Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA.     

Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia

This paper describes a sensitive method for study of the isoelectric point and molecular weight of immunoreactive low density lipoprotein (LDL) receptors of cultured human fibroblasts. The fibroblast receptors are solubilized with Triton X-100, partially purified by batch elution from DEAE-cellulose, and subjected to two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are transferred electrophoretically to nitrocellulose paper which is then incubated with a mouse monoclonal antibody (IgG-C7) directed against the LDL receptor, followed by an 125I-labeled antibody against mouse IgG. The receptor-bound monoclonal antibody is localized by autoradiography. By this technique, the immunodetectable LDL receptors from normal human fibroblasts migrate as a single spot with an isoelectric point of 4.3 and a Mr of approximately 160,000. In one patient with homozygous familial hypercholesterolemia whose cells fail to bind 125I-labeled IgG-C7, no immunoreactive LDL receptor spot was detected after electrophoresis. We also studied LDL receptors from three homozygotes whose cells bind 125I-IgG-C7, i.e. cross-reacting material-positive mutants. Their immunodetectable receptors were indistinguishable from normal receptors in terms of isoelectric point and molecular weight. Similarly, the receptors from one patient with the internalization-defective form of familial hypercholesterolemia showed normal electrophoretic migration. The immunoblotting technique should prove useful in analyzing structural alterations, if they exist, in LDL receptors from other subjects with cross-reacting material-positive forms of familial hypercholesterolemia.     

Rat mesenteric artery endothelial cells in culture secrete ET-1

Endothelial cells were harvested by the collagenase perfusion of isolated mesenteric arteries of rats and cultured. An endothelin peptide was detected in the supernatant of these cells by an antibody which recognizes ET-1 but not "rat" endothelin (ET-3). Culture media was extracted using a C-8 solid phase column and subjected to reverse phase HPLC using a system that separates all known endothelins and immunoreactive endothelins measured using another antibody which recognizes all endothelins. The main immunoreactive peak co-eluted with ET-1. We could not detect any ET-2, ET-3 or Vasoactive Intestinal Contractor. A smaller immunoreactive peak of unknown structure that eluted earlier than ET-1 was also detected. In conclusion, rat endothelial cells secrete a peptide of similar chromatographic and immunoreactive properties as ET-1.     

In vivo interaction of morphine and endogenous opiate-like peptides

In the csf of rats, the concentration of peptides, reacting with antibody to leu-enkephalin, was determined by radioimmunoassay. Intravenous injection of morphine caused a biphasic response, i.e. an immediate and a delayed rise of the level of such immunoreactive peptides in the csf. After i.v. naloxone, only the late peak was observed. Combined administration of morphine and naloxone delayed the appearance of the first peak, expected after morphine injection; the second peak did not show within 2 – 3 hours. The results are interpreted by assuming release of enkephalins and related peptides from two sites, viz. synaptic clefts and intracellular pools.     

Detection and partial characterization of a developmentally regulated nuclear antigen in neural cells in vitro and in vivo

We report that a monoclonal antibody directed against phosphorylated neurofilaments (SMI 31) recognizes nuclear antigens present in embryonic but not in adult neural cells. On Western blots, the antibody reacts with four proteins of apparent MW 35, 37, 52/54, and 250 KD which are found exclusively in developing brain tissue. These nuclear antigens are expressed by glial and neuronal cells. Both nuclear staining and immunoreactive proteins decrease with ongoing in vitro differentiation. A computer search for proteins that share the epitope recognized by antibody SMI 31 did not yield any proteins of known nuclear localization that exhibit the same molecular weights and solubility characteristics as the above immunoreactive proteins. We conclude that antibody SMI 31 recognizes hitherto unknown nuclear proteins which, in neural cells, are developmentally regulated.     

Measurement of tyrosine hydroxylase apoenzyme protein by enzyme-linked immunosorbent assay (ELISA): effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity and content.

A enzyme-linked immunosorbent assay has been developed for tyrosine hydroxylase (TH). The method uses a polyclonal antibody to trap TH, a monoclonal antibody to bind the immobilized TH, a biotinylated, anti-mouse immunoglobulin to bind the monoclonal antibody, and streptavidin covalently coupled to horseradish peroxidase (SA-HRP). The antigen-antibody complex is detected colorometrically following incubation with an HRP substrate. The method detects less than 1 ng (16 fmol) of TH and can be performed in 3 h. The high specificity of the assay is attributed to the use of both polyclonal and monoclonal antibodies, each of which are specific for TH. Data acquisition and reduction is rapid and linked directly to a common desktop computer. Levels of TH protein average 1 ng/microgram protein in striatum and, following treatment with the neurotoxicant MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), are decreased to a similar extent as is catalytic activity. In contrast, MPTP did not alter TH homospecific activity. The monoamine oxidase B inhibitor deprenyl blocked both the decrease in activity and the decrease in immunoreactive protein caused by MPTP.     

Mechanism of the Increase in Catalase Activity through Microbody Development in Wounded Sweet Potato Root Tissue

An increase in catalase activity accompanied by microbody developmentin wounded sweet potato root tissue was investigated with aspecific antibody against sweet potato catalase. The increasewas completely inhibited by cycloheximide. Analysis with singleradial immunodiffusion method showed that protein immunoprecipitatedby the antibody increased in wounded tissue, indicating theinvolvement of de novo synthesis of catalase protein in theactivity-increase. The activity-increase was, however, moreremarkable than the increase in immunoreactive protein and thisresults in an increase in specific catalase activity in woundedtissue, indicating the presence in intact tissue of an inertor less active protein, immunologically analogous to catalase.Actually, immunological analysis showed the presence in intacttissue of an immunoreactive protein which differed from activecatalase protein in the mobility on a polyacrylamide gel andprobably also in the molecular weight of subunit. The immunoreactiveprotein seemed to exist in a significant amount outside themicrobodies in intact tissue cells. Thus, there is a possibilitythat the increase in catalase activity in wounded tissue ispartly due to activation of the immunoreactive protein. (Received October 16, 1982; Accepted February 24, 1983)     

Mechanism by which antibodies inhibit hapten-malate dehydrogenase conjugates. An enzyme immunoassay for morphine.

Antimorphine antibodies inhibit the activity of morphine conjugates of mitochondrial malate dehydrogenase. Conjugation of malate dehydrogenase through tyrosine and amino groups resulted in only moderate losses of enzyme activity. On conjugation through disulfide bonds the enzyme activity first increased but dropped sharply with increasing substitution. Only the former conjugates were inhibited by excess antibodies. The degree of inhibition (up to 86%) was directly related to the number of morphine residues bonded directly to amino groups. The maximum number of antibody binding sites that bind to enzyme was nearly equal to the number of haptens provided there were 16 or less haptens/enzyme. However up to 26 haptens/enzyme became completely bound by antibody on long incubation. Inhibition of enzyme activity was detectably reduced by 2 times 10 minus 9 M morphine or 2 times 10 minus 10 M codeine, thus providing a sensitive assay for these drugs. The data suggest that enzyme inhibition occurs by conformational freezing of the enzyme when antibody binds to a morphine residue attached to one specific amino group.     

Characterization and immunochemistry of pyruvate dehydrogenase complex of Ascaris muscle

Pyruvate dehydrogenase complex and lipoamide dehydrogenase were purified from muscle of Ascaris lumbricoides var. suum which contains relatively a large amount of the complex. Molecular weights of three constituent enzymes of Ascaris pyruvate dehydrogenase complex were as follows; alpha- and beta-subunits of pyruvate dehydrogenase were 42,000 and 37,000, respectively, lipoate acetyltransferase was 76,000 and lipoamide dehydrogenase was 56,000. Furthermore, two unknown polypeptides having molecular weight of 46,000 and 41,000 were detected. Anti-Ascaris lipoamide dehydrogenase antibody precipitated three constituent enzymes and two unknown polypeptides, suggesting that lipoamide dehydrogenase not only binds tightly to complex, but also two unknown polypeptides bind tightly to complex.     

Quality control test for immunoreactivity of radiolabeled antibody

A rapid method for the measurement of the immunoreactive fraction of a radiolabeled monoclonal antibody or antibody fragment has been developed. This may be used as a quality control test prior to patient administration of the radiolabeled antibody preparation. The test employs solid phase antigens and the assay is conducted under conditions of antigen excess. Assay parameters have been evaluated and a standardized procedure has been developed. The assay has been compared to a standard extrapolation method and found to give approximately the same result. The test has been used on four different radiolabeled antibodies currently in clinical trials in patients with colorectal cancer. Mean immunoreactive fractions for these radiolabeled antibodies ranged from 35 to 65% and the variability of the immunoreactive fraction ranged from 140 to 240% for different antibodies. We conclude that the quality, defined as the immunoreactive fraction, of radiolabeled antibodies is both low and highly variable, indicating the need for a quality control test of these radiopharmaceuticals in the clinic prior to patient administration.     

Number of immunoreactive GnRH-containing neurons is heritable in a wild-derived population of white-footed mice (Peromyscus leucopus)

The evolution of mammalian brain function depends in part on levels of natural, heritable variation in numbers, location, and function of neurons. However, the nature and amount of natural genetic variation in neural traits and their physiological link to variation in function or evolutionary change are unknown. We estimated the level of within-population heritable variation in the number of gonadotropin-releasing hormone (GnRH) neurons, which play a major role in reproductive regulation, in an unselected outbred population recently derived (<10 generations) from a single natural population of white-footed mice (Peromyscus leucopus, Rafinesque). Young adult male mice exhibited an approximately threefold variation in the number of neurons immunoreactive for GnRH in the brain areas surveyed, as detected using SMI-41 antibody with a single-label avidin-biotin complex method. Consistent with earlier findings of selectable variation in GnRH neurons in this population, the level of genetic variation in this neuronal trait within this single population was high, with broadsense heritability using full-sib analysis estimated at 0.72 (P<0.05). Either weak selection on this trait or environmental variation that results in inconsistent selection on this trait might allow a high level of variation in this population.     

GABA-like immunoreactivity of identified interneurons in the flight system of the locust,Locusta migratoria

Summary The transmitter content of identified inhibitory interneurons in the flight system of the locust, Locusta migratoria, has been characterized using antibodies raised against protein-conjugated gamma aminobutyric acid. Identified flight neurons were filled with the fluorescent dye, Lucifer Yellow. Serial sections of dye-filled neurons were incubated with an antibody to gamma aminobutyric acid which was subsequently tagged with a fluorescent marker. Excitatory motoneurons to wing muscles and 13 flight interneurons (3 excitatory, 7 inhibitory, and 3 with unknown synaptic effect) were examined. Neither the moto-neurons nor any of the 3 excitatory interneurons contained immunoreactive material. Six of the 7 inhibitory interneurons did contain immunoreactive material. All the neurons which contained immunoreactive material and whose synaptic effect is known were inhibitory. We conclude that most of the inhibitory flight interneurons which have been described use gamma aminobutyric acid as their transmitter. Interestingly, at least 1 set of interneurons known to be inhibitory does not use gamma aminobutyric acid. We predict that the 2 interneurons which do contain immunoreactive material and whose synaptic effect is not yet known will be found to have inhibitory roles in the operation of the flight circuitry.     

Immunochemical studies of ferrochelatase protein: characterization of the normal and mutant protein in bovine and human protoporphyria.

Protoporphyria is a hereditary disorder characterized by a marked decrease in the activity of ferrochelatase, the terminal enzyme in the heme biosynthetic pathway. We have prepared specific polyvalent antibodies against bovine ferrochelatase in rabbits. The specificity of the antibody preparation against ferrochelatase was demonstrated by western blot analysis and immunoprecipitation of ferrochelatase activity. The antibody also cross-reacted weakly with ferrochelatase from human mitochondria. To quantify immunoreactive ferrochelatase in tissue samples, a kinetic-based enzyme-linked immunosorbent assay (k-ELISA) was developed. Ferrochelatase activity and the level of immunoreactive protein were measured in hepatic mitochondria isolated from six normal and nine protoporphyric (homozygous) cattle. Ferrochelatase activity was less than 10% of normal in mitochondria from protoporphyric animals; the amount of immunoreactive material was equivalent to that from normal animals. Similar studies were performed with samples from three normal and two protoporphyric (heterozygous) humans. Ferrochelatase activity was decreased in protoporphyric samples (about 17% of normal, but there was no concomitant decrease in immunoreactive material. These data demonstrate that a normal amount of ferrochelatase protein is present and suggest that bovine and human protoporphyria result from point mutations in the gene encoding ferrochelatase.     

Leucocyte myosin and its location in the cell.

The intracellular location of the binding site of antibody against purified myosin prepared from equine leucocytes was investigated in neutrophils and lymphocytes by electron microscopy using peroxidase-labelled antibody method. The myosin extracted from equine leucocytes could bind skeletal muscle F-actin and the formed complex showed the biophysical and biochemical properties and electron microscopic appearance of actomyosin. On immunodiffusion, the leucocyte myosin formed a single precipitin line with its antibody prepared in rabbits. The antibody also formed single precipitin lines with myosins from lymphocytes and thrombocytes, fusing with each other. The antibody against the leucocyte myosin did not react with myosins from skeletal or arterial smooth muscle. The specificity of the antibody was further established by determination of K+-EDTA-activated ATPase activity remained in the supernate of antigen-antibody mixture. Under electron microscope, the intracellular immunoreactive products of peroxidase labelled antibody were found in cytoplasm of neutrophils and lymphocytes incubated with antibody against leucocyte myosin, but not in neutrophils or lymphocytes treated with IgG from normal rabbits.     

Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.     

Chronic, but not acute, administration of morphine alters antiopiate (Tyr-MIF-1) binding sites in rat brain

Opiate addiction could involve a change in the binding of endogenous antiopiates. A candidate for such a role is Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), a brain peptide that can antagonize exogenous and endogenous opiates and bind to opiate receptors. Its primary action, however, may be through its own binding site in brain, which we now report is altered by chronic administration of morphine. Rats given morphine pellets had reduced binding of both iodinated and tritiated Tyr-MIF-1 on day 5, when substantial tolerance is evident. In contrast, mu and delta opiate receptors were increased. Acute injection of an analgesic dose of morphine did not reduce Tyr-MIF-1 binding, indicating that chronic administration is required for the change. These findings open new approaches to the study of addiction by focusing on antiopiate activity.     

Rat liver mitochondrial contact sites and carnitine palmitoyltransferase-I

In hepatic mitochondria, the outer membrane enzyme, carnitine palmitoyltransferase-I (CPT-I), appears to colocalize with contact sites. We have prepared contact sites that are essentially devoid of noncontact site membranes. The contact site fraction has a high specific activity for CPT-I and contains a protein at 88 kDa that is recognized by antibodies directed at two different peptide epitopes on CPT-I. Similarly long-chain acyl-CoA synthetase (LCAS) specific activity is high in this fraction; a protein at 79 kDa is recognized by an antibody against LCAS. Although activity of carnitine palmitoyltransferase-II (CPT-II) is present, it is not enriched in the contact site fraction, and a protein of 68 kDa weakly reacted with anti-CPT-II antibody. Likewise, carnitine-acylcarnitine translocase (CACT) protein is present, but at a somewhat reduced level. Using an analytical continuous sucrose gradient, we demonstrate that the activities of CPT-I and LCAS and their associated immunoreactive proteins are present in a constant amount throughout the contact site subfractions. The enzymatic activity of CPT-II and its associated immunoreactive protein, as well as immunoreactive CACT, is absent in the lighter density gradient subfractions and is present in the higher density subfractions only in trace amounts. This heterogeneity of the contact site fraction is due to unvarying amounts of outer membrane and increasing amounts of attached inner membrane with increasing density of the subfractions.     

Agglutinating and Bactericidal Properties of Fractions of Rabbit Anti-Vibrio cholerae Serum

The major portion of the agglutinating and bactericidal activity of the sera of rabbits immunized with live Vibrio cholerae or with cholera vaccine was found in the gammaM fractions during the early stages of immunization. After 5 weeks or more, gammaG fractions accounted for more than half of the agglutinating activity. When late antibody was measured as the amount of protein precipitated by somatic antigens, nearly 3 times as much gammaG as gammaM was required for agglutination, and about 30 times as much gammaG as gammaM was required to kill 50% of a standard inoculum in the presence of complement. The ratio of vibriocidal to agglutinin titer of gammaG fractions at different stages of immunization was more variable than that of gammaM fractions. More complement was required for a vibriocidal effect by gammaG than by gammaM. Increasing the amount of complement decreased the amount of both gammaG and gammaM required to kill, but smaller amounts of gammaM required disproportionately larger amounts of complement. Less time was required by gammaM than by gammaG to kill 50% of the inoculum. Removal of the group-reactive antibody from anti-Ogawa serum and serum fractions by absorption with Inaba reduced the vibriocidal titer by more than one-half.     

Identification by radioimmunoassay and HPLC of morphine modulatory peptide-immunoreactivity in rat spinal cord and brain

Two so-called morphine modulatory peptides, an octapeptide and an octadecapeptide, have recently been isolated from bovine spinal cord. We have raised antibodies to the octapeptide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH₂: FF-8), which in radioimmunoassay react with peptides terminating in Arg-Phe-NH₂. This dipeptide is common to both the morphine modulatory peptides and the molluscan neuropeptide FMRF amide. The distribution and molecular forms of immunoreactive peptides were examined in the rat central nervous system and gastrointestinal tract. Highest concentrations of FF-8-like immunoreactivity were found in the dorsal spinal cord, brain stem and hypothalamus. The immunoreactive material in central nervous system extracts was resolved by reversed phase HPLC into three peaks of activity, the two largest peaks eluted in similar positions to the standard octapeptide and octadecapeptide. It appears that previously observed FMRF amide-like immunoreactivity in the rat central nervous system corresponds to peptides immunochemically and chromatographically similar to the two bovine spinal cord peptides.     

Control of severe pain with sustained-release morphine tablets v. oral morphine solution.

Recently a sustained-release morphine sulfate tablet (MS Contin [MSC]) was introduced in Canada. In a randomized double-blind crossover trial we compared MSC given every 12 hours with a morphine sulfate solution (MSS) given every 4 hours to 17 patients suffering from chronic severe pain. After titration of the morphine dosage to optimize the analgesic effect, each patient received 10 days of therapy with either MSC or MSS, then 10 days of therapy with an equal daily dose of the other formulation. Both preparations provided effective pain control, with minimal side effects. There was no significant difference between MSC and MSS in pain scores on a visual analogue scale (VAS), severity scores for tiredness and nausea, amount of supplemental morphine needed for break-through pain or patient preference. The plasma morphine concentrations tended to be greater during treatment with MSC. The study had an 89% probability of detecting a clinically significant difference in VAS pain scores. We conclude that an individualized, twice-daily regimen of MSC is as effective as MSS given every 4 hours for control of severe pain. The twice-daily regimen has several advantages: it provides for an uninterrupted night''s sleep, it is substantially more convenient than the six doses per day required with MSS, and it should help reduce both medication errors and noncompliance.