Depositional records of plutonium and (137)Cs released from Nagasaki atomic bomb in sediment of Nishiyama reservoir at Nagasaki.

In a sediment core of Nishiyama reservoir at Nagasaki city, depth profiles of (240)Pu/(239)Pu isotopic ratio, (239+240)Pu and (137)Cs activities were determined. Sediments containing plutonium and (137)Cs, which were deposited immediately after a detonation of Nagasaki atomic bomb, were identified in the core. Observed below the sediments were macroscopic charcoals, providing evidence for initial deposit of the fallout of the Nagasaki atomic bomb. This is the first entire depositional records of plutonium and (137)Cs released from the Nagasaki atomic bomb together with those from atmospheric nuclear tests.

HMG1-related DNA-binding protein isolated with V-(D)-J recombination signal probes.

In order to isolate cDNA clones for DNA-binding components of the V-(D)-J recombinase, phage libraries from a pre-B-cell line were screened with a radiolabeled probe containing recombination signal sequences (RSS). Among prospective clones, cDNA T160 was analyzed further. It produced a protein of 80.6 kDa which bound to DNA containing RSS but not to DNA in which the RSS had been mutated. A search of a data base revealed that the T160 protein has significant sequence homology (56%) to the nonhistone chromosomal protein HMG1 within the C-terminal region of 80 amino acids. DNA-binding analysis with truncated proteins showed that the HMG homology region is responsible for DNA binding. Using restriction fragment length polymorphisms, the T160 gene was mapped at the proximal end of mouse chromosome 2. Evidence was obtained for genetic linkage between the T160 gene and the recombination activator genes RAG-1 and RAG-2.

Plutonium isotopes derived from Nagasaki atomic bomb in the sediment of Nishiyama reservoir at Nagasaki, Japan.

The source of plutonium in sediments deposited at Nishiyama reservoir at Nagasaki was characterized by their (240)Pu/(239)Pu atom ratio. The average ratio was approximately 0.03, except in two layers. The main source of the plutonium was the Nagasaki atomic bomb. The plutonium continues to flow into the reservoir even now. The (240)Pu/(239)Pu atom ratios in two layers were higher than the average, which showed that plutonium in these layers were made of those of nuclear tests added to those of the atomic bomb.

A synthetic peptide coded for by the pre-S2 region of hepatitis B virus for adding immunogenicity to small spherical particles made of the product of the S gene.

Small spherical particles produced in the non-permissive phase of hepatitis B virus infection, when the viral genome is integrated into the chromosome of hosts, are rich in the product of the S gene, but poor in the product of the pre-S2 region. For the purpose of adding immunogenicity to spherical particles deficient in the pre-S2 region product, they were conjugated with a synthetic peptide of 19 amino acid residues. The peptide reproduced a hydrophilic area of the pre-S2 region product encoded by viral genomes of subtypes adr, ayw and ayr. The spherical particles supplemented with the pre-S2 peptide raised antibody to the pre-S2 region product in mice, in addition to antibody to the product of the S gene. Antibody to pre-S2 region product, prepared from sera of immunized mice by absorption with the S gene product, bound to spherical particles bearing pre-S2 region product, irrespective of adr, adw, ayw or ayr subtype, and agglutinated hepatitis B virions in immune electron microscopy. Based on the results obtained, the synthetic peptide may prove useful in adding protective efficacy to small spherical particles poor in pre-S2 region product.

Antibodies against type II phospholipase A2 prevent renal injury due to ischemia and reperfusion in rats.

This study was performed to determine the involvement of type II phospholipase A2 (PLA2-II) in renal injury caused by ischemia and reperfusion. Ischemia and reperfusion significantly elevated levels of blood urea nitrogen and serum creatinine in rats. These increases were significantly reduced by i.v. administration of rabbit IgG F(ab')2 fragments against rat PLA2-II. Increased levels of acid-stable PLA2 activity in the kidney were caused by ischemia and reperfusion, and were suppressed by administration of anti-PLA2-II F(ab')2. Increased levels of myeloperoxidase activity, a marker of neutrophil infiltration, in the kidney were also reduced after anti-PLA2-II F(ab')2 treatment. These results suggest that PLA2-II plays a pivotal role in pathogenesis of ischemia and reperfusion injury through induction of neutrophil infiltration.

Thrombopoietic activity of recombinant human interleukin-11 in nonhuman primates with ACNU-induced thrombocytopenia.

The effect of recombinant human interleukin-11 (rHuIL-11) on myelosuppressive nimustine (ACNU)-induced thrombocytopenia was assessed in nonhuman primates. A single intravenous (i.v.) injection of ACNU (15 mg/kg) was administered to cynomolgus monkeys on day 0. rHuIL-11 (100 microg/kg/day) or the vehicle was given subcutaneously (s.c.) from day 1 to day 21. In monkeys receiving ACNU, the circulating platelet count decreased to a low of 42 +/- 6 x 10(9)/L by day 21 but returned to pretreatment levels (375 +/- 48 x 10(9)/L) on day 30. Administration of rHuIL-11 prevented severe thrombocytopenia; the platelet count fell only to 138 +/- 23 x 10(9)/L on day 18, and platelet recovery was faster (458 +/- 91 x 10(9)/L by day 27) compared with that of the control animals. The size of bone marrow megakaryocytes from rHuIL-11-treated animals was larger than that of the controls, indicating that rHuIL-11 stimulated megakaryopoiesis in a myelosuppressive condition. Treatment with ACNU also caused leukopenia and moderate anemia. rHuIL-11 transiently and slightly decreased the white blood cell (WBC) and red blood cell (RBC) counts. Conversely, rHuIL-11 accelerated recovery of RBC count in the late administration period. These results support the assertion that rHuIL-11 may be an important therapeutic agent for reducing the severity and duration of thrombocytopenia following cancer chemotherapy.

A pitfall in two-site sandwich 'one-step' immunoassay with monoclonal antibodies for the determination of human alpha-fetoprotein.

Utilizing monoclonal antibodies directed to 2 distinct antigenic determinants of the human alpha-fetoprotein (AFP), Uotila, Ruoslahti and Engvall developed the 2-site sandwich immunoassay. They found that due to the different specificities of monoclonal antibodies, 2 antigen-antibody reactions, AFP with immobilized antibody and AFP with labeled antibody, could be accomplished in a single step. We have found, however, that above a certain concentration, AFP detectable by their method decreased because the labeled antibody tended to bind with AFP that failed to react with the immobilized antibody. Consequently, 2 different AFP concentrations produced the same result, and an extremely high, still clinically expectable concentration gave a false negative result. Such an inhibition in high AFP concentrations was not observed in the conventional '2-step' immunoassay within the range of concentrations tested (0.1 ng-3 mg/ml). On the basis of these observations, the 2-site '1-step' immunoassay for AFP would have to be applied on multiple dilutions of the serum to avoid an erroneous interpretation of the results.

A two-site sandwich radioimmunoassay of beta 2-microglobulin with monoclonal antibodies.

Among 21 batches of monoclonal antibodies raised against beta 2-microglobulin (anti-beta 2m), 6 reacted with soluble beta 2m, as well as with beta 2m present in association with HLA heavy chains on the surface of leukocytes. The remaining 15 anti-beta 2m antibodies bound with soluble beta 2m, but failed to react with beta 2m on the cell surface. No monoclonal anti-beta 2m antibodies revealed precipitin lines when they were tested against beta 2m in immunodiffusion. When 2 anti-beta 2m antibodies with different specificities were mixed together, however, they developed a precipitin line against beta 2m. Based on these observations, there was only 1 epitope on beta 2m that was available for the binding with a monoclonal anti-beta 2m antibody with either specificity. This allowed the development of a solid-phase radioimmunoassay in which beta 2m in test specimens was sandwiched between immobilized anti-beta 2m of 1 specificity and radiolabeled anti-beta 2m of a heterologous specificity in a single step. Monoclonal antibodies with at least 2 different specificities would be required for developing a sandwich-type immunoassay of polypeptides, such as beta 2m, that do not display 2 or more epitopes of the same specificity.

Three-site sandwich radioimmunoassay with monoclonal antibodies for a sensitive determination of human alpha-fetoprotein.

Utilizing monoclonal antibodies against human alpha-fetoprotein, 3 distinct antigenic determinants were identified. These antigenic determinants, provisionally designated a, b and c, were arranged in such a manner that the binding of one determinant with the corresponding antibody did not inhibit, or only barely inhibited the binding of antibodies directed to the other 2 determinants. Monoclonal antibodies with 3 different specificities were, therefore, applied to develop a sandwich-type solid-phase radioimmunoassay of the antigen in which wells were coated with anti-a, and radiolabeled anti-b together with radiolabeled anti-c was employed to detect the bound antigen. The 3-site sandwich radioimmunoassay involving 3 different determinants gave a higher sensitivity than 2-site assays in which only anti-b or anti-c was employed as a radiolabeled reagent, because the radioactivity of the 2 labeled antibodies was added on the antigen bound to immobilized anti-a.

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies.

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.

A solid-phase enzyme immunoassay for the determination of IgM and IgG antibodies against translation products of pre-S1 and pre-S2 regions of hepatitis B virus.

The envelope of hepatitis B virus is coded for by pre-S1, pre-S2 regions and the S gene. A method was developed to determine antibody to the product of pre-S1 region (anti-pre-S1) and antibody to the product of pre-S2 region (anti-pre-S2), either of IgM or IgG class, by a solid-phase enzyme immunoassay. For the determination of anti-pre-S1, tubular particles containing translation products of pre-S1, pre-S2 regions and the S gene were broken into constituent envelope polypeptides and immobilized on a solid support. Serums were absorbed with spherical particles containing translation products of pre-S2 region and the S gene, obtained from plasma positive for hepatitis B e antigen (HBeAg) and deprived of particles carrying pre-S1 product by an affinity column. They were then tested for the binding with tubular polypeptides fixed on a solid support, and the bound antibody representing anti-pre-S1 was detected by monoclonal antibody to human IgM/mu or IgG/gamma labeled with horseradish peroxidase. For the determination of anti-pre-S2, test serums were absorbed with spherical particles containing the product of the S gene, obtained from plasma positive for antibody to HBeAg and deprived of particles bearing pre-S2 product by an affinity column. They were then tested for the binding with polypeptides, fixed on a solid support, composed of products of pre-S2 region and the S gene. The assay was applied to the determination of anti-pre-S1 and anti-pre-S2 of IgM or IgG class in asymptomatic carriers and in persons who had recovered from infection with hepatitis B virus.

Application of hepatitis B core particles produced by human primary hepatocellular carcinoma (PLC/342) propagated in nude mice to the determination of anti-HBc by passive hemagglutination.

Human primary hepatocellular carcinoma (PLC/342), carried by nude mice, produces hepatitis B core particles as well as hepatitis B surface antigen particles. Core particles purified form PLC/342 tumors displayed epitopes of hepatitis B core antigen (HBcAg) but not epitopes of hepatitis B e antigen (HBeAg) on their surface, unlike core particles prepared from Dane particles, derived from plasma of asymptomatic carriers, that expressed epitopes of both HBcAg and HBeAg. Core particles obtained from PLC/342 tumors were applied to the determination of antibody to HBcAg (anti-HBc) by passive hemagglutination. The assay detected anti-HBc not only in individuals with persistent infection with hepatitis B virus and in those who had recovered from transient infection, but also in patients with acute type B hepatitis, indicating that it can detect anti-HBc of either IgG or IgM class. A liberal availability of core particles from tumors carried by nude mice, taken together with an easy applicability of the method, would make the passive hemagglutination for anti-HBc a valuable tool in clinical and epidemiological studies, especially in places where sophisticated methods are not feasible.

A two-site sandwich radioimmunoassay of human gamma interferon with monoclonal antibodies.

Two monoclonal antibodies (nos. 6008 and 6016) were raised against human gamma interferon (IFN-gamma) derived from E. coli harboring the recombinant cDNA for IFN-gamma, and one (3710) against a synthetic peptide representing its C-terminus amino acid sequence of 20 residues. The monoclonal antibody against the synthetic peptide (3710) reacted either with IFN-gamma or the synthetic peptide. One monoclonal anti-IFN-gamma (6008) did not react with the synthetic peptide, while the other (6016) showed a weak binding with the peptide. The binding of the monoclonal antibody against the synthetic peptide (3710) with IFN-gamma was not inhibited by 6008, but to a certain extent by 6016. A 2-site '1-step' radioimmunoassay was developed in which 6008 was fixed on a solid-phase support, and the test sample together with radiolabeled 3710 was added for the binding with it. The assay was rapid with a sensitivity capable of detecting a few ng/ml of IFN-gamma.

A two-site sandwich radioimmunoassay of human fibroblast (beta-) interferon with monoclonal antibodies.

Monoclonal antibodies were produced against human fibroblast (beta-)interferon (IFN-beta). Four of them were directed to the determinant designated a, while the remaining 3 to another determinant named b. An IFN molecule was found to bear one each of a and b determinants arranged in such a manner that the occupation of a with the corresponding antibody did not interfere with the binding of b to its corresponding antibody, and vice versa. This allowed the development of a 2-site sandwich radioimmunoassay in which antibody to b was immobilized on wells of a microtiter plate and the bound antigen was detected by the radiolabeled antibody to a. The 2-site sandwich radioimmunoassay detected, with a high sensitivity, IFN-beta either induced from fibroblast or produced by Escherichia coli harboring the gene of IFN-beta.

Pharmacological studies on a new benzamide derivative, YM-09151-2, with potential neuroleptic properties.

The electrophysiological properties and cataleptogenicity of YM-09151-2 (N-[(2RS, 3RS)-1-benzyl-2-methyl-3-pyrrolidinyl]-5-chloro-2-methoxy-4-methylaminobenzamide) were studied in the cat. This drug inhibited the EEG arousal response to electrical stimulation of the mesencephalic reticular formation with the same potency as that of haloperidol, whereas chlorpromazine revealed a more potent central depressant action. The duration of the afterdischarge induced by electrical stimulation of the amygdaloid nucleus was initially shortened and thereafter prolonged by both YM-09151-2 and haloperidol. However, YM-09151-2 was less effective than haloperidol and chlorpromazine in both augmenting the spindle bursts produced by electrical stimulation of the caudate nucleus and antagonizing the inhibitory effect of L-DOPA on the caudate spindle, indicating a smaller effect of YM-09151-2 on the extrapyramidal dopaminergic system than that of the two neuroleptic drugs. In fact, YM-09151-2 produced no cataleptic behaviour in the cat even at a dose of 5 mg/kg (s.c.), although haloperidol and chlorpromazine caused catalepsy in doses of 0.5 and 5 mg/kg (s.c.), respectively. Sulpiride, chemically related to YM-09151-2, showed much weaker central actions than YM-09151-2. The results indicate that a new benzamide, YM-09151-2, has a potent neuroleptic effect with a slight central depressant activity and that the cataleptogenicity of the compound is weaker than that of haloperidol and of chlorpromazine.

Synthesis and neuroleptic activity of benzamides. Cis-N-(1-benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-(methylamino)benzamide and related compounds.

Three series of benzamides of N,N-disubstituted ethylenediamines (linear alkane-1,2-diamines), 1-substituted 2-(aminomethyl)pyrrolidines, and 1-substituted 3-aminopyrrolidines (cyclic alkane-1,2-diamines) were designed and synthesized as potential neuroleptics. All target compounds were evaluated for their inhibitory effects on apomorphine-induced stereotyped behavior in rats, and a good correlation between structure and activity was found throughout the series. In the linear series (analogues of metoclopramide), introduction of a benzyl group on the terminal nitrogen, rather than an ethyl group, and a methyl group on the p-amino group of metoclopramide both enhanced the activity. The resulting N-[2-(N-benzyl-N-methylamino)ethyl]-5-chloro-2-methoxy-4-(methylamino) benzamide(23) was about 15 times more active than metoclopramide. In the cyclic series, particularly among the benzamides of 1-benzyl-3-aminopyrrolidine, most of the compounds tested were more active than the corresponding linear benzamides. cis-N-(1-Benzyl-2-methylpyrrolidin-3-yl)-5-chloro-2-methoxy-4-(methylamino) benzamide (YM-09151-2, 55) was the most active among all of the compounds tested, being 13 and 408 times more potent than haloperidol and metoclopramide, respectively. Moreover, compound 55 exhibited a fairly high ratio of antistereotypic activity to cataleptogenicity compared with haloperidol and metoclopramide. It is expected that compound 55 may be used as a potent drug with few side effects in the treatment of psychosis.

Synthesis and structure-activity studies of a series of spirooxazolidine-2,4-diones: 4-oxa analogues of the muscarinic agonist 2-ethyl-8-methyl-2,8-diazaspiro[4.5]decane-1,3-dione.

A series of spirooxazolidine-2,4-dione derivatives related to the putative M1 agonist 2-ethyl-8-methyl-2,8-diazaspiro[4.5]decane-1,3-dione (RS86; 1) were synthesized. The compounds were evaluated as cholinergic agents in in vitro binding assays and in in vivo pharmacological tests including antiamnesic effects using scopolamine-treated mice, hypothermia, and salivation in mice. Four compounds (5a,c,f and 17a) exhibited affinity for cortical M1 receptors and reversed scopolamine-induced impairment of mouse passive avoidance tasks, as did 1. Among these compounds, only 5a exhibited M1-receptor stimulating activity in pithed rats. Structural requirements for muscarinic activity in this series of spirooxazolidine-2,4-dione derivatives were as strict as those reported for spirosuccinimide derivatives including 1. The antiamnesic dose of 3-ethyl-8-methyl-1-oxa-3,8-diazaspiro[4.5]decane-2,4-dione (5a) was 2 orders of magnitude lower than the doses inducing hypothermia and salivation, in contrast to 1 for which the former dose was only 5-10-fold lower than the latter. These results suggest that the 8-azaspiro[4.5]decane skeleton represents a useful template for designing new muscarinic agonists as antidementia drugs.

Dopamine D3 and D4 receptor antagonists: synthesis and structure--activity relationships of (S)-(+)-N-(1-Benzyl-3-pyrrolidinyl)-5-chloro-4- [(cyclopropylcarbonyl) amino]-2-methoxybenzamide (YM-43611) and related compounds.

In this study, we synthesized a series of (S)-N-(3-pyrrolidinyl)benzamide derivatives, 1, 2a-d, 5a-1, and 7, and their enantiomers, (R)-1 and (R)-5c-e, and evaluated their binding affinity for cloned dopamine D2, D3, and D4 receptors and their inhibitory activity against apomorphine-induced climbing behavior in mice. The results indicate that D2, D3, and D4 receptors have different bulk tolerance (D4 > D3 > D2) for the substituent of the 4-amino group (R1) on the benzamide nuclei and that cyclopropyl-, cyclobutyl-, and cyclopentylcarbonyl groups likely possess adequate bulkiness with respect to D3 and D4 affinity and selectivity over D2 receptors in this series. The results also suggested that the N-substituent (R2) on the pyrrolidin-3-yl group performs an important role in expressing affinity for D2, D3, and D4 receptors and selectivity among the respective subtypes. One of the compounds, (S)-(+)-N-(1-benzyl-3-pyrrolidinyl)-5-chloro-4-[(cyclopropylcarbonyl+ ++) amino]-2-methoxybenzamide (5c) (YM-43611), showed high affinity for D3 and D4 receptors (Ki values of 21 and 2.1 nM, respectively) with 110-fold D4 selectivity and 10-fold D3 preference over D2 receptors and weak or negligible affinity for representative neurotransmitter receptors. Compound 5c displayed potent antipsychotic activity in inhibiting apomorphine-induced climbing behavior in mice (ED50 value, 0.32 mg/kg sc).

In vitro pharmacological profile of YM-43611, a novel D2-like receptor antagonist with high affinity and selectivity for dopamine D3 and D4 receptors.

1. We investigated some neurochemical properties of a novel benzamide, YM-43611, [(S)-N-(1-benzyl-3-pyrrolidinyl)-5-chloro-4-cyclopropylcarbonylamino+ ++-2- methoxybenzamide] in comparison with putative D2-like receptor antagonists using both rat and human cloned dopamine D2-like receptors in vitro. 2. Receptor binding studies revealed that YM-43611 had appropriately potent affinities for both rat and human D2-like receptors, with moderate selectivity for D3 receptors and high selectivity for D4 receptors over D2 receptors (Ki values (nM) for rat receptors: D2, 165; D3, 35.5; D4, 1.85, and for human receptors: D2, 42.9; D3, 11.2; D4, 2.10). 3. YM-43611 displayed weak or negligible affinity for other neurotransmitter receptors, namely D1, D5, alpha(1), alpha(2), beta, 5-HT1A, 5-HT2A, 5-HT3, H1, M1 and M2 receptors. 4. Dopamine stimulated low-Km GTPase activity on membranes from Chinese hamster ovary (CHO) cells expressing the human D2-like receptor subtype. This response to dopamine of low-Km GTPase activity was inhibited by use of putative D2-like receptor antagonists. YM-43611 showed a moderate selectivity for D3 receptors (Ki = 45.5 nM) and a high selectivity for D4 receptors (Ki = 3.28 nM) over D2 receptors (Ki = 70.6 nM). 5. Dopamine inhibited forskolin-stimulated adenylate cyclase in intact CHO cells expressing the human D2-like receptor subtype. YM-43611 shifted the inhibition curve of dopamine on respective D2-like receptor subtype-mediated cyclic AMP formation to the right in a parallel fashion, showing a pA2 value of 7.42 (38.1 nM) for D2 receptors, a pKB value of 8.06 (8.68 nM) for D3 receptors, and a pA2 value of 8.42 (3.77 nM) for D4 receptors. 6. YM-43611 but not the other D2-like receptor antagonists exhibited good selectivity with respect to dual antagonism for D3 and D4 receptors in both receptor binding and functional assays. 7. These results indicate that YM-43611 is a novel D2-like receptor antagonist with high potency and selectivity for both D3 and D4 receptors. YM-43611 is therefore expected to be valuable in exploration of the physiological role of D3 and D4 receptors.

Hepatitis B surface antigen with an excess or deficiency in subtypic determinants in sera from asymptomatic carriers in Japan.

Using the solid-phase enzyme immunoassay with monoclonal antibodies, hepatitis B surface antigen (HBsAg) was subtyped in sera from 5082 asymptomatic carriers who donated blood units at regional blood centers in Japan. Among them, 5004 sera contained HBsAg of a regular subtype, i.e., adw, adr, ayw or ayr, while 74 contained HBsAg with excessive subtypic determinants, such as adyw, adyr, adwr, aywr, or adywr. The presence of subtypic determinants on the selfsame particle was ascertained by sandwiching HBsAg between two monoclonal antibodies of distinct subtypic specificities. The remaining 4 sera contained HBsAg that possessed only one subtypic determinant, such as ad, ar or aw. HBsAg particles of atypical subtypes would have been given rise to by a point mutation in the S gene involving the codons regulating subtypic specificities.