Role of ouabain-like compound in the rostral ventrolateral medulla in rats.

To determine whether ouabain-like compound (OLC) exerts modulatory influences on the activity of vasomotor neurons in the rostral ventrolateral medulla (RVLM), we examined the effects of microinjecting ouabain, digoxin-specific antibody Fab fragments, and mAb against ouabain on the rat RVLM. Microinjection of ouabain into the unilateral RVLM of anesthetized normotensive rats elicited dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). The pressor and sympathoexcitatory effects of ouabain in the RVLM were reversed by microinjections of an M2 muscarinic antagonist, gallamine, or digoxin-specific antibody Fab fragments. Furthermore, a prior microinjection in the RVLM of gallamine, digoxinspecific antibody Fab fragments, or kainic acid or intravenous injection of hexamethonium all prevented the pressor and sympathoexcitatory effects induced by a subsequent microinjection of ouabain. Microinjections of either digoxinspecific antibody Fab fragments or gallamine per se significantly decreased baseline MAP and RSNA. Injection of digoxin-specific antibody Fab fragments attenuated the effects of a subsequent injection of gallamine. Microinjection of mAb against ouabain, but not nonspecific IgG, also significantly decreased baseline MAP and RSNA. These results suggest that OLC in the RVLM contributes to the tonic activity of vasomotor neurons in anesthetized normotensive rats, and the action of OLC in the RVLM is at least partly mediated by M2 muscarinic mechanisms.

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver.

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.

Stress-induced elevation of ouabainlike compound in rat plasma and adrenal.

Recent observations demonstrate the presence of neurosteroids and their rapid increase in response to acute stress. In view of a steroidal nature of ouabainlike compound, we tested the hypothesis that ouabainlike compound may participate in a homeostatic response to acute stress. Male Wistar rats were subjected to acute stress by swimming in water (22 degrees C) for 10 minutes. The levels of ouabainlike compound in plasma, hypothalamus, pituitary, and adrenal at 10, 40, and 70 minutes (n = 8 for each) after the end of swim stress were compared with nonstressed control levels (n = 10). Ouabainlike compound was measured by a radioimmunoassay for ouabain. Plasma levels of corticosterone and catecholamines were also measured. Plasma corticosterone concentrations increased rapidly at 10 minutes (P < .01) and then declined. A trend for a rise in plasma catecholamines was found at 10 minutes. Adrenal levels of ouabainlike compound concomitantly increased at 10 minutes (P < .01, control: 58.9 +/- 5.9 pmol ouabain equivalents per gram; 10 minutes: 92.5 +/- 4.8; 40 minutes: 47.3 +/- 9.6; 70 minutes: 45.1 +/- 6.3). In contrast, the response of plasma ouabainlike compound was slow and doubled at 40 minutes (P < .01, control: 115 +/- 12 pmol ouabain equivalents per liter; 10 minutes: 132 +/- 23; 40 minutes: 226 +/- 53; 70 minutes: 117 +/- 16). Ouabainlike compound levels in hypothalamus and pituitary remained unaltered. These findings suggest that ouabainlike compound may function as a stress hormone.

Elevation of ouabainlike compound levels with hypertonic sodium chloride load in rat plasma and tissues.

A major biologically active endogenous digitalis-like factor in the mammalian body may be an isomer of ouabain (ouabainlike compound, OLC). However, the exact role of OLC in sodium homeostasis is still unclear, and acute isotonic volume expansion does not enhance the secretion of OLC. We tested the hypothesis that OLC may be more important in the response to acute hypertonic NaCl load rather than isotonic volume expansion. We injected intraperitoneally 2 mL of 20% NaCl solution into male Wistar rats (n=34) and measured OLC levels in plasma, hypothalamus, pituitary, and adrenal at baseline (n=10) and 1, 2, and 4 hours (n=8 for each). In response to hypertonic NaCl loading, plasma Na-K ratio was elevated at 2 and 4 hours (P<.01). OLC levels in pituitary increased (P<.01) at 1 hour. Thereafter, plasma OLC levels increased at 2 and 4 hours (P<.05; basal, 75+/-11 pmol/L [+/-SEM]; 1 hour, 55+/-11; 2 hours, 130+/-24; 4 hours, 156+/-20). Concomitantly, OLC levels in adrenal increased at 2 and 4 hours (P<.01; basal, 1.7+/-0.2 pmol/g; 1 hour, 4.5+/-0.9; 2 hours, 5.0+/-0.7; 4 hours, 6.8+/-2.2). A significant correlation was observed between OLC levels in plasma and adrenal (P<.05). Plasma Na-K ratio positively correlated with OLC levels in plasma (r=.51, P<.01) and adrenal (r=.48, P<.01). Similar injection of physiological saline solution or hypertonic sucrose solution in physiological saline did not increase OLC levels in plasma and tissues. These findings indicate the elevation of OLC levels in plasma, pituitary, and adrenal in response to acute hypertonic NaCl load in rats and suggest that OLC may be involved in the response to the hypernatremic state.

CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line.

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.

Distribution and characterization of tumor necrosis factor-alpha-like immunoreactivity in the murine central nervous system.

Tumor necrosis factor-alpha (TNF alpha) is a protein released from macrophages during infection and inflammation. Recent studies suggest that it has several effects within the central nervous system, including generation of fever, enhancement of slow wave sleep, and stimulation of pituitary hormone secretion. We have proposed that TNF alpha may be synthesized by neurons in the CNS and used as a neuromodulator in the pathways involved in the central control of these activities. To test this hypothesis, we have used an antiserum raised against recombinant murine (rm) TNF alpha with an indirect immunoperoxidase technique to stain the murine CNS immunohistochemically. Western blot analysis of mouse brain homogenates revealed one band with electrophoretic mobility identical to that of rmTNF alpha. We identified TNF alpha-like immunoreactive (ir) neurons in the hypothalamus, in the bed nucleus of the stria terminalis, in the caudal raphe nuclei, and along the ventral pontine and medullary surface. TNF alpha ir innervation was widespread within the CNS, particularly in areas involved in autonomic and endocrine regulation, including the hypothalamus, amygdala, bed nucleus of the stria terminalis, parabrachial nucleus, dorsal vagal complex, nucleus ambiguus, and thoracic sympathetic preganglionic cell column. Our data suggest that TNF alpha may serve as a neuromodulator in central pathways involved in the regulation of the autonomic, endocrine and behavioral components of the acute-phase response to inflammation and infection.

Production and characterization of monoclonal antibodies to human tumor necrosis factor.

Five stable hybridoma cell lines secreting antibodies to recombinant human tumor necrosis factor (TNF) were established. All monoclonal antibodies belong to the IgG1 subclass. One monoclonal antibody (MAB) (designated as 3B10) neutralizes the L929 cytotoxic activity of natural and recombinant human TNF. Other MABs bind to human TNF but do not neutralize cytotoxic activity. A sandwich enzyme immunoassay specific to human TNF molecule has been developed using 3B10. This assay measures only biologically active human TNF molecules and is as sensitive as a bioassay which measures the cytotoxic activity on L929 cells. These results show that 3B10 is quite useful in studying the biological functions of TNF.

Characterization of Norwalk virus GI specific monoclonal antibodies generated against Escherichia coli expressed capsid protein and the reactivity of two broadly reactive monoclonal antibodies generated against GII capsid towards GI recombinant fragments.

BACKGROUND: Norwalk virus causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. In a previous study, the capsid protein of recombinant Norwalk virus genogroup II was expressed in an E. coli system and monoclonal antibodies were generated against it. The analysis of the reactivity of those monoclonal antibodies suggested that the N-terminal domain might contain more antigenic epitopes than the C-terminal domain. In the same study, two broadly reactive monoclonal antibodies were observed to react with genogroup I recombinant protein. RESULTS: In the present study, we used the recombinant capsid protein of genogroup I and characterized the obtained 17 monoclonal antibodies by using 19 overlapping fragments. Sixteen monoclonal antibodies recognized sequential epitopes on three antigenic regions, and the only exceptional monoclonal antibody recognized a conformational epitope. As for the two broadly reactive monoclonal antibodies generated against genogroup II, we indicated that they recognized fragment 2 of genogroup I. Furthermore, genogroup I antigen from a patient's stool was detected by sandwich enzyme-linked immunosorbent assay using genogroup I specific monoclonal antibody and biotinated broadly reactive monoclonal antibody. CONCLUSION: The reactivity analysis of above monoclonal antibodies suggests that the N-terminal domain may contain more antigenic epitopes than the C-terminal domain as suggested in our previous study. The detection of genogroup I antigen from a patient's stool by our system suggested that the monoclonal antibodies generated against E. coli expressed capsid protein can be used to detect genogroup I antigens in clinical material.

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats.

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Constitutive testosterone 6 beta-hydroxylase in rat liver.

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of the histaminergic neuron system in the central nervous system of rats; a fluorescent immunohistochemical analysis with histidine decarboxylase as a marker.

The distribution of histidine decarboxylase-like immunoreactivity (HDCI) in the rat central nervous system was studied by the indirect immunofluorescence technique. HDCI cell bodies were concentrated in the posterior hypothalamic area, such as in the tuberal magnocellular nucleus, caudal magnocellular nucleus, posterior hypothalamic nucleus and lateral hypothalamus just lateral to the fasciculus mammillothalamicus at the level of the posterior hypothalamic nucleus. Extensive networks of HDCI fibers of various densities were found in many areas of the brain; they were particularly dense in the hypothalamus but were also found in the following areas: rostrally in the cerebral cortex, olfactory nuclei, medial amygdaloid nucleus, n. tractus diagonalis, and bed nucleus of the stria terminalis, and caudally in the central gray matter of the midbrain and pons, auditory system, n. vestibularis medialis, n. originis nervi facialis, n. parabrachialis, n. commissuralis, n. tractus solitarii, and n. raphe dorsalis.

Production and partial characterization of anti-cord factor (trehalose-6,6'-dimycolate) IgG antibody in rabbits recognizing mycolic acid subclasses of Mycobacterium tuberculosis or Mycobacterium avium.

An ELISA with cord factor (trehalose-6,6'-dimycolate) is useful for the serodiagnosis of tuberculosis. To clarify the exact antigenic epitope in cord factor, recognized by a rabbit anti-cord factor IgG antibody, and to ascertain the most sensitive and specific diagnostic test antigen, rabbits were immunized with two kinds of cord factors isolated from Mycobacterium tuberculosis or Mycobacterium avium and the reactivities of the sera were tested against cord factors or the component mycolic acid methyl esters by ELISA. The serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against M. tuberculosis cord factor, but less reactive against M. avium cord factor. In contrast, the serum from rabbits immunized with M. avium cord factor was highly reactive against M. avium cord factor but less reactive against M. tuberculosis cord factor. Moreover, the serum from rabbits immunized with M. tuberculosis cord factor reacted against mycolic acid methyl esters, especially methoxy mycolic acid methyl ester. On the other hand, the serum from rabbits immunized with M. tuberculosis cord factor was less reactive against trehalose-6-monomycolate and not reactive against sulfolipid (2,3,6,6'-tetraacyl trehalose 2'-sulfate). From these results, it was concluded that the anti-cord factor IgG antibody, produced experimentally in rabbits, recognized the differences in the cord factor structures, i.e. the hydrophobic moiety rather than the carbohydrate moiety. It was also noted that the serum from rabbits immunized with M. tuberculosis cord factor was highly reactive against methoxy mycolic acid as an epitope. This paper is the first to describe how the anti-cord factor IgG antibody can recognize the mycolic acid subclasses, which differ according to the species of mycobacteria.

Evidence for the presence of a histaminergic neuron system in the rat brain: an immunohistochemical analysis.

Histamine-containing cells in rats were identified by indirect immunofluorescent histochemistry using an antibody raised against histidine decarboxylase (HDC), the enzyme forming histamine, which was purified from fetal rat liver. HDC-like immunoreactive (HDCI) structures could be detected in the brain as well as in peritoneal mast cells and basal-granulated cells in deep crypts of the gastric mucosa of rats. Numerous HDCI neurons were found in the posterior hypothalamic area and HDCI nerve fibers with a varicose appearance of fluorescence were widely distributed in various regions of the brain.

Detection of endotoxin antibody in long-term dialysis patients.

Endotoxins are often seen in dialysate. They are derived from Gram-negative bacteria especially Pseudomonas, E. coli and Serratia. Endotoxins are large-molecular-weight substances with an average molecular weight of 10(8). These large units can be divided into subunits down to a molecular weight of 10,000 which are thought to pass through dialyzer membranes. To investigate this, endotoxin antibody levels were measured in two groups of patients on chronic regular hemodialysis, a low-flux group using cellulosic membrane dialyzers (cuprophan and cuproammonium rayon (CAR) and a high-flux group using synthetic polymer membrane dialyzers (PMMA, EVAL). Using an ELISA based on standard endotoxin antibodies the percentages of patients in the low flux group with endotoxin antibodies were 26.9% with Cuprophan and 25% with CAR, not significantly different from a normal control group. In the PMMA and EVAL groups, it was 53.6% and 68.4% respectively. Back filtration of dialysate into blood is understood as the main reason for the entry of endotoxin in patients treated with high-flux dialyzers.

An anti-platelet activating factor antibody and its effects on platelet aggregation.

Production of antibodies against platelet activating factor (PAF) has been difficult, probably because of the low antigenicity of PAF, a low-molecular-weight phospholipid. We therefore used colloidal gold as a hapten carrier to produce anti-PAF polyclonal and monoclonal antibodies. Both antibodies reacted with PAF, lyso PAF, and L-alpha-lysophosphatidyl choline palmitoyl (lyso PCP), but they did not react phosphorylcholine chloride (PCC). Their affinities were higher for PAF than for lyso PAF and lyso PCP. When the antibodies were tested on PAF-induced platelet aggregation, they suppressed aggregation in a dose-dependent manner.

Development of the insulin-like growth factor-I assay system.

A high speed full automatic ELISA system for measurement of insulin-like growth factor-I (IGF-I) was established by using magnetic particle-linked monoclonal antibody and enzyme-labeled monoclonal antibody. A standard curve was obtained, and the effect of dilution on the assay system was investigated. An IGF-I spike recovery test of human serum samples and a study of the correlation with a radioimmunoassay system were performed, and good results were obtained from all studies. The assay range was 0.5-50 ng/ml, and the time required for the full automatic measurement was 15 minutes. This assay system will play a central role in the clinical approach to IGF-I.

Characterization of catch-relaxing peptide (CARP) immunoreactive neurons in the Helix nervous system.

Both small and large diameter of CARP immunoreactive neurons could be observed in the different ganglia of CNS of Helix but not in the foot musculature. The immunoreactivity is the strongest in the varicose segments of immunoreactive fibers. The present findings suggest a transmitter or modulatory role of CARP in both central and peripheral regulatory processes.