Contrasting effects of an angiotensin converting enzyme inhibitor and a calcium antagonist on calcium transients in isolated rat cardiac myocytes.

OBJECTIVE: The aim was to examine the effects of an angiotensin converting enzyme (ACE) inhibitor and a calcium antagonist on intracellular calcium transients in isolated cardiac myocytes from a monocrotaline induced right ventricular hypertrophy model. METHODS: One week after monocrotaline injection, Sprague-Dawley rats were given either an ACE inhibitor (delapril-HCl) or a calcium antagonist (nilvadipine) for two weeks. Using fura-2/AM, calcium transients were measured in single myocytes separated from the right ventricle. RESULTS: The severe right ventricular hypertrophy observed in untreated rats was significantly reduced in drug treated animals. The inhibitory effects of delapril were more prominent than those of nilvadipine, although both drugs reduced right ventricular pressure to the same extent. Calcium transients in delapril treated rats were similar to those in control rats. On the other hand, the calcium transient in nilvadipine treated rats was decreased and its time course was prolonged. The changes were similar to those found in monocrotaline treated rats. The responsiveness of calcium transients to isoprenaline in delapril treated rats was similar to that in control rats. The responsiveness in nilvadipine treated rats was decreased, and was similar to that in monocrotaline treated rats. Delapril improved developed tension and the beta adrenoreceptor responsiveness of developed tension to isoprenaline. CONCLUSIONS: Although delapril and nilvadipine inhibited cardiac hypertrophy in monocrotaline treated rats, significant improvement of contractile function and beta adrenoreceptor responsiveness was observed only in the delapril treated rats. This improvement was partially due to the improvement in calcium transients and the restoration of the beta adrenoreceptor responsiveness of the calcium transient to beta adrenergic stimulation.

Prominent inhibitory effects of tranilast on migration and proliferation of and collagen synthesis by vascular smooth muscle cells.

To obtain some ideas about prevention of restenosis after percutaneous transluminal coronary angioplasty (PTCA), we examined the effects of transilast (anti-allergic agent) on migration and proliferation of, and collagen synthesis by, cultured vascular smooth muscle cells (VSMC) from the thoracic aorta of WKY rats. Tranilast was added to culture medium containing 10% fetal calf serum (FCS). The cultures were pulse-labeled with 3H-thymidine (TdR) or 3H-proline (Pro). TdR and Pro uptake into VSMC were measured. The effect of tranilast on migration of VSMC was examined by using culture dishes of an original design. We also examined the inhibitory effects of various drugs, such as a Ca antagonist, an angiotensin converting enzyme (ACE) inhibitor, a phosphodiesterase inhibitor, elastase, colchicine, and mitomycin C, on proliferation and migration of VSMC. Our data showed that the inhibitory effects of tranilast on migration and proliferation of, and collagen synthesis by, VSMC were prominent. Maximal percentage inhibition of proliferation, migration and collagen synthesis was 60.8 +/- 2.3%, 52.7 +/- 14.7% and 62.1 +/- 8.1%, respectively. On the other hand, the inhibitory effects of other drugs, with the exception of colchicine and mitomycin C, on proliferation and/or migration of VSMC were not very strong. Although the inhibitory effects of colchicine and mitomycin C were strong in vitro, their clinical usefulness may be limited by systemic side-effects. These results indicate the potential usefulness of tranilast for prevention of restenosis of coronary arteries after PTCA.

Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans.

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.

Different effects of an angiotensin converting enzyme inhibitor and a calcium antagonist on protein metabolism in rats with right ventricular hypertrophy.

OBJECTIVE: We examined the effects of a calcium antagonist and an angiotensin converting enzyme (ACE) inhibitor on contractile and non-contractile protein metabolism and cardiac function in a monocrotaline-induced right ventricular hypertrophy model, in order to define the effects of these drugs on cardiac hypertrophy. METHODS: One week after monocrotaline injection, male Sprague-Dawley rats were given either a calcium antagonist (nilvadipine; 3 mg/kg per day) or an ACE inhibitor (delapril-HCl; 30 mg/kg per day) for 2 weeks. Right ventricular pressure, the right ventricle: (left ventricle + interventricular septum) ratio, myosin isoenzymes, collagen concentration, collagen types and contractility of right ventricular free wall were examined. RESULTS: In untreated rats significant monocrotaline-induced right ventricular hypertrophy with an increase in the proportion of collagen types III and V was observed. There were no significant changes in collagen concentration. Both drugs reduced right ventricular pressure to the same degree and decreased right ventricular hypertrophy. However, the inhibitory effect of delapril on right ventricular hypertrophy was stronger than that of nilvadipine. Nilvadipine reduced the collagen concentration and reversed changes in collagen types, whereas delapril did not have any significant effect on collagen concentration or collagen types. Cardiac contractility was improved by delapril, but not by nilvadipine. CONCLUSIONS: The results show that a calcium antagonist disproportionately inhibited contractile and non-contractile protein metabolism, whereas an ACE inhibitor proportionally inhibited them and improved cardiac function in a model of right ventricular hypertrophy. The improvement in cardiac function may be due partly to the proportional inhibition of contractile and non-contractile proteins elicited by an ACE inhibitor.

Cytokine dependent growth of human TF-1 leukemic cell line in human GM-CSF and IL-3 producing transgenic SCID mice.

Although severe combined immunodeficient (SCID) mice are considered useful as an animal model for human hematopoietic diseases, the complete reconstruction of human hematopoietic cells can not be established even in these mice. This appears to be because human cytokines, adhesion molecules and extracellular matrices which support differentiation and growth of human hematopoietic cells differ from those in animals. To improve this animal model, we attempted to produce transgenic (Tg) mice producing human interleukin 3 (hIL-3) and human granulocyte macrophage colony stimulating factor (hGM-CSF) with the homozygote of the scid gene. We established two Tg mouse lines, one releasing both 0.5-1 ng/ml of hIL-3 and 0.05-0.2 ng/ml of hGM-CSF in their sera and another releasing only high (2-10 ng/ml) levels of hGM-CSF. When human cytokine-dependent myeloid cell line, TF-1, was subcutaneously transplanted into these two Tg-SCID mouse lines, TF-1 could be successfully engrafted and grew in all lines of Tg-SCID mice but not in control mice. We also observed that TF-1 grows in GM-CSF Tg-SCID mice in a dose dependent manner in vivo and IL-3 shows an additive effect on its growth. These results indicated that these Tg-SCID mice were an useful in vivo model for investigating human leukemogenesis, especially the role of IL-3 and GM-CSF in leukemogenesis.

Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the flavoprotein (Fp) subunit of liver mitochondria.

Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle, and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms. In this study, homology probing with mixed primers for the polymerase chain reaction and subsequent sequence analysis were successfully applied to clone cDNA for the flavoprotein (Fp) subunit of human liver complex II. The isolated clone contains an open reading frame of 1,992 nucleotides and encodes a mature protein of 621 amino acids with a molecular weight of 68,011. The amino acid sequence was highly homologous with that of bovine heart Fp (93.2%) and was quite different from the partial sequence of human placental Fp reported previously [Malcovati et al. (1991) in Flavins and Flavoproteins 1990, pp. 727-730], which showed striking homology to that of Bacillus subtilis. To solve this discrepancy, the partial cDNA sequences of the stomach and placental Fp subunits of human complex II were determined in addition to the full length cDNA of liver. The sequence data, sensitivity to thiol reagents and antigenic properties indicated that the major from of FP subunit in human complex II is unique at least among the three tissues analyzed, and is more similar to the Fp subunit of bovine heart than to that of B. subtilis.

Developmental changes in enzyme activities and in structural features of rat masticatory muscle mitochondria.

The functional ability of a muscle is closely related to the activities of the mitochondria, which are energy-producing organelles in muscle cells. The development of the mammalian masticatory muscle progresses dramatically when feeding behavior changes from suckling to mastication, but it is unclear how the energy-producing systems of the mitochondria change. In this paper, the development of rat masticatory muscle mitochondria was investigated in terms of enzyme activities of the mitochondrial respiratory chain and the structural and numerical development of mitochondria, especially regarding the change in feeding behavior from suckling to mastication. Using isolated mitochondria from the masticatory muscle, we measured succinate dehydrogenase, NADH dehydrogenase, succinate-O2 oxidoreductase, and NADH-O2 oxidoreductase. These were found to be increased in the 15-day postnatal rat compared with the 0- to 10-day postnatal rat. The structural development of mitochondria was gradual in the 0- to 15-day postnatal rat. However, a notable increase was found in the cross-sectional area of mitochondria between 10 and 15 days postnatally. The number of mitochondria per muscle fiber was apparently constant during the same period. We demonstrated that the change in feeding behavior was well-correlated with an increase in mitochondrial enzyme activity, also supported by the early structural development of mitochondria.

Gou-teng (from Uncaria rhynchophylla Miquel)-induced endothelium-dependent and -independent relaxations in the isolated rat aorta.

Gou-teng is a drug used for treatment of hypertension in Chinese medicine. Its antihypertensive action has been previously confirmed in the spontaneously hypertensive rat (SHR). Here, its vasorelaxing effect and the mechanisms of actions were studied in vitro. Gou-teng extract (GTE) relaxed the norepinephrine (NE)-precontracted aortic ring preparations isolated from Wistar Kyoto rats (WKY) with and without intact endothelium; the latter was significantly less sensitive than the former. The GTE-induced endothelium-dependent relaxation was significantly inhibited by NG-monomethyl-L-arginine (NMMA) in a dose-dependent manner while indomethacin did not affect the relaxation. Atropine inhibited the acetylcholine (ACh)-induced endothelium-dependent relaxation but did not the GTE-induced one. Furthermore, once GTE was applied, the following NE-induced contraction was significantly reduced even after repeated washout. NMMA effectively reduced and rather reversed this residual effect of GTE. From these results, it is concluded that GTE relaxes the NE-precontracted rat aorta through endothelium-dependent and, to lesser extent, -independent mechanisms. The endothelium-dependent component would be mediated by EDRF/NO pathway in which the muscarinic cholinoceptors were not involved. Thus, GTE appears to be a potent and long-lasting vasodilator mainly through EDRF/NO release.

Non-ulcerative cutaneous lesion in immunodeficient mice with Leishmania amazonensis infection.

Cutaneous leishmaniasis begins as papules or nodules at the site of promastigote inoculation. The next key pathogenic event in this disease is the formation of an ulcer at this site. Leishmania infection in immunodeficient mice, however, showed non-ulcerative cutaneous lesions suggesting the involvement of the immune system in ulcer formation. Severe combined immunodeficient (SCID), recombination-activating gene 2 knockout (RAG-2-/-), and immunocompetent mice were inoculated subcutaneously with cultured L. amazonensis promastigotes. Macroscopic nodules appeared at the inoculation site within 2 weeks of infection in all the mice and gradually extended to the surrounding skin tissue. Although nodules of immunocompetent mice ulcerated within 6 weeks, immunodeficient mice did not form ulcers even after 25 weeks of inoculation. These results strongly suggest the importance of functional T and B cells in ulcer formation of cutaneous leishmaniasis and are consistent with clinical features of non-ulcerative cutaneous leishmaniasis in some AIDS patients. The present study also indicates that the L. amazonensis-infected immunodeficient mouse model might be suitable for studying the mechanisms of ulcer formation in cutaneous leishmaniasis.

Phylogenetic identification of Sparganum proliferum as a pseudophyllidean cestode by the sequence analyses on mitochondrial COI and nuclear sdhB genes.

Sparganum proliferum is a larval cestode for which the adult stage is unknown. It is characterized by the continuous branching and budding when parasitized to humans, and causes fatal human sparganosis. However, the biological features of S. proliferum, including its taxonomic status, still remain obscure. Our previous investigation suggested that S. proliferum might be phylogenetically distinct from Spirometra erinaceieuropaei, by the analysis on mitochondrial NADH dehydrogenase subunit 3 (ND3) gene. However, mitochondrial DNA sequence in Platyhelminth is known to have heteroplasmy within a species. Therefore, in the present study, we have investigated the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit I (COI) gene and the partial nucleotide sequences of nuclear coded succinate dehydrogenase iron-sulfur protein subunit gene (sdhB). The results clearly demonstrated that S. proliferum is a distinct species from S. erinaceieuropaei, and that S. proliferum belongs to the order Pseudophyllidea.

Three new reduced anthracycline related compounds from pathogenic Nocardia brasiliensis.

Three new metabolites were isolated from a pathogenic bacterium, Nocardia brasiliensis IFM 0075 strain, a producer of a new anthracycline antibiotic (SO-075R1) and its mutant strain (IFM 0075-13-1). The structural studies showed that they are reduced anthracyline related compounds. Some biosynthetic routes of these metabolites were discussed.

Comparative study and cDNA cloning of the flavoprotein subunit of mitochondrial complex II (succinate-ubiquinone oxidoreductase: fumarate reductase) from the dog heartworm, Dirofilaria immitis.

Mitochondrial complex II functions as a fumarate reductase (FRD), the reverse reaction of succinate dehydrogenase (SDH), and plays an important role in the anaerobic respiratory chain of parasitic helminths. In this study, complex II from the dog heartworm, Dirofilaria immitis adult, which is thought to act as a homolactatic fermenter, was examined in terms of its enzymatic features and primary structure in order to investigate the possible role of mitochondria in this filaria. Mitochondria from D. immitis adult showed high FRD activity when the enzymatic assay was performed using methylviologen as an artificial electron donor. The ratio of SDH to FRD in D. immitis was comparable to that in Ascaris suum adult, which is known to have an anaerobic mitochondrial respiratory chain with a high FRD activity of complex II. The FRD activity of D. immitis mitochondria was inhibited by the sulfhydryl reagent N-ethylmaleimide (NEM), while that of A. suum complex II was resistant to this inhibitor. The presence of the flavoprotein (Fp) subunit, which contains the substrate binding active site, was confirmed in D. immitis mitochondria by immunoblotting using a monoclonal antibody against the A. suum Fp subunit. By homology probing with the polymerase chain reaction, the entire cDNA for the D. immitis adult Fp was cloned and sequenced. The deduced amino acid sequence showed significant homology to that of A. suum and other mitochondrial Fps, in contrast to much less similarity to bacterial FRD, even though the D. immitis complex II showed high FRD activity. These results are the first indication of the presence of a functional complex II in D. immitis mitochondria.

Three epiphyseal fractures (distal radius and ulna and proximal radius) and a diaphyseal ulnar fracture in a seven-year-old child's forearm.

SUMMARY: The authors report a rare case of fracture separations at both ends of the radius combined with an epiphyseal and diaphyseal fracture of the ipsilateral ulna. A seven-year-old girl fell one story and sustained a closed injury of her forearm. A closed reduction was unsuccessful, and an open reduction was performed with three of the four fractures being secured with Kirschner wires. These wires were removed one month later, and range-of-motion exercises were started. Thirty months after surgery, both forearms were equal in length, although the proximal radial epiphyseal line appeared partially closed. Joint motions, including forearm rotation, were normal. Radiologically, the ulnar diaphysis and the radial neck were posteriorly convex 20 degrees and 18 degrees, respectively.

Pneumocystis carinii cysts are susceptible to inactivation by chemical disinfectants.

The inactivation efficacy of eight disinfectants commonly used in laboratories and animal rooms to inactive Pneumocystis carinii cysts was estimated by experimental infection in C.B-17-scid mice. The disinfectants examined in this study were 70% ethyl alcohol, 10% iodoform, 0.5% hypochlorous acid, two 1% quanternary ammonium salts, 3% hydrogen peroxide, sodium chlorite and 1% cresol soap. The lung homogenates from P. carinii infected C.B-17-scid mice were treated with each disinfectant for 15 min at room temperature, washed with saline, and inoculated into C.B-17-scid mice. Eight weeks after inoculation, lungs from these mice were examined by staining with toluidine blue O to detect P. carinii cysts. PCR amplifying 346 bp of P. carinii specific mitochondrial ribosomal RNA large segments was also performed using DNA extracted from the lungs of the mice. As a result, seven disinfectants, excepting for 0.5% hypochlorous acid, were effective in the inactivation of P. carinii cysts. These results suggest that P. carinii cysts were sensitive to chemical disinfectants even though they have been commonly considered as insensitive.

Lack of B cell leakiness in BALB/cA-nu, scid double mutant mice.

BALB/cA mice homozygous for both nu and scid mutations (BALB/cA-nu/nu, scid/scid) were developed by mating between BALB/cA-scid and BALB/cA-nu. These mice have greater longevity than C.B-17-scid because no thymic lymphoma occurs in them unlike in the latter. C.B-17-scid is known to show the leaky phenomenon in which a few clones of functional T and B cells develop in aged C.B-17-scid. Unexpectedly, the leaky B cells and T cells were absent or suppressed in BALB/cA-nu, scid mice when cytokine expressions were determined by RT-PCR, lymphocyte phenotypes by flow cytometry and serum immunoglobulin levels by ELISA. These results indicate that B cell leakiness may be induced by leaked T cells. BALB/cA-nu, scid mice may be useful as a recipient in allo- and xeno-transplantation experiments because of the absence of both thymic lymphomas and leakiness, in addition to lack of hair.

Serological studies on porcine Pneumocystis carinii pneumonia: kinetics of the antibody titers in swine herds and the association of porcine reproductive and respiratory syndrome virus infection.

Serological titers to Pneumocystis carinii (Pc) and porcine reproductive and respiratory syndrome virus (PRRSV) were measured on a herd with epidemic Pc pneumonia (case herd) and two comparison herds, by an indirect fluorescent-antibody technique. In the case herd, the geometric mean titer (GMT) for Pc were 1:80 in pigs 1 week old, 1:10 in pigs 5 weeks old, and 1:80 to 1:190 in pigs over 6 weeks old. GMTs for PRRSV were > 1:145 in most of age groups over 7 weeks old. In comparison herds, Pc and PRRSV antibody titers were low in weanling pigs. The results clarified the kinetics of antibodies to Pc and concurrent infection of PRRSV in the case herd.

Cross-reactivity between human and feline Ia antigens, using a monoclonal antibody HLA-D.m1.

A murine monoclonal antibody HLA-D.m1 produced against human Ia antigens which react with 10% to 30% of human peripheral-blood mononuclear cells was found to react with greater than 90% of feline mononuclear cells. The antigen was not expressed on granulocytes. By direct immunofluorescence, 7% to 41% of feline lymphocytes expressed surface immunoglobulins. In mixed-lymphocyte reaction, the addition of antibody HLA-D.m1 to cultures abolished proliferation. These studies indicate that most feline peripheral-blood mononuclear cells express Ia-like antigens.

Characterization of feline T and B cells.

Feline peripheral-blood lymphocyte populations (n = 22) were examined for the following markers: rosette formation with guinea pig erythrocytes (GPE-T cells), rosette formation with human RBC (HRBC-T cells), rosette formation with sheep RBC, mixed rosette formation with GPE-T cells and HRBC-T cells (total T cells), erythrocyte antibody-complement rosettes, and surface immunoglobulin. An average of 28% +/- 7% (range, 16% to 39%) of the feline lymphocytes formed rosettes with GPE-T cells, and 27% +/- 7% (range, 11% to 36%), with HRBC-T cells. An average of 57% +/- 9% (range, 33% to 75%) of the lymphocytes formed mixed rosettes. The erythrocyte antibody-complement rosette-forming cells and surface immunoglobulin-bearing cells were found in peripheral blood lymphocytes (10% +/- 6% and 24% +/- 8%, respectively). The murine monoclonal antibodies OKT 11 and HuLy-m1, specific for a framework determinant of human E-rosette receptor antigens, cross-reacted with feline cell membrane molecules recognizing a bimolecular complex (45,000 to 50,000 daltons) similar to that described in persons. We investigated the distribution of these E-rosette receptor-like antigens on feline lymphocytes. By complement-mediated lymphocytotoxicity, about 30% of the feline lymphocytes expressed the antigens. When lymphocytes were treated with HuLy-m1 antibody, spontaneous rosette formation with HRBC-T cells was significantly inhibited.

Humanization and simultaneous optimization of monoclonal antibody.

Antibody humanization is an essential technology for reducing the potential risk of immunogenicity associated with animal-derived antibodies and has been applied to a majority of the therapeutic antibodies on the market. For developing an antibody molecule as a pharmaceutical at the current biotechnology level, however, other properties also have to be considered in parallel with humanization in antibody generation and optimization. This section describes the critical properties of therapeutic antibodies that should be sufficiently qualified, including immunogenicity, binding affinity, physiochemical stability, expression in host cells and pharmacokinetics, and the basic methodologies of antibody engineering involved. By simultaneously optimizing the antibody molecule in the light of these properties, it should prove possible to shorten the research and development period necessary to identify a highly qualified clinical candidate and consequently accelerate the start of the clinical trial.

Engineered antibody Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131).

Engaging inhibitory FcγRIIb by Fc region has been recently reported to be an attractive approach for improving the efficacy of antibody therapeutics. However, the previously reported S267E/L328F variant with enhanced binding affinity to FcγRIIb, also enhances binding affinity to FcγRIIa(R131) allotype to a similar degree because FcγRIIb and FcγRIIa(R131) are structurally similar. In this study, we applied comprehensive mutagenesis and structure-guided design based on the crystal structure of the Fc/FcγRIIb complex to identify a novel Fc variant with selectively enhanced FcγRIIb binding over both FcγRIIa(R131) and FcγRIIa(H131). This novel variant has more than 200-fold stronger binding affinity to FcγRIIb than wild-type IgG1, while binding affinity to FcγRIIa(R131) and FcγRIIa(H131) is comparable with or lower than wild-type IgG1. This selectivity was achieved by conformational change of the C(H)2 domain by mutating Pro to Asp at position 238. Fc variant with increased binding to both FcγRIIb and FcγRIIa induced platelet aggregation and activation in an immune complex form in vitro while our novel variant did not. When applied to agonistic anti-CD137 IgG1 antibody, our variant greatly enhanced the agonistic activity. Thus, the selective enhancement of FcγRIIb binding achieved by our Fc variant provides a novel tool for improving the efficacy of antibody therapeutics.