the neurohypophysis, adrenal medulla, adenohypophysis, pars intermedia, and in sporadic neuroedocrine cells of the lung. Expression of NSP-C was found in several small cell lung cancer (SCLC) cell lines, in non-SCLC cell lines with neuroendocrine features, but not in typical non-SCLC cell lines. Also, in a neuroblastoma cell line NSP-C expression was observed. Immunoblotting and immunoprecipitation studies with RNL-4 and POL-8 identified the 23 kDa NSP-C polypeptide in these cell lines. Immunofluorescence microscopy showed that also in these cell lines NSP-C is located at the endoplasmic reticulum, as shown before for NSP-A and NSP-B. In some of the cell lines coexpression of NSP-A and NSP-C was observed, while in others only one of the two could be detected. The differential expression of NSP-A and NSP-C in these cell lines is confirmed by immunoblotting and was also evident at the mRNA level. When NSP-A and NSP-C were coexpressed, the number of NSP-C-positive cells was always less than the number of NSP-A-positive cells. A partial colocalization of NSPs was observed in the endoplasmic reticulum. Cell fractionation studies revealed that both proteins are retained in the membranous fraction of the cell, from which they can be solubilized by Triton X-100. Immunoprecipitation analyses under native conditions indicate that NSP-C does not need to associate with NSP-A to form high molecular weight NSP-reticulons.

Subcellular localization and supramolecular organization of neuroendocrine-specific protein B (NSP-B) in small cell lung cancer.

We have recently isolated and characterized a novel gene that is expressed in a neuroendocrine-specific fashion and was therefore designated neuroendocrine-specific protein (NSP)-gene. The NSP-gene encodes three transcripts of different size, with unique 5'-sequences and completely overlapping 3'-sequences. The resulting proteins have an apparent molecular mass of 135 kDa as determined for NSP-A and 23 kDa as found for NSP-C. In the present study we focused on the biochemical characterization and subcellular localization of NSP-B, so far only found to be expressed in the neuroendocrine lung cancer cell line NCI-H82, and its relation to NSP-A. Transfection studies with the NSP-B transcript in COS-1 cells, followed by immunoprecipitation, resulted in a set of proteins ranging in molecular mass from 35 to 45 kDa, identical to NSP-Bs detected by immunoblotting in NCI-H82. In this cell line a major NSP-B triplet in the 43 to 45 kDa range and a 35 kDa NSP-B were consistently detected. Only the 45 kDa NSP-B was found to be phosphorylated. The observed pI values of the 43 to 45 kDa triplet ranged from 4.8 to 5.0, while the 35 kDa NSP-B has a more basic pI value of 5.7. Gel filtration studies show that NSP-A and NSP-B form supramolecular aggregates with a molecular mass of over 500 kDa, present to a minor extent in the phosphate buffered saline soluble cell fraction, but mainly occurring in the membranous pellet fraction from which they can be solubilized by Triton X-100.(ABSTRACT TRUNCATED AT 250 WORDS)

Cluster-10 lung-cancer antibodies recognize NSPs, novel neuro-endocrine proteins associated with membranes of the endoplasmic reticulum.

We have identified a novel gene (the NSP gene) encoding 3 transcripts and coding for 3 neuroendocrine-specific proteins (NSPs), by screening a cDNA expression library of the small-cell lung-cancer (SCLC) cell line NCI-H82 with the cluster-10 lung-cancer antibodies RNL2 and RNL3. The 3 transcripts code for NSPs with apparent molecular weights of 135 kDa (NSP-A), 43 to 45 and 35 kDa (NSP-B) and 23 kDa (NSP-C). NSP-A and NSP-B are recognized by antibodies RNL2 and RNL3, while second-generation antibodies, specifically recognizing NSP-A and NSP-C, have been produced after immunization with a hybrid protein obtained after bacterial expression of the largest NSP-transcript or with a synthetic peptide specific for NSP-C. The NSPs exhibit a highly restricted distribution pattern and are found mainly in neural and neuro-endocrine cell types, and in neuro-endocrine tumours. Of the different types of lung tumours, mainly SCLC and carcinoids were positive in immunocytochemical assays using the anti-NSP antibodies, while non-SCLC were in general negative. The subcellular distribution of the NSPs was studied in human SCLC cell lines. They do not co-localize with components typical of neuro-endocrine granules, such as synaptophysin and chromogranin. The use of NSP antibodies in the immunofluorescence technique applied to cultured SCLC cells, made it obvious that these proteins localize in the endoplasmic reticulum. Cell fractionation procedures, monitored by immunoblotting assays, indicated an association of the NSPs with the microsomal fraction, from which they could be solubilized with Triton X-100. Gel filtration studies with this solubilized fraction revealed that NSPs form supramolecular aggregates with a molecular weight of more then 500 kDa.

Monoclonal antibody MON-114: detection of a marker for neuroendocrine differentiation in human lung cancer.

Mouse monoclonal antibody MON-114 was generated upon immunization with a human small cell lung carcinoma cell line GLC-19. Immunohistochemical analysis of normal tissues with MON-114 showed staining of the adrenal gland, brain and peripheral nerves. With respect to human lung carcinomas, 7 out of 8 small cell lung carcinomas were positively stained as well as 5 out of 5 carcinoid tumors, whereas only 4 out of 31 squamous cell carcinomas and 3 out of 19 adenocarcinomas were weakly stained. Furthermore, 1 large cell carcinoma was negative for MON-114 staining. Apparently, MON-114 stains cells of neuroendocrine differentiation.

Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin.

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

MON-150, a versatile monoclonal antibody against involucrin: characterization and applications.

A monoclonal antibody, designated MON-150, was found serendipitously to react strongly with the granular layer of normal human epidermis and with the upper spinous layers of psoriatic epidermis. From analysis by flow cytometry of cultured human keratinocytes, it appeared that the percentage of MON-150-positive cells strongly increased when the cells reached confluence and the growth fraction declined. To identify the antigen recognized by MON-150, a lysate of human keratinocytes was subjected to affinity chromatography using a MON-150 Sepharose column. This yielded a single protein of approximately 350 kDa as measured on Superose 6 FPLC gel permeation chromatography using non-denaturing conditions. In Western blot analysis under denaturing and reducing conditions, a 140-kDa protein was detected. The subcellular localization and the molecular weight of the antigen recognized by MON-150 suggested that the antigen involved might be involucrin. This was confirmed using a commercial polyclonal antiserum against involucrin. We conclude that MON-150 is a new, versatile antibody against human involucrin.

Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal antibodies against the 7B2 neuroendocrine protein.

Three mouse monoclonal antibodies (Moabs) have been obtained with specificity for the 7B2 protein, a proposed member of the granin family of neuroendocrine proteins. Bacterially produced hybrid proteins of 7B2 were used as immunogens. The Moabs were designated MON-100, MON-101, and MON-102. Furthermore, we report the construction of 35 deletion mutants of the glutathione S-transferase-7B2 (GST-7B2) fusion-gene using recombinant DNA technology. The hybrid proteins encoded by eleven of these mutants were used in epitope mapping experiments and the results of these studies strongly suggested that recognition of 7B2 by all three Moabs involved the same 16 amino acid region of 7B2 (from amino acid residue 128-135). This was further substantiated by the observation that MON-101 and MON-102 specifically recognized a conjugate between bovine serum albumin and the synthetic peptide Phe-Glu-Pro-Glu-His-Asp-Tyr-Pro-Gly-Leu-Gly-Lys based upon the deduced amino acid sequence of the predicted epitope region in 7B2. In an approach to generate a series of 7B2-specific Moabs targeted against another epitope region in the 7B2 protein, the hybrid protein encoded by deletion mutant pPV32 was used as the immunogen. This protein lacked the epitope region recognized by the first series of Moabs. A second series of three Moabs, designated MON-142, MON-143, and MON-144, was obtained and, in all three cases, the region of 7B2 from amino acid residue 64-94 appeared to be involved in specific recognition by the Moabs. The whole panel of six anti-7B2 antibodies appeared to be useful in immunoprecipitation and Western blot analysis of the 7B2 protein and specifically stained neuroendocrine cells in immunohistochemical experiments. Using a double determinant sandwich enzyme immunoassay, 7B2 protein levels in rat pituitary were determined as 20 ng/mg tissue.

Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein.

Mouse monoclonal antibody MON-100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON-100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON-100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON-100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON-100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of Mr 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti-7B2 monoclonal antibody MON-100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments.