New ways for the standardization of autoantibody assays: chimeric monoclonal antibodies.

Recombinant production of human autoantigens and new assay methodologies have created new opportunities for the detection of autoantibodies in autoimmune disease situations. However, the standardization of test results remains unsatisfactory which can be traced to supply and batch variation problems of the patient sera used as standard materials. Human monoclonal autoantibodies can be used as novel standards, but are difficult to generate and produce routinely. We present a strategy based on atransgenic mouse strain producing chimeric human IgG1 antibodies after immunization. Together with traditional mouse hybridoma technology this approach allows creation of large panels of chimeric monoclonal autoantibodies for standardization purposes.

Functional properties of CYP2D6 1 (wild-type) and CYP2D6 7 (His324Pro) expressed by recombinant baculovirus in insect cells.

The debrisoquine/sparteine or CYP2D6 genetic polymorphism of drug oxidation is a common cause for interindividual variability in drug response. We recently identified a mutant allele, designated CYP2D6-E or CYP2D6*7, which is associated with the poor metabolizer phenotype and occurs in Caucasian populations with a frequency of about 1%. In contrast to other loss-of-function alleles, a full length protein with a single amino acid substitution. His324Pro, is encoded by the CYP2D6*7 allele. To functionally analyze this mutant protein form of CYP2D6, recombinant baculoviruses were constructed to express the CYP2D6 cDNA. Up to 0.33 nmol of spectrally detected P450/mg of cell protein were produced in Spodoptera frugiperda cells, whereas Trichoplusia ni 5B1-4 cells reproducibly produced 0.8 nmol/mg (4% of total cell protein). Insect cell membranes were functionally characterized with cumene hydroperoxide or after reconstitution with purified rat NADPH:cytochrome P450 reductase. Km values for the substrates bufuralol and sparteine and other enzymatic properties were almost identical to those of human liver microsomes. The H324P mutation was introduced into the cDNA by site-directed mutagenesis and recombinant baculovirus was obtained. Expression under a variety of conditions demonstrated that mutant protein amounts comparable to the wild-type enzyme were produced. However, no spectrally detectable P450 was formed and no catalytic activity was detected. Furthermore, in contrast to the wild-type protein the mutant protein was almost exclusively located in a detergent-insoluble insect cell fraction. These results demonstrate that the H324P mutation is responsible for the in vivo poor metabolizer phenotype associated with the CYP2D6*7 allele by preventing normal protein folding and heme incorporation.

Phytoene synthase from Narcissus pseudonarcissus: functional expression, galactolipid requirement, topological distribution in chromoplasts and induction during flowering.

A cDNA coding for the carotenoid biosynthetic enzyme phytoene synthase was cloned from a Narcissus pseudonarcissus flower cDNA library, and the corresponding protein was overexpressed in insect cells using the baculovirus lipofection system. The full-length overexpressed enzyme exhibited very reduced catalytic activity compared with an overexpressed N-truncated form, with its transit sequence removed by site-directed mutagenesis. The shortened form readily bound quantitatively to lipid bilayers. Although it was active with liposomes prepared from plastid lipids, with phospholipid liposomes it was not, even though association took place. In this latter case, free galactose was capable of substituting for galactolipids, resulting in enzymatic activity. It is concluded that galactolipids are involved in catalytic activity, but do not serve as a membrane anchor. Antibodies raised against the recombinant enzyme made it possible to distinguish between a membrane-bound and a soluble, protein-complexed inactive form of phytoene synthase, present in the chromoplast stroma. These findings and data on phytoene synthase mRNA and protein expression presented here are discussed in terms of a possible regulatory role in color formation during chromoplast (flower) development.

A novel, soluble form of phytoene desaturase from Narcissus pseudonarcissus chromoplasts is Hsp70-complexed and competent for flavinylation, membrane association and enzymatic activation.

A cDNA coding for the carotenoid biosynthetic enzyme phytoene desaturase from Narcissus pseudonarcissus was cloned and the corresponding protein expressed in insect cells using the baculovirus system. Polyclonal antibodies raised against the recombinant protein allowed the detection of soluble and tightly membrane-bound populations of phytoene desaturase in the chromoplasts isolated from petals. The soluble form is enzymatically inactive and a constituent of a larger Hsp 70-containing protein complex in the stroma, whereas the membrane-bound form is functional. In vitro, the soluble form is able to associate on to/into protein-free liposomal membranes made from chromoplast lipids, thereby gaining activity by binding added flavine adenine dinucleotide (FAD). Once bound to membranes, activated phytoene desaturase works independently of any added FAD, employing membrane-bound electron acceptors. FAD, however, exerts no positive effect on the membrane-association process. Its role is confined to enzymatic activation. Although carotenoid accumulation is strongly induced during flower development, only very low concentrations of phytoene desaturase transcripts are detectable, while the corresponding protein accumulates in low, but measurable amounts, appearing in soluble and membrane-bound states. Post-transcriptional mechanisms contribute significantly to carotenoid accumulation, as do factors determining the enzymatic activity of phytoene desaturase, for example by influencing the redox-state of membrane-bound electron acceptors.

Significance of P53 in human thyroid tumors.

Mutational changes in the p53 tumor suppressor gene are the most frequent genetic alterations in human malignant tumors. Studies have shown a correlation of p53 expression in breast cancer with tumor prognosis. In contrast to mutational activation of ras and GSP in thyroid tumors, little is known about the role of p53 in thyroid tumor development. Therefore thyroid tumors and thyroid tumor cell lines were studied for the presence of p53 mutations. Snap-frozen tissues from 57 differentiated thyroid carcinomas (DTCs) and 5 goiters were studied by immunohistochemical methods. A panel of six antibodies (pAb 240, 421, 1620, 1801, DO7, and CM1) was employed by using the ABC technique. Five cell lines from DTCs (FTC133, 236, 238, PTC337, MTC164) were examined by the same technique. Additionally, genomic DNA from the cells was amplified by the polymerase chain reaction (PCR) and the PCR product studied for p53 mutations (R273H) by mutation-specific oligonucleotide hybridization (MOH) and temperature gradient gel electrophoresis (TGGE) for the p53 exon 8. None of the benign thyroid tumors and 7 of 57 (12%) DTCs strongly express p53 with a heterogeneous distribution in the tumor tissue. All seven patients have metastatic disease or dedifferentiated tumors G3 (three of seven). CM1 was positive in two cell lines (FTC-133, PTC-337), questionable in FTC-238, and negative in FTC-236 and MTC-164. All three follicular cell lines, however, and the original tumor tissue showed the same p53 mutation (R273H) in MOH analysis and TGGE. P53 mutations are rare in thyroid tumors, but the presence of p53 mutations indicates a poor prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of autoantibodies to the 65-kD isoform of glutamate decarboxylase by radioimmunoassay.

Autoantibodies (AAb) to glutamate decarboxylase (GAD) occur with a high prevalence in sera of newly diagnosed type I (insulin-dependent) diabetic patients. The aim of this study was to establish a GAD-AAb radioimmunoassay using 125I-labelled GAD65 and to evaluate this assay in a cross-sectional study with newly diagnosed type I diabetic patients (diabetes duration < 6 weeks). Furthermore, subjects at high risk of developing type I diabetes and individuals suffering from other autoimmune diseases were examined in this assay. For GAD-AAb detection, 125I-labelled GAD65 was incubated with 10 microliters of human serum overnight on ice. Thirty of 51 (59%) type I diabetic patients but none of the 54 healthy blood donors tested were found to be positive. A displacement step using 100,000 g supernatant from rat brain containing or not containing GAD showed the specificity of the binding of 125I-GAD65. Concerning the individuals at high risk of developing diabetes. 9/12 (75%) islet cell antibody (ICA)-positive non-diabetic and 4/34 (12%) ICA-negative subjects with metabolic abnormalities were GAD-AAb positive. These results show the association between type I (insulin-dependent) diabetes mellitus and the occurrence of GAD65-AAb, which possibly predicts a risk of developing the disease.

Autoantibodies against GAD65 rather than GAD67 precede the onset of type 1 diabetes.

The enzyme glutamate decarboxylase (GAD) is considered one of the major Beta cell antigens in Type 1 diabetes mellitus. The GAD autoantibody (GAD-AAb) prevalence in newly diagnosed Type 1 diabetic patients has been described up to 80%, depending on the detection method used. The aim of this study was to evaluate a simple, specific, and sensitive radioimmunoassay (RIA) method for detection of AAb against both isoforms of the enzyme, GAD65 and GAD67, in a cross-sectional study using sera from newly diagnosed Type 1 diabetic patients and in a longitudinal study using sera from prediabetic patients and individuals at risk of developing the disease. The 125I-labelled full-length human recombinant proteins of GAD65 and GAD67 expressed in SF9 cells were used as the antigen source. The prevalence of GAD65-AAb in newly diagnosed Type 1 diabetic patients was found to be 73% (112/153), in contrast to 19% (14/72) of GAD67-AAb. Only one patient produced AAb restricted to GAD67. Furthermore, GAD65-AAb could also be detected in 73% (11/15) of prediabetic patients (up to 122 months before clinical manifestation of the disease), whereas only 27% (4/15) of them were positive for GAD67-AAb. In the group at risk of developing Type 1 diabetes, these prevalences were 77% (10/13) and 46% (6/13), respectively. In all GAD67-AAb-positive patients investigated in the longitudinal study, AAb to GAD65 were detectable. In 47% of patients positive for both GAD65-AAb and ICA, the GAD65-AAb appeared by up to 46 months before the occurrence of ICA was detected. The data illustrated that GAD65 is the main immunogenic isoform of the enzyme in the preclinical and clinical stages. The RIA detecting AAb against this isoform may facilitate the screening for individuals at risk of developing the disease.

Analysis of the tumor suppressor activity of the K-rev-1 gene in human tumor cell lines.

Overexpression of the human K-rev-1 gene in v-Ki-ras-transformed NIH 3T3 cells has been reported to result in the reversal of transformation and tumor suppression. To address whether human K-rev-1 is a tumor suppressor gene of human tumor cells, we have systematically transfected epitope-tagged wild-type or activated mutant K-rev-1 complementary DNA expression vectors into a series human tumor cell lines that express an activated ras oncogene, namely HT1080, EJ, and SW480. Using the epitope-tag-specific monoclonal antibody, it is shown that the K-rev-1 protein localizes to the medial/trans-Golgi network. Ectopic expression of the wild-type or activated mutant K-rev-1 protein did not significantly affect the morphology or in vitro growth of any clones. Furthermore, all clones expressing the wild-type or activated mutant K-rev-1 protein were tumorigenic. Western blot analysis of tumor reconstitutes demonstrated that there was no decrease or loss of introduced K-rev-1 protein expression. The results in the present study demonstrate that expression of K-rev-1 does not reverse the transformed phenotype or significantly affect the tumorigenic phenotype of human tumor cell lines that express endogenous ras oncogenes.

Prevalence of autoantibodies to the 65- and 67-kD isoforms of glutamate decarboxylase in insulin-dependent diabetes mellitus.

We investigated the presence of autoantibodies to baculovirus-expressed human recombinant 65- and 67-kD isoforms of glutamate decarboxylase (GAD65 and GAD67) in insulin-dependent diabetes mellitus (IDDM). In the immunoprecipitation test using [35S]methionine-labeled GADs antibodies to GAD65 were detected in 13/15 (87%) islet cell antibody (ICA)-positive and in 1/35 (2.9%) ICA-negative first-degree relatives of patients with IDDM, in 6/11 (54.5%) ICA-positive nondiabetic schoolchildren, and in 35/50 (70%) patients with newly diagnosed IDDM. GAD67 antibodies were positive only in five (33%) of the ICA-positive relatives (P < 0.05) and in nine (18%) IDDM patients at onset (P < 0.00001). After onset of IDDM antibodies to GAD65 and GAD67 declined but were still positive in 25 and 9.4% of subjects with long-standing IDDM (> 10 yr). In all study groups antibodies to GAD67 were only detected in GAD65 antibody-positive sera. An immunotrapping enzyme activity assay for GAD65 antibodies was positive in 64/75 (85.3%) of sera that were GAD antibody positive in the immunoprecipitation test (r = 0.870, P < 0.0001). In two (2.7%) sera GAD65 antibodies that block GAD enzyme activity were found. Our data suggest that antibodies to GAD65 but not to GAD67 represent sensitive markers for preclinical and overt IDDM. The immunotrapping assay here described represents a valuable technique for specific and sensitive screening for GAD antibodies.

Baculovirus-mediated expression of human 65 kDa and 67 kDa glutamic acid decarboxylases in SF9 insect cells and their relevance in diagnosis of insulin-dependent diabetes mellitus.

cDNAs coding for the full-length human 65 and 67 kDa glutamic acid decarboxylases (GAD65 and GAD67) were amplified from pancreas and hippocampus cDNA libraries by polymerase chain reaction, respectively. Both cDNAs were inserted into a baculovirus vector which mediated highly efficient expression of the human GAD65 and GAD67 with histidine-hexapeptides as affinity ligands at their C-termini in Spodoptera frugiperda (Sf9) cells. The recombinant GAD proteins were purified to homogeneity by affinity chromatography using a metal-chelating matrix. The infected Sf9 insect cells expressed the recombinant human GAD65 and GAD67 with natural-like conformations, as confirmed by measurement of their enzyme activities as well as their fully restored autoantigenicities. Immunoprecipitation of metabolically labeled infected Sf9 cells demonstrated the autoantigenic potential of the recombinant GAD proteins. The practicability of using recombinant GAD65 and GAD67 derived from the baculovirus expression system for the development of an immunoassay for the diagnosis of insulin-dependent diabetes mellitus is discussed.

Expression of recombinant human thyroid peroxidase by the baculovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease.

The cDNAs coding for human full-length and soluble thyroid peroxidase (TPO) were constructed, cloned into a baculovirus transfer vector and used for infection of Spodoptera frugiperda (Sf9) cells. The soluble TPO lacking 87 amino acids of the C-terminal transmembrane and intracisternal domains was designed as a fusion protein with a histidine-hexapeptide as an affinity ligand at its C-terminus. Whereas the recombinant full-length TPO was expressed mainly in an insoluble form in Sf9 cells, the recombinant soluble TPO was almost completely secreted into the culture medium. Both the full-length and the soluble TPO were purified by conventional methods and by a specific affinity chromatography using metal chelating matrix respectively, and tested for their autoantigenicity towards anti-TPO autoantibodies. The ELISA established with the purified recombinant soluble TPO as antigen demonstrated its specificity, practicability and reproducibility in screening of anti-TPO autoantibodies in sera of autoimmune thyroid patients. High correlation (r = 0.89, n = 175) was obtained between the soluble TPO and natural TPO prepared from human thyroid glands. Pathological sera (n = 200) were positively assayed with a significance of 91%.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21.

The neurofibromatosis type 1 (NF1) protein contains a region of significant sequence similarity to ras p21 GTPase-activating protein (GAP) and the yeast IRA1 gene product. A fragment of NF1 cDNA encoding the GAP-related domain (NF1 GRD) was expressed, immunoaffinity purified, and assayed for effects on N-ras p21 GTPase activity. The GTPase of wild-type ras p21 was stimulated by NF1 GRD, but oncogenic mutants of ras p21 (Asp-12 and Val-12) were unaffected, and the GTPase of an effector mutant (Ala-38) was only weakly stimulated. NF1 GRD also down-regulated RAS function in S. cerevisiae. The affinity of NF1 GRD for ras p21 was estimated to be 250 nM: this is more than 20-fold higher than the affinity of GAP for ras p21. However, its specific activity was about 30 times lower. These kinetic measurements suggest that NF1 may be a significant regulator of ras p21 activity, particularly at low ras p21 concentrations.

Structural and functional analysis of ypt2, an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a Sec4 protein homologue.

Using the cloned Saccharomyces cerevisiae YPT1 gene as hybridization probe, a gene, designated ypt2, was isolated from the fission yeast Schizosaccharomyces pombe and found to encode a 200 amino acid long protein most closely related to the ypt branch of the ras superfamily. Disruption of the ypt2 gene is lethal. The bacterially produced ypt2 gene product is shown to bind GTP. A region of the ypt2 protein corresponding to but different from the 'effector region' of ras proteins is also different from that of ypt1 proteins of different species but identical to the 'effector loop' of the S.cerevisiae SEC4 gene product, a protein known to be required for vesicular protein transport. The S.pombe ypt2 gene under control of the S.cerevisiae GAL10 promoter is able to suppress the temperature-sensitive phenotype of a S. cerevisiae sec4 mutant, indicating a functional similarity of these GTP-binding proteins from the two very distantly related yeasts.

The ras-related mouse ypt1 protein can functionally replace the YPT1 gene product in yeast.

The protein-coding region of the essential Saccharomyces cerevisiae YPT1 gene coding for a ras-related, guanine-nucleotide-binding protein was exchanged in chromosome VI by the protein-coding segment of either the mouse ypt1 gene or the v-Ki-ras gene, and different chimeric YPT1-v-Ki-ras genes. The mouse ypt1 protein with 71% of identical residues compared with the yeast Ypt1 protein could functionally fully replace its yeast homologue as long as the mouse gene was overexpressed under transcriptional control of the inducible GAL10 promoter. In contrast, neither the viral Ki-ras nor the hybrid proteins were able to substitute for the loss of YPT1 gene function. This study suggests that different parts of the yeast Ypt1 protein are required for the interaction with cellular targets and that these essential parts are conserved in the mammalian ypt1 protein.

The ras-related ypt protein is an ubiquitous eukaryotic protein: isolation and sequence analysis of mouse cDNA clones highly homologous to the yeast YPT1 gene.

The YPT1 gene of the yeast Saccharomyces cerevisiae codes for a guanine nucleotide-binding protein which is essential for cell viability. Using as hybridization probe cloned yeast YPT1 gene sequences, we have isolated from cDNA libraries prepared from RNA of mouse F9 and C3H10T1/2 cells several overlapping cDNA clones with identical sequence in the regions of overlap. The cDNAs were derived from a gene, designated ypt1, which codes for a protein of 205 amino acids with 71% homology to the yeast YPT1 gene product. Amino acid sequences typical for guanine nucleotide-binding proteins and characteristic for ypt proteins are perfectly conserved in the mouse ypt1 protein. Two mRNAs of 1600 and 3200 nucleotides, originating from the mouse ypt1 gene and differing in the length of their 3'-non-translated region, were identified in mouse F9 cells and in all mouse tissues examined. A monoclonal antibody specifically recognizing the 23.5-kd yeast YPT1 protein cross-reacted with a protein of identical size on protein blots of mouse, rat, pig, bovine and human cell lines.

Effects of calcium entry blocker emopamil on postischemic energy metabolism of the isolated perfused rat brain.

The effects of emopamil on postischemic energy metabolism and electroencephalographic (EEG) recovery were investigated in the isolated rat brain perfused at either constant pressure or, alternatively, at constant flow rate. Flow rate and perfusion pressure were monitored continuously. The brains were perfused with a fluorocarbon emulsion for 30 min, and after 30 min of ischemia, perfusion was reinstituted for 5, 30, or 60 min. Global cerebral perfusion rate was increased by emopamil throughout the perfusion period and, accordingly, in brains perfused at a constant flow rate, perfusion pressure was reduced by the drug. At constant pressure perfusion, after 5 min after ischemia, cortical levels of creatine-phosphate, adenosine triphosphate (ATR), glucose, glucose-6-phosphate, and fructose-6-phosphate were higher in emopamil-treated brains than in controls, although the levels of adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were reduced. When brains were perfused at constant flow rate, however, emopamil exhibited no effect on brain energy metabolism in the early reperfusion period. Postischemic restoration of high-energy phosphates proved to depend on the flow rate used. After 30 min of postischemic reperfusion, cortical levels of lactate were lower in emopamil-treated brains compared to controls at both constant pressure and constant volume perfusion. Postischemic lactate levels were independent of flow rate and were also reduced when emopamil was only present during reperfusion. The postischemic restoration of cortical EEG activity was improved by the calcium entry blocker.(ABSTRACT TRUNCATED AT 250 WORDS)