Biomedical subjects

E K Chan

Publications and source records attributed to E K Chan.

At least 37 records · Page 2Linked to original sources

PubMed2000

The Golgi apparatus segregates from the lysosomal/acrosomal vesicle during rhesus spermiogenesis: structural alterations.

The acrosome is an acidic secretory vesicle containing hydrolytic enzymes that are involved in the sperm's passage across the zona pellucida. Imaging of the acrosomal vesicle and the Golgi apparatus in live rhesus monkey spermatids was accomplished by using the vital fluorescent probe LysoTracker DND-26. Concurrently, the dynamics of living spermatid mitochondria was visualized using the specific probe MitoTracker CMTRos and LysoTracker DND-26 detected the acrosomal vesicle from its formation through spermatid differentiation. LysoTracker DND-26 also labeled the Golgi apparatus in spermatogenic cells. In spermatocytes the Golgi is spherical and, in round spermatids, it is localized over the acrosomal vesicle, as confirmed by using polyclonal antibodies against Golgin-95/GM130, Golgin-97, and Golgin-160. Using both live LysoTracker DND-26 imaging and Golgi antibodies, we found that the Golgi apparatus is cast off from the acrosomal vesicle and migrates toward the sperm tail in elongated spermatids. The Golgi is discarded in the cytoplasmic droplet and is undetectable in mature ejaculated spermatozoa. The combined utilization of three vital fluorescent probes (Hoechst 33342, LysoTracker DND-26, and MitoTracker CMTRos) permits the dynamic imaging of four organelles during primate spermiogenesis: the nucleus, the mitochondria, the acrosomal vesicle, and the Golgi apparatus.

Acrosin↗

PubMed2000

Identification and characterization of a novel Golgi protein, golgin-67.

In the course of screening a lambdagt11 human leukemic T-cell cDNA expression library with an antibody specific to the mitotic target of Src, Sam68, we identified and cloned a cDNA encoding a novel protein with a predicted molecular mass of 51.4 kDa. Polyclonal antibodies raised to a His(6)-tagged construct of this protein, detected a approximately 67-kDa protein in immunoprecipitation experiments, and cytological studies showed that this protein localized to the Golgi complex, through colocalization experiments with specific Golgi markers. Therefore, we designated this protein golgin-67. Sequence analysis revealed that golgin-67 is a highly coiled-coil protein, with potential Cdc2 and Src kinase phosphorylation motifs. It has sequence homologies to other Golgi proteins, including the coatamer complex I vesicle docking protein, GM130. Structurally, golgin-67 resembles, golgin-84, an integral membrane Golgi protein with an N-terminal coiled-coil domain and a single C-terminal transmembrane domain. The C-terminal region of golgin-67, which contains a predicted transmembrane domain, was demonstrated to be essential for its Golgi localization.

Amino Acid Sequence↗

PubMed2000

Optimal parameters for laser tissue soldering: II. Premixed versus separate dye-solder techniques.

BACKGROUND AND OBJECTIVE: Laser tissue soldering by using an indocyanine green (ICG)-doped protein solder applied topically to the tissue surface and denatured with a diode laser was investigated in Part I of this study. The depth of light absorption was predominantly determined by the concentration of the ICG dye added to the solder. This study builds on that work with an in vitro investigation of the effects of limiting the zone of heat generation to the solder-tissue interface to determine whether more stable solder-tissue fusion can be achieved. STUDY DESIGN/MATERIALS AND METHODS: An alternative laser tissue soldering technique was investigated, which increased light absorption at the vital solder-tissue interface. A thin layer of ICG dye was smeared over the surface to be treated, the protein solder was then placed directly on top of the dye, and the solder was denatured with an 808-nm diode laser. Because laser light at approximately 800 nm is absorbed primarily by the ICG dye, this thin layer of ICG solution restricted the heat source to the space between the solder and the tissue surfaces. A tensile strength analysis was conducted to compare the separate dye-solder technique with conventional techniques of laser tissue soldering for which a premixed dye-solder is applied directly to the tissue surface. The effect of hydration on bond stability of repairs formed by using both techniques was also investigated using tensile strength and scanning electron microscopy analysis. RESULTS: Equivalent results in terms of tensile strength were obtained for the premixed dye-solder technique using protein solders containing 0.25 mg/ml ICG (liquid solder, 220 +/- 35 N/cm(2); solid solder, 602 +/- 32 N/cm(2)) and for the separate dye-solder technique (liquid solder, 228 +/- 41 N/cm(2); solid solder, 578 +/- 29 N/cm(2)). The tensile strength of native bovine thoracic aorta was 596 +/- 31 N/cm(2). Repairs created by using the separate dye-solder technique were more stable during hydration than their premixed dye-solder counterparts. The conventional premixed dye-solder was simpler and approximately twice as fast to apply. The separate dye-solder technique, however, increased the shelf-life of the solder, because the dye was mixed at the time of the experiment, thus conserving its spectral absorbency properties. CONCLUSION: Two laser-assisted tissue soldering techniques have been evaluated for repairing aorta incisions in vitro. The advantages and disadvantages of each of these techniques are discussed.

Absorption↗

PubMed2000

Human autoantibodies to a novel Golgi protein golgin-67: high similarity with golgin-95/gm 130 autoantigen.

Autoantibodies to subcellular organelles have been described in patients with various systemic rheumatic diseases and our laboratories have been focused on studies of the Golgi complex as the autoimmune target. We have previously isolated and described four of the five known Golgi autoantigens reported to date. During the characterization of Golgi autoantigen golgin-95/gm130, another human cDNA that shared a significant degree of similarity in both nucleotide and amino acid sequences was identified. Analysis of cDNAs from different libraries suggested that this is a distinct gene encoding a protein of 67 kDa which has four regions with sequence identity to gm130, ranging between 42 and 60%. In this report, we describe the complete cDNA encoding a closely related Golgi protein provisionally named golgin-67. Among a group of 84 human anti-Golgi sera, five (6%) were shown to recognize golgin-67. Anti-golgin-67 human sera and affinity purified rabbit antibody to the recombinant protein gave predominant Golgi staining. Golgin-67 is thus the smallest member of a growing family of Golgi autoantigens rich in alpha-helical coiled-coil domain. The current hypothesis for the generation of autoimmune antibody to the Golgi complex is discussed.

Amino Acid Sequence↗

PubMed2000

Structure and characterization of the murine p80 coilin gene, Coil.

Cajal bodies (coiled bodies, CBs) are nuclear organelles of unknown function and are characterized by a wide variety of components including various basal transcription and cell cycle proteins, the nucleolar proteins fibrillarin and Nopp140, numerous small nuclear ribonucleoproteins, the survival motor neuron protein complex, and the marker protein, p80 coilin. To gain insight into the role of p80 coilin in CBs, we have cloned the murine gene Coil and have mapped it to the distal portion of chromosome band 11D. The approximately 2.6-kb transcript is detectable in all tissues analyzed, with the highest levels in brain and testis. Sequence analysis shows that, like its human counterpart, the mouse coilin gene is composed of seven exons and spans nearly 30 kb of genomic DNA. The predicted amino acid sequence reveals two conserved N- and C-terminal domains, and comparison with the Xenopus SPH-1 protein reveals that these three genes are indeed orthologous. These results should facilitate gene disruption experiments aimed at creating a genetic model system to study CBs.

Amino Acid Sequence↗

PubMed2000

Autoantibodies to DFS 70 kd/transcription coactivator p75 in atopic dermatitis and other conditions.

BACKGROUND: Sera of patients with atopic dermatitis (AD) were found to have autoantibodies that reacted with tissue culture cell substrates in immunohistochemistry to display a characteristic pattern of nuclear distribution of dense fine speckles. The sera also recognized a 70-kd protein on Western immunoblots, and the antigen was termed dense fine speckles 70 kd (DSF70). OBJECTIVE: Because spontaneously occurring autoantibodies could be immune responses to proteins that might be participating in the disease process, it was of interest to identify the antigens driving the autoimmune antibody response. METHODS: A serum containing high-titer antibodies to DFS70 was used to immunoscreen a complementary (c)DNA expression library to isolate cDNA encoding the antigen. After the cDNA was isolated, this was used to express recombinant protein to determine the prevalence of antibody in AD and other conditions. RESULTS: Thirty percent of patients with AD were found to have antibody to recombinant DFS70 in Western immunoblots. Sixteen percent of patients with asthma and 9% of patients with interstitial cystitis had antibodies of the same specificities. The cDNA encoding DFS70 was identical to a transcription coactivator called p75, which had been shown to be required for RNA polymerase II-dependent transcription. Another important finding was that IgE antibodies to DFS70 were also present in AD sera. CONCLUSION: It is suggested that a common basis for the presence of autoantibodies to DFS70 might be related to AD in asthma, interstitial cystitis, and other conditions. A possible role of this antigen-antibody system in pathogenesis remains to be demonstrated, but it appears to be a marker for a subset of patients with AD.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Vesicular traffic and golgi apparatus dynamics during mammalian spermatogenesis: implications for acrosome architecture.

Vesicular membrane trafficking during acrosome biogenesis in bull and rhesus monkey spermatogenesis differs from the somatic cell paradigm as imaged dynamically using the Golgi apparatus probes beta-COP, giantin, Golgin-97, and Golgin-95/GM130. In particular, sorting and delivery of proteins seemed less precise during spermatogenesis. In early stages of spermiogenesis, many Golgi resident proteins and specific acrosomal markers were present in the acrosome. Trafficking in both round and elongating spermatids was similar to what has been described for somatic cells, as judged by the kinetics of Golgi protein incorporation into endoplasmic reticulum-like structures after brefeldin A treatment. These Golgi components were retrieved from the acrosome at later stages of differentiation and were completely devoid of immature spermatozoa. Our data suggest that active anterograde and retrograde vesicular transport trafficking pathways, involving both beta-COP- and clathrin-coated vesicles, are involved in retrieving Golgi proteins missorted to the acrosome and in controlling the growth and shape of this organelle.

Acrosome Reaction↗

PubMed2000

Coagulum index predicts coagulum formation in right atrial radiofrequency energy delivery to ablate atrial fibrillation.

An ongoing multicenter U.S. clinical study evaluates the safety and effectiveness of creating linear lesions with radiofrequency (RF) energy using a 3.7 Fr microcatheter used with a 9 Fr steerable guiding catheter in the right atrium (RA) to treat paroxysmal atrial fibrillation (PAF). Study entry criteria were symptomatic, drug refractory PAF. RF energy was delivered in 60-second episodes at 30 W or 50 W power settings. Electrode tissue contact was ascertained by sharp electrograms of high amplitude. Coagulum Index (C.I.), a new calculation to predict the probability of coagulum development, was derived analytically from measurements of RF power, RF current, and time to reach maximum temperature in each ablation episode. The RF data from 398 separate ablation episodes in 15 patients were used to calculate C.I. Coagulum presence was determined by postablation visual inspection of the microcatheter electrodes. A logistic model was used to estimate the probability of coagulum occurrence, with C.I. as the predictive variable. When C.I. > or = 12, the probability of coagulum formation increased significantly in this model (P < 0.0001). Prolonging the power delivery rise time and limiting maximum RF power setting to 30 W effectively lowers C.I. and minimizes coagulum formation. These results should also apply to left atrium ablation, where minimizing coagulum formation may decrease the risk of stroke or mortality.

Atrial Fibrillation↗

PubMed2000

Autoantibodies from patients with idiopathic ataxia bind to M-phase phosphoprotein-1 (MPP1).

In an attempt to identify unique disease-related autoantibodies, the serum from an ataxia and sensory neuropathy patient was used as a probe to isolate a 2.5-kd cDNA from a HeLa expression library. The nucleotide sequence was 99% identical to MPP1, a cell-cycle-related nuclear protein phosphorylated during mitosis. Expression of the cDNA in an in vitro translation system yielded a recombinant protein that migrated in SDS-PAGE at approximately 97 kd. This protein was immunoprecipitated by the prototype human serum, by an immune guinea pig anti-MPP1 serum, but not by normal human serum or preimmune guinea pig serum. Western blot analysis of HeLa cell proteins showed that the prototype human serum and immune guinea pig antiserum recognized an approximately 225-kd protein, suggesting that the isolated clone contained a partial cDNA. By indirect immunofluorescence, the affinity-purified antibody and a guinea pig antiserum reacted with nuclei of interphase HEp-2 cells and the cytoplasm of certain neuronal cells. Sera from 10 of 25 unselected patients with ataxia, 1 of 30 patients with peripheral neuropathy, 1 of 50 multiple sclerosis patients, 0 of 20 amyotrophic lateral sclerosis, 0 of 10 children with postviral ataxia, 0 of 10 systemic lupus erythematosus patients, 0 of 3 patients with hereditary cerebellar ataxia, 0 of 8 with ataxia telangiectasia, and 0 of 30 age- and gender-matched controls immunoprecipitated the recombinant MPP1 protein. None of the patients with anti-MPP1 antibodies had evidence of malignancy. This is the first report of MPP1 as a target autoantigen in patients with idiopathic ataxia.

Adult↗

PubMed1999

A novel cytoplasmic protein with RNA-binding motifs is an autoantigen in human hepatocellular carcinoma.

In hepatocellular carcinoma (HCC), autoantibodies to intracellular antigens are detected in 30-40% of patients. Patients with chronic hepatitis or liver cirrhosis develop HCC, and when this occurs, some patients exhibit autoantibodies of new specificities. It has been suggested that these novel autoantibody responses may be immune system reactions to proteins involved in transformation-associated cellular events. One HCC serum shown to contain antibodies to unidentified cellular antigens was used to immunoscreen a cDNA expression library, and a full length cDNA clone was isolated with an open reading frame encoding 556 amino acids with a predicted molecular mass of 62 kD. The 62-kD protein contained two types of RNA-binding motifs, the consensus sequence RNA-binding domain (CS-RBD) and four hnRNP K homology (KH) domains. This protein, provisionally called p62, has close identity (66-70%) to three other proteins at the amino acid sequence level, and all four proteins may belong to a family having CS-RBD in the NH2-terminal region and four KH domains in the mid-to-COOH- terminal region. The homologous proteins are: KH domain-containing protein overexpressed in cancer (Koc); zipcode binding protein, a protein which binds to a conserved nucleotide element in chicken beta-actin mRNA (ZBP1); and a protein which binds to a promoter cis element in Xenopus laevis TFIIIA gene (B3). p62 protein is cytoplasmic in location, and autoantibodies were found in 21% of a cohort of HCC patients. Patients with chronic hepatitis and liver cirrhosis, conditions which are frequent precursors to HCC, were negative for these autoantibodies, suggesting that the immune response might be related to cellular events leading to transformation. However, the possible involvement of p62 autoantigen as a factor in the transformation process remains to be elucidated.

Amino Acid Sequence↗

PubMed1999

Use of an agent to reduce scattering in skin.

BACKGROUND AND OBJECTIVE: A method to increase light transport deeply into target areas of tissue would enhance both therapeutic and diagnostic laser applications. The effects of a hyperosmotic agent on the scattering properties of rat and hamster skin were investigated. STUDY DESIGN/MATERIALS AND METHODS: A hyperosmotic agent, glycerol, was applied in vitro and in vivo to rat and hamster skin to assess the changes in tissue optical properties. Changes in the reduced scattering coefficient after application of the agent in vitro to rat skin and after the skin has been rehydrated were assessed to evaluate the effect of the agent on tissue. RESULTS: Experimental results showed a transient change in the optical properties of in vitro rat skin. A 50% increase in transmittance and decrease in diffuse reflectance occurred within 5-10 min after the introduction of anhydrous glycerol. In addition, reduction of light scattering with this technique increased depth of visibility with optical coherence tomography. Injection of glycerol under the skin allowed in vivo visualization of blood vessels. CONCLUSIONS: The application of the agent reduces the amount of refractive mismatch found in the tissue and markedly reduces random scattering, thereby making the skin less turbid for visible wavelengths for a controlled period of time.

Administration, Topical↗

PubMed1999

Optimal parameters for laser tissue soldering. Part I: tensile strength and scanning electron microscopy analysis.

BACKGROUND AND OBJECTIVES: The use of liquid and solid albumin protein solders to enhance laser tissue repairs has been shown to significantly improve postoperative results. The published results of laser-solder tissue repair studies have, however, indicated inconsistent success rates. This can be attributed to variations in laser irradiance, exposure time, solder composition, chromophore type, and concentration. An in vitro study was performed using indocyanine green-doped albumin protein solders in conjunction with an 808 nm diode laser to determine optimal laser and solder parameters for tissue repair in terms of tensile strength and stability during hydration. STUDY DESIGN/MATERIALS AND METHODS: Twenty-five different combinations of laser irradiance (6.4, 12.7, 19.1, 25.5, 31.8 W/cm2) and exposure time (20, 30, 40, 50, 100 or 40, 60, 80, 100, 200 seconds) were used. The effect of changing bovine serum albumin (BSA) concentration (25% and 60%) and indocyanine green (ICG) dye concentration (2.5 mg/ml and 0.25 mg/ml) of the protein solder on the tensile strength of the resulting bonds was investigated. The effect of hydration on bond stability was also investigated using both tensile strength and scanning electron microscopy analysis. RESULTS: Tensile strength was observed to decrease significantly with increasing irradiance. An optimum exposure time was found to exist where further irradiation did not improve the tensile strength of the bond. Tensile strength was found to be greatly improved by increasing the BSA concentration. Finally, the lower ICG dye concentration increased the penetration depth of the laser light in the protein solder leading to higher tensile strengths. The strongest repairs were formed by using 6.4 W/cm2 irradiation for 50 seconds with a protein solder composed of 60% BSA and 0.25mg/ml ICG. In addition, the solid protein solder provided more stable adhesion to the tissue than did the liquid protein solder when the tissue was submerged in a hydrated environment. CONCLUSIONS: This study greatly enhances the current understanding of the various factors affecting the soldering process. It provides a strong basis for optimization of the laser light delivery parameters and the solder constituents to achieve strong and reliable laser tissue repairs.

Animals↗

PubMed1999

Defining a novel 75-kDa phosphoprotein associated with SS-A/Ro and identification of distinct human autoantibodies.

Mothers of children with neonatal lupus erythematosus (NLE) and heart block, as well as patients with Sjögren's syndrome (SS) and systemic lupus erythematosus, have serum autoantibodies that recognize SS-A/Ro autoantigens including the 60-kDa ribonucleoprotein. By yeast 2-hybrid screening, we identified a novel 75-kDa protein (pp75) that interacts with the carboxyl 70% of 60-kDa SS-A/Ro. The specificity of interaction was confirmed using mammalian 2-hybrid and chemical crosslinking studies. Immunoprecipitation with radiolabeled HeLa cell extracts showed that pp75 was phosphorylated and associated with 2 other phosphoproteins of 64 kDa. In Northern blot analysis, pp75 was expressed in all tissues analyzed; the highest expression was in the human heart. Based on immunofluorescence of transfected HeLa cells, pp75 is localized predominantly in the cytoplasm, an observation confirmed by immunohistochemistry in untransfected cells. Based on Western blot and ELISA assays, sera from 14 of 84 mothers of children with NLE recognized pp75, including 1 mother in whom anti-SS-A/Ro antibodies were not detected. In addition, sera from 5 of 80 patients with SS were positive for anti-pp75 antibody. Identification of molecular partners is a first step toward elucidating the functions and possible involvement in pathogenesis of long-recognized autoantigens such as 60-kDa SS-A/Ro, which are at present poorly understood.

Adaptor Proteins, Signal Transducing↗

PubMed1999

mRNA and protein expression of SSA/Ro and SSB/La in human fetal cardiac myocytes cultured using a novel application of the Langendorff procedure.

Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric alpha-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 x 10(6) cells was obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17beta-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contributing to the development of congenital heart block. Differential constitutive and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the marked discordance of clinically detectable injury in these two target tissues.

Abortion, Induced↗

PubMed1999

Early endosome antigen. 1: An autoantigen associated with neurological diseases.

BACKGROUND: We have identified 36 human sera sent for autoantibody analyses that produce a unique vesicular staining pattern of the cytoplasm of tissue culture cells. The purpose of this study was to identify the autoantigens that are recognized by the sera that produce this staining pattern and determine if the patients have common clinical features. METHODS: A serum from one of the patients (MS) with rapidly progressive demyelinating polyneuropathy was used to isolate a approximately 4.5 kb cDNA insert from a HeLa expression library. The purified cDNA (MS-5.1) was characterized by a poly A tail and an open reading frame that encoded 1329 amino acids. The derived amino acid sequence was found to be 99% identical to a 180 kd peripheral endosomal protein named early endosome antigen (EEA1). RESULTS: Antibodies from rabbits immunized with the recombinant protein and the prototype human serum produced an identical distinctive speckled cytoplasmic staining pattern. These sera also precipitated the in vitro translated recombinant protein and reacted with the isolated recombinant protein in a Western immunoblot. Of the 36 sera that produced an identical staining pattern as the prototype and immune rabbit sera, 8 (22%) had IgG antibodies that recognized the recombinant EEA1 protein when tested by immunoblotting and immunoprecipitation assays. Of the 8 patients with anti-EEA1 antibodies 4 were females, 4 were males, and the mean age was 69 years (range 48 to 86 years). CONCLUSIONS: Diagnoses included: polyneuropathy, lower motor neuron disease, pigmented retinitis, seronegative polyarthritis, interstitial pulmonary fibrosis, Raynaud's phenomenon, Wegener's granulomatosis, and proteinuria. Three of the eight patients with EEA1 autoantibodies died within 1 year after EEA1 antibodies were identified.

Aged↗

PubMed1998

Induction of antibodies reactive with SSA/Ro-SSB/La and development of congenital heart block in a murine model.

To correlate the arrhythmogenic effects of maternal autoantibodies with the genesis of congenital heart block, female BALB/c mice were immunized with human recombinant 48-kDa SSB/La, 60-kDa SSA/Ro, 52-kDa SSA/Ro (52alpha), and 52beta (amino acids 169-245 deleted) as well as with murine recombinant 52-kDa SSA/Ro. Control animals received beta-galactosidase or a polypeptide encoded by pET-28 alone. Following primary immunization and two boosters, high titer responses to the respective Ags were established by ELISA, immunoblotting, and immunoprecipitation. Sera from mice immunized with either human 52alpha or 52beta immunoprecipitated murine 52Ro. mRNA and protein expression of 52Ro was demonstrated in the newborn murine heart. A spectrum of atrioventricular nodal conduction abnormalities was identified by electrocardiogram. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48La, 20% of 54 pups born to 60Ro-immunized mothers, 6% of 56 pups born to 52alpha-immunized mothers, 7% of 86 pups born to 52beta-immunized mothers, and 9% of 22 pups born to mothers immunized with murine 52Ro. Advanced conduction abnormalities were only identified in offspring of 52alpha- or 52beta-immunized mice. In the 52alpha group, one pup had complete block and another had second-degree block (Wenckebach type); in the 52beta group, five pups had complete block. Maternal Abs to the primary immunogens were detected in the pups. No control had any conduction abnormalities. This Ab-specific animal model provides strong evidence for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro, in the development of congenital heart block. The range and frequency of conduction defects suggest that additional factors promote disease expression.

Animals↗

PubMed1998

Accessibility of SSA/Ro and SSB/La antigens to maternal autoantibodies in apoptotic human fetal cardiac myocytes.

Access of intracellular Ags SSA/Ro and SSB/La to cognate maternal autoantibodies is unexplained despite their strong association with congenital heart block. To investigate the hypothesis that apoptosis facilitates surface accessibility of these Ags, human fetal cardiac myocytes from 16- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusing the aorta with collagenase. Confirmation of cardiac myocytes included positive staining with antisarcomeric alpha-actinin and contractility induced by 1.8 mM calcium. Incubation with 0.5 microM staurosporine or 0.3 mM 2,3-dimethoxy-1,4-naphthoquinone induced the characteristic morphologic and biochemical changes of apoptosis. The cellular topology of Ro and La was evaluated with confocal microscopy and determined in nonapoptotic and apoptotic cardiocytes by indirect immunofluorescence. In permeabilized nonapoptotic cardiocytes, Ro and La were predominantly nuclear, and propidium iodide (PI) stained the nucleus. In early apoptotic cardiocytes, condensation of the PI- and Ro- or La-stained nucleus was observed, accompanied by Ro/La fluorescence around the cell periphery. In later stages of apoptosis, nuclear Ro and La staining became weaker, and PI demonstrated nuclear fragmentation. Ro/La-stained blebs emerged from the cell membrane, a finding observed in nonpermeabilized cells, supporting an Ab-Ag interaction at the cell surface. In summary, induction of apoptosis in cultured cardiocytes results in surface translocation of Ro/La and recognition by Abs. Although apoptotic cells are programmed to die and do not characteristically evoke inflammation, binding of maternal Abs and subsequent influx of leukocytes could damage surrounding healthy fetal cardiocytes.

Antigen-Antibody Reactions↗

PubMed1998

Molecular remedy of complex I defects: rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells.

The NDI1 gene encoding rotenone-insensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae mitochondria was cotransfected into the complex I-deficient Chinese hamster CCL16-B2 cells. Stable NDI1-transfected cells were obtained by screening with antibiotic G418. The NDI1 gene was shown to be expressed in the transfected cells. The expressed Ndi1 enzyme was recognized to be localized to mitochondria by immunoblotting and confocal immunofluorescence microscopic analyses. Using digitonin-permeabilized cells, it was shown that the transfected cells, but not nontransfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrate. The activities were inhibited by flavone, antimycin A, and KCN but not by rotenone. Added NADH did not serve as the substrate, suggesting that the expressed Ndi1 enzyme was located on the matrix side of the inner mitochondrial membranes. Furthermore, although nontransfected cells could not survive in a medium low in glucose (0.6 mM), which is a substrate of glycolysis, the NDI1-transfected cells were able to grow in the absence of added glucose. When glycolysis is slow, either at low glucose concentrations or in the presence of galactose, respiration is required for cells to survive. The mutant cells do not survive at low glucose or in galactose, but they can be rescued by Ndi1. These results indicated that the S. cerevisiae Ndi1 was expressed functionally in CCL16-B2 cells and catalyzed electron transfer from NADH in the matrix to ubiquinone-10 in the inner mitochondrial membranes. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

Animals↗