A rapid ELISA-based serum assay for myelin basic protein in multiple sclerosis.

We have developed a sensitive, ELISA-based assay to detect autoantibodies to myelin basic protein (MBP) in human serum. Autoantibody levels were measured in 98 normal healthy adults (age range 20-66) and 94 clinically definite multiple sclerosis (MS) cases (age range 18-63). Of the MS patients, 77% had elevated levels of MBP autoantibodies (IgG) whereas only five normal individuals had antibody levels increased over normal. From the receiver-operator curve (ROC), the mean+/-2SD as clinical decision limit offers high sensitivity (77%) and specificity (95%). No change in assay performance was observed when hemoglobin, triglycerides or bilirubin were added to serum samples. The success of the assay is dependent on the use of heparin, an anionic molecule, which neutralizes the positive charge on the highly cationic MBP.

Heterogeneity of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) at the level of trimeric subunits.

To study organization of the main light-harvesting chlorophyll a/b-protein complex of photosystem II (LHCII) from spinach thylakoid membranes at the level of trimeric subcomplexes, we have applied non-denaturing isoelectric focusing (ndIEF) in vertical, slab polyacrylamide gels. When analyzed by two consecutive ndIEF/electroelution runs, spinach BBY membrane preparations (PSII(alpha)-enriched, stacked thylakoid membranes) were resolved into nine fractions of 100% purity, labelled 1-9 in order of decreasing pI values. Seven of these fractions (3-9) were shown by absorption spectroscopy to stand for LHCII subcomplexes. The subcomplexes were established - by monitoring their circular dichroism spectra and comparing them to the spectra of native LHCII trimers and monomers - to be structurally intact trimers. The analysis of polypeptide composition of the subcomplexes in terms of apparent molecular masses and Lhcb genes' products led us to the conclusion that each of the subcomplexes might be a mixed population of closely similar individual trimers, comprising of permutations of Lhcb1 and Lhcb2 (subcomplexes 3-7) or Lhcb1, Lhcb2 and Lhcb3 (subcomplexes 8 and 9).

Identification of Lhcb1/Lhcb2/Lhcb3 heterotrimers of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II).

Using non-denaturing isoelectric focusing in polyacrylamide vertical slab gel, we have purified to homogeneity three trimeric subcomplexes of LHC II from Arabidopsis thylakoid membranes. The polypeptide composition of the subcomplexes were studied by immunoblotting. Our results indicate the existence in vivo of LHC II heterotrimers containing Lhcb1, Lhcb2 and Lhcb3 gene products.

An Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins.

An Arabidopsis thaliana cDNA clone encoding a novel 110 amino acid thylakoid protein has been sequenced. The in vitro synthesized protein is taken up by intact chloroplasts, inserted into the thylakoid membrane and the transit peptide is cleaved off during this process. The mature protein is predicted to contain 69 amino acids, to form one membrane-spanning alpha-helix and to have its N-terminus at the stromal side of the thylakoid membrane. The protein showed similarity to the LHC, ELIP and PsbS proteins of higher plants, but more pronounced to the high-light-inducible proteins (HLIPs) of cyanobacteria and red algae, to which no homologue previously has been detected in higher plants. As for HLIP and ELIP, high light increases the mRNA levels of the corresponding gene. Sequence comparisons indicate that the protein may bind chlorophyll and form dimers in the thylakoid membrane. The level of expression of the protein seems to be far lower than that of normal PSI and PSII subunits.

Preparation of Schiff base adducts of phosphatidylcholine core aldehydes and aminophospholipids, amino acids, and myoglobin.

We have prepared Schiff base adducts of the core aldehydes of phosphatidylcholine and aminophospholipids, free amino acids, and myoglobin. The Schiff bases of the ethanolamine and serine glycerophospholipids were obtained by reacting sn-1-palmitoyl(stearoyl)-2-[9-oxo]nonanoyl-glycerophosphocholine (PC-Ald) with a twofold excess of the aminophospholipid in chloroform/methanol 2:1 (vol/vol) for 18 h at room temperature. The Schiff bases of the amino acids and myoglobin were obtained by reacting the aldehyde with an excess of isoleucine, valine, lysine, methyl ester lysine and myoglobin in aqueous methanol for 18 h at room temperature. Prior to isolation, the Schiff bases were reduced with sodium cyanoborohydride in methanol for 30 min at 4 degrees C. The reaction products were characterized by normal-phase high-performance liquid chromatography and on-line mass spectrometry with electrospray ionization. The amino acids and aminophospholipids yielded single adducts. A double adduct was obtained for myoglobin, which theoretically could have accepted up to 23 PC-Ald groups. The yields of the products ranged from 12 to 44% for the aminophospholipids and from 15-57% for the amino acids, while the Schiff base of the myoglobin was estimated at 5% level. The new compounds are used as reference standards for the detection of high molecular weight Schiff bases in lipid extracts of natural products.

Removal of endotoxin from recombinant protein preparations.

OBJECTIVES: To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications. DESIGN AND METHODS: Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer. In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions. RESULTS: Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies. CONCLUSION: Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.

Analytical performance and clinical utility of a sensitive immunoassay for determination of human cardiac troponin I.

OBJECTIVES: To determine the serum and plasma level of human cardiac troponin I (cTnI) resulting from myocardial damage, we have developed a sensitive and specific one-step enzyme immunoassay to measure cardiac troponin I. DESIGN AND METHODS: The COBAS cTnI assay is a semi-automated one-step solid phase immunoassay compatible with the COBAS Core. The assay is performed in a sandwich type format using a polyclonal goat antibody capture and two highly specific horseradish peroxidase conjugated monoclonal antibody detectors directed against different epitopes of the cTnI molecule. Calibrators were made with purified recombinant cTnI. RESULTS: The level of cTnI was determined in 84 healthy donors with no evidence of myocardial injury, resulting in a lower limit of detection (LLD) of 0.09 microgram/L. The upper reference limit (URL) of the normal reference range was calculated as 0.20 microgram/L. The dynamic range of the consequent EIA was between 0.09 and 6.0 micrograms/L with a total assay time of 45 min. Intra-assay and inter-assay variances (CVs) were < or = 4%. Cross-reactivity with fast and slow skeletal troponin I was absent in concentrations up to 2.0 mg/L. Common interferents yielded negative results in the cTnI assay. Clinical utility was confirmed by measuring the circulating serum or plasma levels of cardiac troponin I in serial samples from marathon runners, clinical samples from trauma patients, and patients presenting to the Emergency Department with complaints of chest pain. Results were further evaluated using clinical diagnosis at discharge and quantified concentrations of other cardiac markers by a Stratus analyzer and ELISA procedures. CONCLUSIONS: Results from normal and clinical samples assayed in house for cTnI concentrations indicate that the Spectral EIA is a highly sensitive means of quantifying cTnI levels in serum and plasma for acute cardiac syndrome. The cardiac specificity of cTnI over other well-known cardiac markers is reflected in experimental results and parallel clinical diagnosis.

Evaluation of cardiac STATus CK-MB/myoglobin device for rapidly ruling out acute myocardial infarction.

The Cardiac STATus CK-MB/Myoglobin device is highly sensitive and has a high negative predictive value within 3 hours of patient presentation. The device may play a role in the re-triage of patients from the CCU to less intensive settings, resulting in a net cost savings.

Use of enzyme immunoassay for measurement of skeletal troponin-I utilizing isoform-specific monoclonal antibodies.

OBJECTIVE: To determine the serum level of fast skeletal troponin I (fsTnl) resulting from skeletal muscle damage, we have developed a sensitive two-site enzyme immunoassay to measure skeletal troponin I. DESIGN AND METHODS: Twelve monoclonal antibodies were raised against human fsTnl. Of these antibodies, 8 were fsTnl-specific and the remaining 4 reacted with both skeletal and cardiac troponin I (cTnl). Two monoclonals were utilized for a development of this fsTnl immunoassay. Standards were made with purified recombinant human fsTnl for the range of 0-25 micrograms/mL. RESULTS: Total assay variance (CV) ranged from 1.7% to 9.6%. The upper limit of the normal reference range was established as 0.2 microgram/L by determining fsTnl concentration in sera of 108 healthy donors without evidence of muscle damage. Purified human cTnl up to 500 micrograms/L and cTnl-positive clinical serum samples yielded negative results in the fsTnl assay. The serum levels of fsTnl were determined in trauma patients, patients with chronic degenerative muscle disease, and marathon runners. In the study populations, the serum levels of fsTnl were correlated with other biochemical markers that are traditionally used to monitor striated muscle damage. CONCLUSIONS: In the present preliminary studies, measuring the serum levels of fsTnl in patients with various forms of muscle damage is more accurate than using the classical non muscle-specific biochemical markers.

Advantages of backscatter electron imaging scanning electron microscopy for intracellular localization of cardiac analytes by gold conjugated antibody.

Myoglobin and myosin light chain 1 (MLC1) are intracellular human cardiac marker proteins which are released as a consequence of ischemia. Human cardiomyocytes were isolated from fresh biopsies and also maintained for several passages in cell culture. The cardiomyocytes were fixed in 100% methanol at -20 degrees C, and labeled. The immunolocalization of intracellular antigen by fluorescence conjugated imaging was compared with scanning electron microscopy (SEM) backscatter electron (BSE) imaging of gold conjugated antibody. Ultra-violet light microscopy showed the intracellular distribution of both proteins to be mainly in the nuclear envelope, the cytoplasm immediately surrounding the nucleus and along portions of the cell membrane. To confirm this observed distribution of myoglobin and MLC1, labeling was repeated with antimyoglobin and anti-MLC1 monoclonal antibodies conjugated to colloidal gold particles. The advantage of colloidal gold labeling is that the intracellular antigen-antibody complexes may be more precisely located because of the significant improvement in resolution provided by BSE imaging in the SEM. BSE imaging confirmed the presence and subsarcolemma localization of myoglobin in cardiomyocytes directly isolated from fresh biopsies. The distribution of colloidal gold-conjugated antibodies did not coincide with the intracellular distribution of the two proteins in the cardiomyocytes grown in cell culture as indicated by immunofluorescence. A relatively random, intracellular gold particle distribution was confirmed by x-ray microanalysis. BSE imaging resulted in consistent auto-backscatter labeling patterns very similar to the labeling patterns obtained with immunofluorescent labeling. X-ray microanalysis confirmed that these auto-backscatter labeling patterns were formed by concentrations of intracellular phosphate. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and subsequent Western blotting indicated that myoglobin and MLC1 were no longer present in detectable quantities in these cells after several passages. Polymerase chain reaction (PCR) amplification of mRNA for human myoglobin and cardiac MLC1 confirmed the absence of their transcripts. Electrophoretic analysis of proteins in cardiomyocytes grown in cell culture confirmed an increasing presence of alkaline phosphatase. Staining of this enzyme with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium showed that alkaline phosphatase was distributed in the same intracellular pattern as the fluorescence conjugated anti-body and the phosphatase auto-backscatter. These results indicate that high-resolution backscatter SEM imaging may be used as necessary control to confirm fluorescence light microscope intracellular labeling of antigens.

Specific replacement of consecutive AGG codons results in high-level expression of human cardiac troponin T in Escherichia coli.

The adult isoform of human cardiac troponin T (TnT) contains 288 amino acids, 14 of which (4.9%) are encoded by the rarely used arginine codons (12 AGG, 2 AGA) in Escherichia coli genes. To generate sufficient quantity of TnT protein for antibody production, we cloned the corresponding cDNA and expressed it in E. coli. A low-level expression of TnT that comprised only about 1% of total cell protein was initially observed with the use of the native cDNA. The existence of two pairs of consecutive AGG codons AGG(165) AGG(166) and AGG(215) AGG(216) in the cDNA was suspected to be the main cause for this low-level expression. These two pairs of consecutive AGG codons were successively replaced with the major synonymous codon CGT by site-directed mutagenesis. As suspected, a 10-fold increase in TnT expression was obtained when one pair of the rare arginine codons was replaced and a 40-fold increase was achieved when both pairs of the rare codons were replaced. Our finding demonstrates the importance of consecutive rare codons in the suppression of high-level expression of heterologous proteins in E. coli and suggests that in order to maximize protein expression, a similar approach may be taken with other genes which contain consecutive rare codons.

The resolution and biochemical characterization of subcomplexes of the main light-harvesting chlorophyll a/b-protein complex of Photosystem II (LHC II).

LHC II isolated from carnation leaves has been solubilized and resolved by a newly developed, vertical-bed non-denaturing isoelectric focusing in polyacrylamide slab gels to yield three trimeric subcomplexes focusing at pH 4.52, 4.42 and 4.37 (designated a, b and c, respectively), comprising approximately 38%, 24% and 38% of the chlorophyll. The spectroscopic data demonstrated a close similarity among LHC II subcomplexes concerning their chlorophyll content and organization. The most alkaline and the most acidic subcomplex contained the 27 kDa polypeptide of LHC II while the intermediate pI fraction contained both LHC II polypeptides, i.e. 27 kDa and 26 kDa ones associated at 2:1 stoichiometry. The 27 kDa polypeptide could be resolved by denaturing isoelectrofocusing into 10 pI molecular isoforms covering 5.90-4.20 pH range. Three of the isoforms were found in the subcomplexes a and b and eight in the subcomplex c. The 26 kDa polypeptide comprised the unique pI molecular isoform focusing at pH 5.61.

Analysis of the upstream regulatory region of human ventricular myosin light chain 1 gene.

To explore the mechanisms regulating expression of ventricular myosin light chain 1, the human gene including 5'-flanking DNA was cloned and characterized by Southern blot and restriction mapping. A 2 kb 5'-flanking DNA was sequenced and linked to a chloramphenicol acetyltransferase reporter gene. The constructs then were transfected into cultured human and rat cardiomyocytes as well as rat aortic endothelial cells. Deletion analysis of constructs revealed that the basal promoter sequences, which were located within 62 base pairs of the cap site, could direct high levels of chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. The region between -62 to -312 base pairs strongly repressed the chloramphenicol acetyltransferase gene expression in the cardiomyocytes and endothelial cells. Positive elements were found between -312 and -2000 base pairs of the cap site. These results are indicative, among other possibilities, that the human ventricular myosin light chain 1 gene is turned on in cardiomyocytes by the presence of trans-acting factors that are bound to upstream positive elements and is turned off in non-muscle cells by the presence of repressor-binding proteins. But this mechanism remains to be established.

Expression of ventricular myosin subunits in the atria of children with congenital heart malformations.

The presence of ventricular myosin light chains in the atria of children with congenital heart disease was demonstrated by two-dimensional polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis. Ventricular myosin light chains were present in 27% of biopsies from 91 children with different forms of congenital heart disease. Perimembranous ventricular septal defects and tetralogy of Fallot were associated with the presence of ventricular myosin light chains in 50% of patients. The presence of ventricular myosin light chains in these atria did not correlate with pressure or volume overload. Analysis of myosin heavy chain isotype in the same biopsies by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and Western blot analysis indicated that there was no detectable expression of ventricular myosin heavy chain (beta-subunit), suggesting that the genes for the myosin heavy chains and light chains are not expressed coordinately.

Comparison of ion-exchange chromatography, isoelectric precipitation and reversed-phase high-performance liquid chromatography for the separation of individual cardiac myosin light chains.

Three modified procedures for the separation of cardiac myosin light chains are carefully compared. Ion-exchange chromatography gives a purified cardiac myosin light chain 1, whereas light chain 2 is always contaminated by light chain 1. Reversed-phase high-performance liquid chromatography gives the best resolution of these light chains and needs only 20 min for each run. However, it requires pure preparation of myosin light chains before separation. Isoelectric precipitation is the simplest procedure and suitable for large quantities of material. Although it gives the highest yield the separation is not adequate. A modified and rapid procedure for the isolation of cardiac and skeletal total myosin light chains is also presented.

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells.

P19 embryonal carcinoma cells are multipotential stem cells that differentiate into striated muscle as well as some other cell types when aggregated and exposed to dimethyl sulfoxide (DMSO). Immunofluorescence experiments using monospecific antibodies indicated that the majority of muscle cells were mononucleate and contained four myosin isoforms normally found in cardiac muscle; atrial and ventricular myosin heavy chains, ventricular myosin light chain 1, and atrial myosin light chain 2. Northern blot analysis of RNA isolated from differentiating cultures indicated that cardiac actin and skeletal actin mRNAs were expressed at similar levels and with identical kinetics during the differentiation of P19-derived myocytes. These results demonstrate that most of the P19-derived myocytes are of the cardiac type and suggest that they closely resemble the cells of the early embryonic myocardium.

Comparison of the biosynthesis and composition of polyglycerophosphatides and phosphatidylinositols in mitochondria and microsomes isolated from neonatal and adult rat heart and liver.

The level of biosynthesis and the composition of polyglycerophosphatides (phosphatidylglycerol, phosphatidyglycerolphosphate, and diphosphatidylglycerol or cardiolipin) and phosphatidylinositols were examined in mitochondria and microsomes, respectively, isolated from neonatal and adult rat heart and liver. Biosynthesis of [3H]polyglycerophosphatides [( 3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate) was 4.5 times higher in neonatal than in adult heart mitochondria, whereas in the respective liver mitochondria this synthesis was only 15% higher in neonatal mitochondria. The biosynthesis of [3H]phosphatidylinositol was twice as high in neonatal as in adult heart microsomes, but very similar in the respective liver microsomes. The major biosynthesized polyglycerophosphatide was [3H]phosphatidylglycerol. The accumulation of [3H]phosphatidylglycerolphosphate depended on the origin of the mitochondria. Under our experimental conditions [3H]phosphatidylinositol was the only synthesized phosphoinositide in all microsomes. The biosynthesis of cardiolipin depended on the origin of the mitochondria and was highest in adult rat liver mitochondria and lowest in adult heart mitochondria. In all cases the biosynthesized [14C,3H] cardiolipin from [14C]phosphatidylglycerol and [3H]CDP-diglycerides had a ratio of 14C/3H around unity. The biosynthesis of [3H]CDP-diglycerides, the key precursor for the biosynthesis of phosphatidylglycerol, phosphatidylinositol, and cardiolipin, was 30% higher in neonatal than in adult heart microsomes and very similar in the respective liver microsomes. The subcellular localization of the enzymes required for the biosynthesis of the lipids and liponucleotides examined was found to be the same in membranes isolated from neonatal and adult rat heart and liver.

Effect of oxygen tension on the anti-oxidant enzyme activities of tetralogy of Fallot ventricular myocytes.

Since the chronically cyanotic tetralogy of Fallot (TOF) myocardium is more sensitive to reperfusion injury after cardiac surgery than the adult myocardium, we decided to study the regulation of myocardial superoxide dismutase (SOD), catalase and glutathione peroxidase by oxygen tension. TOF myocytes were cultured at a Po2 of 150 mmHg for 30 days to establish the culture. The cells were then cultured at Po2 of 150 and 40 mmHg and the myocyte antioxidant enzymes measured at days 3, 7, 14 and 21. On day 21 the myocytes cultured at Po2 of 40 mmHg were then cultured at 150 mmHg and SOD and catalase activities measured on days 28 and 35. Although there were no differences in the rates of incorporation of 35S-methionine into the myocytes at either Po2 on these days, the myocytes scavenger enzyme levels were significantly higher by day 14 when cultured at a Po2 of 150 mmHg than at a Po2 of 40 mmHg. With the increase in oxygen tension from 40 to 150 mmHg, SOD and catalase activities increased significantly by day 35. The myocytes cultured at Po2 40 mmHg were more sensitive by day 7 to an hypoxanthine-xanthine oxidase generated free radical injury than the Po2 150 mmHg cultured cells. The regulation of these enzyme activities by oxygen tension and the increased sensitivity to free radical injury of the myocytes cultured at an oxygen tension of 40 mmHg provide putative evidence that the chronically cyanotic myocardium may be less well protected than the normally perfused myocardium against oxygen-mediated free radical injury and be at higher risk for cardiovascular surgery.