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Background: Recently, vasodilators have been increasingly being recognized as useful for the treatment of acute heart failure syndromes (AHFS). Although carperitide (alpha-human atrial natriuretic peptide) has vasodilatory, diuretic and organ-protective effects, its efficacy and safety for the first-line drug treatment of AHFS have not been reported., Methods and Results: A prospective observational study was performed in AHFS patients with preserved systolic blood pressure (SBP >or=120 mmHg), pulmonary congestion and dyspnea who were receiving carperitide monotherapy. The analysis was conducted in 1,832 patients (male: 52.7%; mean age: 75.1+/-12.7 years). The initial SBP was 151.1+/-25.7 mmHg; 62.0% were diagnosed as having acutely decompensated chronic heart failure and 78.8% were assessed as functional class III-IV according to New York Heart Association classification. Carperitide was administered at an initial dosage of 0.025-0.05 microg x kg(-1) x min(-1) in 50.4% of patients. In 1,524 patients (83.2%), carperitide monotherapy restored the acute phase and improved the degree of dyspnea as assessed using the modified Borg scale. The incidence of adverse drug reactions was 4.64%; the most frequently reported adverse reaction was hypotension (3.55%)., Conclusion: In the present study, following carperitide monotherapy, 83.2% of AHFS patients recovered from the acute phase. Based on these findings, carperitide seems useful for the first-line drug treatment of AHFS in patients with pulmonary congestion and preserved blood pressure.

For the quantitative analysis of vitamin D-dependent 28-kDa calcium-binding protein (calbindin-D) in the CNS, we have established a highly sensitive immunoassay method. The antisera were raised in rabbits with purified calbindin-D from rat kidneys, and the antibodies were purified with a calbindin-D-coupled Sepharose column. The purified antibodies were specific for calbindin-D, showing a single band on the immunoblot with the extract of rat kidney or cerebellum. The sandwich-type immunoassay system was prepared by the use of purified monospecific antibodies, and the minimum detection limit of the assay was 0.1 pg or 3.6 amol of calbindin-D, which was sufficiently sensitive for the measurement of calbindin-D content in isolated Purkinje cell bodies at the level of single cells. The average content of calbindin-D in a single Purkinje cell was 0.05 pg. Calbindin-D was detected in most of the rat tissues examined, but it was present predominantly in the kidney and CNS, especially in the cerebellum. Calbindin-D was detected at a similarly low level in the cerebral cortex, cerebellum, and brainstem of rat embryos of 15 gestational days, and it increased gradually but differently in these regions, reaching the respective adult levels by 4-5 weeks of postnatal age. In contrast, kidney calbindin-D increased sharply between 15 gestational days and 3 postnatal days, reaching the adult level by 6 days of age. Calbindin-D levels in the adult rat CNS were affected little by age, whereas the concentrations in human cerebral cortices were significantly low in the aged brain as compared with those in the young brain.(ABSTRACT TRUNCATED AT 250 WORDS)

For the quantitative analysis of the A subunit of alpha crystallin (alpha A) in the lens and for the survey of possible existence of alpha A in the non-lenticular tissues, we have established a highly sensitive and specific immunoassay method for alpha A. Antisera to alpha A were raised in rabbits with alpha A purified from bovine lens, or the C-terminal decapeptide (EEKPSSAPSS) of alpha A (alpha Apep). The antibodies to alpha A and alpha Apep were purified by the use of an alpha A-coupled Sepharose 4B column. The F(ab')2 fragments of purified anti-alpha A IgG were immobilized on polystyrene balls and the Fab' fragments of purified anti-alpha Apep IgG were labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the sandwich-type immunoassay using the two antibody preparations was less than 10 pg alpha A without any cross-reactivity with alpha B. By employing the present methods, it was found that a significant amount of immunoreactive alpha A was present in rat spleen and thymus. Very low levels of immunoreactive alpha A were detected in the rectum, caecum, liver, kidney, adrenal, cerebellum and brainstem. The immunoreactive alpha A in the spleen extract was purified partially (about 50% purity) by the use of anti-alpha Apep-coupled Sepharose. The concentration of alpha A in the spleen was less than 1 ng/mg protein before 3 weeks of age. After 5 weeks of age, however, it increased lineally reaching about 20 ng/mg protein by 18 weeks of age. Immunohistochemically, the alpha A was localized in the reticular cells in the spleen and thymus.

A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.

In order to determine the quantitative distribution of alpha B crystallin (alpha B) in non-lenticular tissues, we have established a sensitive immunoassay system for specific measurement of alpha B. Antisera were raised in rabbits by injecting alpha B purified from bovine lenses, or C-terminal decapeptide (KPAVTAAPKK) of alpha B (alpha Bpep). The antibodies to alpha B and alpha Bpep were purified by the use of alpha B-coupled Sepharose column. The F(ab')2 fragments of antibody IgG to alpha B were immobilized on polystyrene balls and the Fab' fragments of antibody IgG to alpha Bpep were labeled with beta-D-galactosidase from Escherichia coli. The sandwich-type enzyme immunoassay consisted of the above two antibodies was sensitive, and the minimum detection limit of the assay was 10 pg alpha B without any crossreactivity with alpha A. By using the assay method, it is revealed that the alpha B was distributed in most of the tissues examined. Among the non-lenticular tissues, alpha B was present at high levels in the heart and striated muscles, especially in the soleus muscle, and kidney. High levels of alpha B in the muscle tissues were also seen in various animals. Developmental increases of alpha B in rat muscle tissues and kidney were observed from 16 days of gestational age to 1 or 5 weeks of postnatal age. In contrast, the alpha B in the brain kept a low level during the same period. After 5 weeks of age, alpha B concentrations in the brain increased sharply, reaching the adult levels at 9 weeks of age. Immunohistochemical staining with anti-alpha Bpep revealed that alpha B was positive not only in glial cells, in the central nervous tissues, but also in some neurons of spinal cord, brainstem, hippocampus, and olfactory bulb. Spermatocytes in the testis were also immunopositive for alpha B.

Concentrations of nervous tissue-related proteins, including S-100 proteins (alpha and beta), enolase isozymes (alpha and gamma), superoxide dismutase (SOD) isozymes (Cu/Zn SOD and Mn SOD), and GTP-binding proteins (alpha subunits of GO and Gi2) were determined in the four cerebrocortical regions (superior frontal gyrus of frontal lobe, parahippocampal gyrus of temporal lobe, superior parietal lobule of parietal lobe, and calcarine area of occipital lobe) of patients with Alzheimer's disease, and age-matched control and young control patients by means of enzyme immunoassay methods. Although the temporal cortex of some patients with Alzheimer's disease (4/7) showed apparently enhanced S-100 beta with decreased gamma-enolase, concentrations of neuronal (neuron-specific gamma-enolase and the alpha subunit of GO) and glial (S-100 beta, S-100 alpha, and alpha-enolase) marker proteins, and both SODs in each region were not significantly different between patients with Alzheimer's disease and the age-matched controls. Concentrations of Gi2 alpha also showed similar values in the cerebral cortices of young and aged controls and patients with Alzheimer's disease. However, when compared with young controls, S-100 beta in the four regions of patients with Alzheimer's disease and aged controls, and Cu/Zn SOD in frontal cortex of patients with Alzheimer's disease were significantly enhanced (P less than 0.01).

A sensitive sandwich-type enzyme immunoassay for measurement of human Mn superoxide dismutase (Mn SOD) was developed using purified antibodies specific to Mn SOD. The antisera were raised in rabbits by injecting Mn SOD purified from human liver. The antibody IgG, purified by the use of Mn SOD-coupled Sepharose, showed a single band on the immunoblotting test with a crude liver extract. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg human Mn SOD/assay tube. Serum Mn SOD concentrations of healthy adults (77.5 +/- 18.0 ng/ml (1 SD), n = 120, 16-64 yr old) were not related to age or sex. Immunoreactive Mn SOD was detectable in most tissues examined except for erythrocytes. The concentrations of immunoreactive Mn SOD and Cu/Zn SOD in the cerebral cortex were not different among the patients with Alzheimer's disease, and the age matched and young patients without neurological disorders.

A sensitive sandwich-type enzyme immunoassay for the aldolase isozyme, A4, was developed using purified antibodies specific to the A subunit of aldolase. The antibodies were raised in sheep being immunized with purified aldolase A4 and then purified by immunoaffinity chromatography on a column of aldolase A4-coupled Sepharose. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody F(ab')2 fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was sensitive enough to detect 10 pg/tube of aldolase A4. The assay was specific to the A subunit of aldolase (aldolase A). It cross-reacted about 40% to aldolase A3C, 7% to A2C2 and 0.3% to AC3, but not cross-reacted with C4 nor B4. Coefficients of variation in intra- and inter-assay were less than 16%. Serum aldolase A levels were determined in healthy adults, which were about 200 ng/ml. The distribution and concentrations of immunoreactive aldolase A in various human tissues were also determined. High concentrations of aldolase A were found in skeletal muscle, heart muscle, cerebrum and lymphatic tissue.

A highly sensitive enzyme immunoassay method for measurement of human Cu/Zn superoxide dismutase (SOD) was established. Antisera were raised in rabbits by injecting SOD purified from human erythrocytes, and antibodies to SOD were purified by the use of Cu/Zn SOD-coupled Sepharose 4B. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive, and the minimum detection limit was 3 pg or 0.1 fmol/assay tube. Serum Cu/Zn SOD levels of normal healthy subjects (36.3 +/- 15.6, n = 120, 16-64 years old) were not related to age and sex. It was confirmed that Cu/Zn SOD levels in erythrocytes and blood plasma were significantly enhanced in patients with Down syndrome. Immunoreactive Cu/Zn SOD was detectable in all the tissues examined and was present at high concentrations in brain, adrenal gland, liver, heart and kidney.

The human gene encoding the beta subunit of S-100 protein (S-100 beta) was mapped on chromosome 21. In order to confirm the expression of gene-dosage effect of S-100 beta in patients with Down's syndrome (DS), concentrations of immunoreactive S-100 alpha and S-100 beta proteins were determined in the blood plasma and lymphocytes fraction of the patients and control subjects. Cu/Zn-superoxide dismutase (SOD), a protein that is known to show the gene-dosage effect on the trisomy of chromosome 21, also was immunoassayed in the same blood samples as control proteins. In blood plasma, S-100 beta protein as well as Cu/Zn SOD was enhanced (P less than 0.001) in the patients (160 +/- 70 pg S-100 beta/ml and 87 +/- 83 ng SOD/ml, N = 44) as compared with control individuals (76 +/- 25 pg/ml, and 18 +/- 11 ng/ml, respectively, N = 28). However, concentrations of S-100 alpha in blood plasma of DS patients were similar to those of normal subjects. Concentrations of S-100 beta in lymphocyte fractions of DS patients (24.7 +/- 10.9 ng/mg protein) were also higher (P less than 0.001) than those of control subjects (10.1 +/- 5.8 ng/mg protein). These results indicate that gene-dosage effect of S-100 beta levels are expressed in DS patients.

We determined tissue concentrations of brain-type glycogen phosphorylase in normal kidney and renal cell carcinoma by enzyme immunoassay; we also localized it immunohistochemically. Tissue concentration of brain-type glycogen phosphorylase in the renal cortex (n = 13) was 1430 +/- 709 ng/mg protein (mean +/- standard deviation) and that in the medulla (n = 13) was 1270 +/- 635 ng/mg protein. On the other hand, the concentration in renal cell carcinoma (n = 26) was 2530 +/- 1540 ng/mg protein, ranging from 520 to 6860 ng/mg, significantly higher than those in renal cortex and medulla. Clear cell type tumors contained slightly higher levels of the phosphorylase (2600 +/- 1430 ng/mg protein) than granular cell type tumors (2100 +/- 1520 ng/mg protein). In renal tissues, brain-type glycogen phosphorylase was immunohistochemically localized in epithelial cells of proximal and distal tubules, collecting tubules, thick and thin limbs of loops of Henle, and Bowman's capsules. In renal cell carcinoma, the phosphorylase was immunohistochemically demonstrated in 97% (34/35) of cases, including one sarcomatoid variant. These findings indicate that renal cell carcinoma cells contain enhanced tissue levels of brain-type glycogen phosphorylase.

The pharmacokinetics of AT-2266 (1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-1,8-naphthyridine- 3-carboxylic acid) were studied in various experimental animals and compared in a number of aspects with those of norfloxacin. Both agents were administered orally. The mean peak plasma levels of AT-2266 in mice, rats, and dogs (given a single dose of 50 mg/kg for mice and rats and 25 mg/kg for dogs) were 2.39, 1.63, and 5.00 mug/ml, respectively, with elimination half-lives of 2.24, 2.81, and 5.76 h. The respective mean plasma levels of norfloxacin at similar dosages were 0.510, 0.410, and 0.700 mug/ml; elimination half-lives were 1.40, 2.35, and 6.06 h. In dogs repeatedly dosed with 25 mg of AT-2266 per kg every 12 h, the mean peak plasma levels after the third and fifth doses were about 1.4 times those after the first dose. The binding rates of AT-2266 and norfloxacin to plasma of mice, rats, and dogs and to human serum ranged from 27.6 to 40.2% and 39.8 to 44.2%, respectively. In rats receiving a single dose of 50 mg/kg, the respective mean peak levels of AT-2266 in plasma, lung, muscle, and kidney were 2.47, 4.60, 5.35, and 33.9 mug/ml or g, whereas those of norfloxacin were 0.234, 0.390, 0.272, and 2.05 mug/ml or g. AT-2266 was widely distributed in tissues of dogs and monkeys after repeated dosage. The respective 24-h recoveries of AT-2266 from urine of mice, rats, and dogs after single doses of 50, 50, and 25 mg/kg were 56.6, 40.5, and 64.1%, and recoveries of norfloxacin at these doses were 4.40, 2.91, and 5.34%. The respective 24-h recoveries of AT-2266 from bile and feces of rats given a single dose of 50 mg/kg were 2.47 and 52.7%. Bioautography of plasma and urine indicated that AT-2266 was metabolized to but a slight degree. The results indicate that AT-2266 is better than norfloxacin in oral absorption and similar to the latter in stability to metabolic inactivation.

Pipemidic acid was absorbed well by the oral route. Its peak levels in plasma ranged from 4 to 12 mug/ml at an oral dose of about 50 mg/kg in mice, rats, dogs, monkeys, and men. The protein binding of pipemidic acid was about 20% in dog plasma and about 30% in human serum. Pipemidic acid was distributed to most of the organs and tissues tested at the concentrations comparable to or higher than the plasma level. Its concentrations in bile and urine were much higher than the plasma level. About 25 to 88% of orally administered pipemidic acid was excreted into urine in a bacteriologically active form, the percentage depending on the animals and doses employed. The remainder was excreted into feces in men. The main active principle in vivo was unchanged pipemidic acid itself. The mean lethal dose of pipemidic acid after a single oral dose was more than 16,000 mg/kg in mice. No abnormalities were observed in mice orally receiving pipemidic acid once a day for 4 weeks at doses of 1,000, 2,000, and 4,000 mg/kg per day, and in rats orally receiving the drug once a day for 2 weeks at doses of 400 and 1,600 mg/kg per day.

Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity. Antisera were developed in rabbits with purified phosphorylase as the immunogen. Antibodies were first affinity-purified with a column of brain phosphorylase-coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase-coupled Sepharose to remove antibodies reactive also with muscle phosphorylase. By using the specific antibodies, a sandwich-type immunoassay system for measurement of brain phosphorylase was prepared. The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was sensitive and specific to the brain phosphorylase. The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross-reactivity of the assay with muscle phosphorylase was less than 1%. Tissue concentrations of immunoreactive brain-type phosphorylase were estimated. The phosphorylase was present in the heart at as high a level as in the brain. The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis. Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex.

A highly sensitive enzyme immunoassay system for measurement of aldolase B subunit (aldolase B) was established. Antisera were raised in rabbits by injecting aldolase B4 purified from human liver, and specific antibodies to aldolase B were purified by the use of a column of aldolase B4-coupled Sepharose. The purified antibody IgG was digested with pepsin to obtain the F(ab)' fragments. The antibody F(ab)' fragments were immobilized noncovalently on polystyrene balls, and the same antibody Fab' fragments were labeled with beta-D-galactosidase from Escherichia coli. The sandwich-type assay system using these reagents was sensitive and specific to aldolase B, showing no cross-reactivity with aldolase-A or aldolase-C. The minimum detection limit of the assay was 3 pg aldolase B4/assay tube. The immunoreactive aldolase B was present at high levels in the liver and kidney, and considerably in the small intestine. It was detected in all the tissues examined. Immunohistochemically, aldolase B is localized in hepatocytes, proximal renal tubular cells and epithelial cells of small intestine. Serum levels of aldolase B in healthy subjects were ranged from 33 to 202 ng/ml.