Niemann-Pick disease experimental model: sphingomyelinase reduction induced by AY-9944.

Organs of rats treated with the drug A Y-9944 for 5 days showed a significant reduction in sphingomyelinase activity. Evidence is presented which suggests that the reduction is due to impaired enzyme synthesis.

Multilineage potential of side population cells from human amnion mesenchymal layer.

Side population (SP) cells were isolated by FACS from a human amnion mesenchymal cell (AMC) layer soon after enzyme treatment. The yield of SP cells from AMC layer (AMC-SP cells) was about 0.1-0.2%. AMC-SP cells grew well with cell doublings of 40-80 days of culture. FACS profiles and immunocytostaining showed that AMC-SP cells were composed of two different cells immunologically: HLA I(-)/II(-) and HLA P/II(-). Oct-3/4 was detected in the nucleus of AMC-SP cells, when the culture was examined at the third, sixth, and 10th passages. RT-PCR showed that AMC-SP cells expressed the Oct-4, Sox-2, and Rex-1 genes. Immunocytochemistry revealed that all AMC-SP cells were vimentin+, CK19+, and nestin+. In addition, flow cytometry analysis showed that SP cells had high expression of CD13, CD29, CD44, CD46, CD49b, CD49c, CD49e, CD59, CD140a, and CD166 but low expression of CD 49d, and CD51. No evidence of expression was obtained for CD34, CD45, CD49a, CD56, CD90, CD105, CD106, CD117, CD133, CD271, or Flk-1. Upon appropriate differentiation protocols, AMC-SP cells differentiated to several cell lineages such as neuroectodermal, osteogenic, chondrogenic, and adipogenic cells. These results indicate that AMC-SP cells have multilineage potential to several cell lineages with unique immunological characteristics such as HLA I(-)/II(-) or HLA I+/II(-). AMC-SP cells should be of considerable value for regenerative medicine because they do not induce acute rejection after allotransplantation, they do not cause ethical issues, and there is no limit of supply.

Uptake and decarboxylation of L-3,4-dihydroxyphenylalanine in cultured monkey placenta amniotic epithelial cells.

In this study we tested the ability of monkey amniotic epithelial cells (MAEC) to take up and decarboxylate l-3,4-dihydroxyphenylalanine (l-DOPA) by incubating the cells in buffer containing l-DOPA under different experimental conditions followed by assaying cellular dopamine (DA) content using high performance liquid chromatography with electrochemical detection. Cellular contents of DA were significantly increased in a time- and l-DOPA-concentration-dependent manner, suggesting the uptake of l-DOPA by MAEC and indicating the presence of aromatic l-amino acid decarboxylase (AADC). This was confirmed by the decreased DA content in the presence of benserazide, an AADC inhibitor. Neither d-DOPA nor DA uptake blockers such as mazindol and GBR 12935 significantly affected l-DOPA uptake and hence DA levels. Further, synthesis of DA from l-DOPA was decreased in the presence of the amino acids tyrosine, phenylalanine and tryptophan, whereas the amino acids glycine and proline were without any significant effect. These findings suggest that MAEC have the capacity to selectively take up and decarboxylate l-DOPA with subsequent production of DA.

Niemann-Pick disease: coupling and uncoupling of inhibited sphingomyelinase activity and exogenous cholesterol esterification in fibroblasts by ionophore treatment.

In order to elucidate a biochemical relationship between sphingomyelin and cholesterol metabolisms, we examined the effects of several ionophores (monensin, nigericin, A23187, ionomycin, lasalocid) on sphingomyelinase activity and cholesterol esterification in cultured human fibroblasts. Phase-contrast microscopy showed the presence of foamy cells with monensin and nigericin treatments only. Electron microscopic examination revealed lamellated membranous bodies and cytoplasmic vacuoles in cells treated with monensin and nigericin. Monensin and nigericin treatments led to reduction of acid sphingomyelinase activity and disturbance of the esterification of lipoprotein-derived cholesterol in cultured fibroblasts, which is compatible with the biochemical changes of Niemann-Pick disease, type C. A23187, ionomycin, and lasalocid treatments showed only sphingomyelinase reduction in treated fibroblasts. Experimental models in this culture system could be produced in these ways, mimicking subtypes of Niemann-Pick disease, type A, B and type C.

Effects of dimethylsulfoxide on sphingomyelinase activities in normal and Niemann-Pick type A, B and C fibroblasts.

The effects of dimethylsulfoxide (DMSO) on sphingomyelinase activity measured at pH range 3.5-8.0 were examined in normal and Niemann-Pick disease type A, B and C fibroblasts culture. In normal cells, a minor activity was observed at pH 7.5, which was 3- to 4-fold lower than a major one at pH 5.0. Both activities at pH 5.0 and 7.5 were Mg2+-independent and localized to lysosomes. Niemann-Pick type C cells had 30-50% residual sphingomyelinase activity at both pH 5.0 and 7.5, as compared to normal control cells, whereas type A and B cells exhibited virtually no activity over the entire pH range examined. Treatment with 2% DMSO caused a marked increase in sphingomyelinase activities at pH 5.0 and 7.5 in normal and Niemann-Pick disease type C cells, while in type A and B cells, both activities remained virtually unchanged after DMSO treatment. The increase in sphingomyelinase activity at pH 5.0 induced in normal cells by DMSO resulted in an increase in the Vmax without a substantial change in the Km and was inhibited by the simultaneous addition of 10 micrograms/ml of cycloheximide. By comparison, a less than 2-fold increase in other lysosomal hydrolase activities was observed after DMSO treatment in all cell lines examined.

Synergistic stimulating effect between cyclic AMP and phorbol ester on plasminogen activator inhibitor type 2 production in human promyelocytic leukemia cell line PL-21.

We investigated the effect of agents which raise intracellular cyclic AMP (cAMP) and protein kinase C activators on the production of plasminogen activator inhibitor type-2 (PAI-2) by cultured human promyelocytic leukemia cell line, PL-21. As previously reported, PMA, a protein kinase C activator, showed a strong stimulating effect on the PAI-2 production. 1-oleoyl-2-acetyl-sn-glycerol (OAG), another synthetic protein kinase C activator, also showed a stimulating effect, which was, however, much less than that of PMA. The agents which raise intracellular cAMP, dibutyryl cAMP, 8-bromo cAMP, prostaglandin E1, and 3-isobutyl-1-methyl-xanthine, little increased the PAI-2 production when tested alone, but showed significant synergistic effects with PMA or OAG. The synergistic effect between PMA and dibutyryl cAMP was further verified by SDS-PAGE followed by immunoblotting using a monoclonal antibody against the PAI-2. It is interesting that the up-regulation of PAI-2 by cAMP and the synergistic effect with PKC activators forms a contrast to the previous reported bi-directional regulation of endothelial PAI-1 secretion by PKC activator and cAMP.

Cloning of mouse prolidase cDNA: predominant expression of prolidase mRNA in kidney.

We cloned mouse prolidase cDNA from a mouse liver cDNA library. Homology to human prolidase is 83.2% at the nucleotide level and 87.2% at the amino acid level. Northern blot analysis showed that while prolidase mRNA was transcribed in brain, heart, liver, and muscle, it was predominantly transcribed in kidney.

Calcium spirulan as an inducer of tissue-type plasminogen activator in human fetal lung fibroblasts.

Calcium spirulan (Ca-SP), a novel sulfated polysaccharide isolated from the blue-green alga Spirulina platensis, has been found to have antiviral and heparin cofactor II-dependent antithrombin activities. We have obtained evidence that Ca-SP is a potent inducer of tissue-type plasminogen activator (t-PA) production. The addition of Ca-SP to a culture of IMR-90 human fetal lung fibroblasts increased t-PA concentrations in the conditioned medium, in a dose- and time-dependent manner, but in the cell lysate, t-PA concentrations were unchanged, suggesting that t-PA induced by Ca-SP is easily secreted into the conditioned medium. The amount of newly synthesized t-PA in IMR-90 cells, as measured by labeling with [35S]methionine and subsequent immunoprecipitation of t-PA from conditioned medium, was significantly increased by Ca-SP-stimulation. However, Ca-SP did not increase the t-PA mRNA levels. As previously reported, thrombin stimulated t-PA gene transcription in IMR-90 cells, and the simultaneous treatment with Ca-SP and thrombin caused further enhancement of t-PA production, in a synergistic manner. It would thus appear that Ca-SP increases t-PA production through post-transcriptional processes. IMR-90 cells also produce plasminogen activator inhibitor type-1 (PAI-1), but Ca-SP showed little effect on the PAI-1 production. H-SP, which was obtained by removing the calcium from Ca-SP, had no effect on the t-PA production. Na-SP, which was prepared by replacement of the calcium with sodium, stimulated the t-PA production similarly to Ca-SP. Thus, Ca-SP specifically induces t-PA production, and the molecular conformation of Ca-SP maintained by Ca or Na may be essential for the stimulation of t-PA synthesis.

Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8.

We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.

Inhibition of thrombin by sulfated polysaccharides isolated from green algae.

Eight different sulfated polysaccharides were isolated from Chlorophyta. All exhibited thrombin inhibition through a heparin cofactor II (HCII)-dependent pathway, and their effects on the inhibition of thrombin were more potent than those of heparin or dermatan sulfate. In particular, remarkably potent thrombin inhibition was found for the sulfated polysaccharides isolated from the Codiales. In the presence of these sulfated polysaccharides, both the recombinant HCII (rHCII) variants Lys(173)-->Leu and Arg(189)-->His, which are defective in interactions with heparin and dermatan sulfate, respectively, inhibited thrombin in a manner similar to native rHCII. This result indicates that the binding site of HCII for each of these eight sulfated polysaccharides is different from the heparin- or dermatan sulfate-binding site. All the sulfated polysaccharides but RS-2 significantly stimulated the inhibition of thrombin by an N-terminal deletion mutant of HCII (rHCII-Delta74). Furthermore, hirudin(54-65) decreased only 2-5-fold the rate of thrombin inhibition by HCII stimulated by the sulfated polysaccharides, while HD22, a single-stranded DNA aptamer that binds exosite II of thrombin, produced an approximately 10-fold reduction in this rate. These results suggest that, unlike heparin and dermatan sulfate, the sulfated polysaccharides isolated from Chlorophyta activate HCII primarily by an allosteric mechanism different from displacement and template mechanisms.

Adenovirus-mediated transfer of human acid maltase gene reduces glycogen accumulation in skeletal muscle of Japanese quail with acid maltase deficiency.

Acid maltase deficiency (AMD) causes a lysosomal glycogenosis inherited as an autosomal recessive trait. The infantile type of AMD (Pompe disease) leads to early death due to severe dysfunction of cardiac and respiratory muscles and no effective therapy is available. Replication-defective adenovirus vectors offer a promising tool for in vivo gene delivery and gene therapy. We constructed a recombinant adenovirus containing the human acid maltase (AM) cDNA downstream of the CAG promoter, composed of modified chicken beta-actin promoter and CMV IE enhancer (AxCANAM). Japanese quail with AMD was used for this study as an animal model for human AMD. When cultured fibroblasts from AMD quail were infected with AxCANAM, AM activity in the cells increased in proportion to the multiplicity of infection (MOI). When AxCANAM (4.5 x 10(8) PFU) was injected into unilateral superficial pectoral muscle of AMD quail, PAS staining showed that glycogenosomes disappeared and stainability of acid phosphatase was reduced in the injected area as compared with the contralateral muscle of the same birds. Biochemically, AM activity increased and glycogen content decreased in the injected muscle. Western blot analysis showed that AMD quail muscle injected with AxCANAM expressed human AM protein processed to active forms. These results suggest that the human AM cDNA transferred by an adenovirus vector was sufficiently expressed, leading to a marked reduction of the glycogen accumulation in the skeletal muscle of AMD quail.

Binding of heparin or dermatan sulfate to thrombin is essential for the sulfated polysaccharide-accelerated inhibition of thrombin by heparin cofactor II.

Heparin cofactor II (HC II) and thrombin were chemically modified with pyridoxal 5'-phosphate, and their effects on the inhibition of thrombin by HC II in the presence of heparin or dermatan sulfate were studied. The inhibition of thrombin by HC II was enhanced about 7000-fold in the presence of heparin or dermatan sulfate. However, this enhancement by heparin dwindled to 110- and 9.6-fold when the modified HC II and the modified thrombin, respectively, were substituted for native proteins. Essentially identical results were obtained from the experiments using dermatan sulfate. These results indicate that the binding of heparin or dermatan sulfate to both thrombin and HC II is required for the sulfated polysaccharide-dependent acceleration of the thrombin inhibition by HC II, and the binding to thrombin is more essential for the reaction.

A comparative study of myelin fractions from metachromatic and globoid leukodystrophies.

Myelin was isolated post mortem from brains of patients with metachromatic and globoid forms of leukodystrophy. In the leukodystrophies, isolates were obtained from central white matter and subcortical "U" fiber areas. Myelin also was obtained from age-matched human controls, and the method yielded light and heavy myelin fractions. In both disorders, the pathologic process affected the heavy isolates to a relatively greater degree. Comparative chemical analyses were made of the myelin isolates, and we concluded that a similar pathogenesis--the production of an unstable membrane in later stages of myelin formation--affects both disorders.

Frequent mutations in Japanese patients with acid maltase deficiency.

We screened 22 Japanese patients with acid maltase deficiency (seven with the infantile type, eight with the juvenile type and seven with the adult type) for three previously described mutations, D645E, S529V and R672Q, and a novel mutation, R600C. Although D645E has been reported to be common in Chinese patients with the infantile type, only three of 44 alleles (two of 14 infantile type alleles) from Japanese patients harbored the D645E mutation. The S529V mutation was identified in six of 14 alleles from adult-onset patients. None of the infantile or juvenile patients harbored the S529V mutation. Therefore, S529V apparently results in the adult type disease and is common in Japanese adult-onset patients. R672Q was identified in two pairs of siblings with the juvenile type. A novel mutation, R600C, was identified in eight of 22 patients (nine of 44 alleles). Therefore, R600C is another common Japanese mutation occurring at a CpG dinucleotide "hot spot". Homozygosity for this mutation apparently results in the infantile phenotype. Genetic diagnosis by detecting these four mutations might be feasible for most Japanese patients with acid maltase deficiency.

Factor VII Toyama (Thr 359 Met): a homozygous missense mutation causing severe type I deficiency.

We performed a DNA analysis on a patient with severe type I factor VII deficiency by the polymerase chain reaction amplification and a direct DNA sequencing method. The proband was a 66-year-old Japanese woman who had recurrent episodes of excessive bleeding after dental extraction. The functional and antigenic levels of plasma factor VII markedly reduced to 1.6% and 2% of normal, respectively. However, she had no serious symptoms such as intracranial or intraarticular hemorrhage. The analysis revealed that the patient was homozygous for a missense mutation, Thr (ACG) to Met (ATG) at codon 359 in the catalytic domain. Her deceased parents were first cousins, and their consanguineous marriage presumably resulted in the homozygosity in her. This patient was the first case of homozygote for the Thr359Met mutation, though heterozygotes for the mutation were previously found in an Italian family.

An immunological method for detection of the carrier of hemophilia B.

This paper presents the results of an immunological study of hemophilia B and its carriers which used two kinds of antibodies, a heterologous antibody of high specificity and a homologous antibody developed in a patient with severe hemophilia B. 1. The factor IX related antigen for hemophilia B could be determined by a neutralization test and not by a precipitation reaction. 2. The activity of factor IX in a definite carrier of hemophilia B was significantly lower than that of factor VIII in a definite carrier of hemophilia A. 3. In hemophilia B, there were no differences between the factor IX antigen determined by either the heterologous rabbit antibodies or the homologous antibodies. And there was no discrepancy between the factor IX antigen and the factor IX activity. Therefore, we cannot use this discrepancy for the detection of a carrier of hemophilia B. 4. However, there was a discrepancy between the factor IX antigen determined by the neutralization test and the factor IX procoagulant activity in the patients of both hemophilia BM and hemophilia B+. The identical results were obtained in the cases of carriers of both hemophilia BM and hemophilia B+. These facts are very useful for the detection of carriers of hemophilia BM and hemophilia B+.

The production of heparin cofactor II is not regulated by inflammatory cytokines in human hepatoma cells: comparison with plasminogen activator inhibitor type-1.

Using the Northern blot technique, we screened 6 human hepatoma cell lines to investigate the regulation mechanism of heparin cofactor II (HC II) biosynthesis. We found that HuH-7 and Hep G2 cells constitutively expressed the HC II gene. In conditioned medium, HuH-7 cells constantly produced HC II that was functionally active and formed a complex with thrombin in the presence of dermatan sulfate. HC II is thought be an acute phase reactant, and, therefore, we examined the effects of the major inflammatory cytokines, IL-6, IL-1 beta, and TNF-alpha, on the regulation of HC II production in HuH-7 and Hep G2 cells. In HuH-7 cells, the antigen and mRNA levels of plasminogen activator inhibitor type-1 (PAI-1), an acute phase protein produced by hepatocytes, were increased in response to stimulation with either IL-6 or IL-1 beta or both, but HC II antigen and mRNA levels were not changed by the same stimulation. Even when Hep G2 cells were treated with a combination of three cytokines, IL-6, IL-1 beta, and TNF-alpha, HC II antigen and mRNA levels were not changed; however, PAI-1 antigen and mRNA levels were clearly increased. These results suggest that the production of HC II in hepatoma cells is not regulated by the major inflammatory mediators, IL-6, IL-1 beta, and TNF-alpha.

Modulation of urokinase-type plasminogen activator gene expression by inflammatory cytokines in human pre-B lymphoma cell line RC-K8.

We examined the effects of inflammatory cytokines, such as interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), transforming growth factor-beta (TGF beta) and lipopolysaccharide (LPS), on the urokinase-type plasminogen activator (uPA) gene expression in RC-K8 human pre-B lymphoma cells. Recombinant IL-1 alpha, recombinant IL-1 beta and LPS but not recombinant IL-6, recombinant TNF alpha and TGF beta dose-dependently increased uPA accumulation in the conditioned medium. Northern blot analysis revealed that uPA mRNA levels rapidly increased with a peak induction at 2 h after stimulation with IL-1 alpha and IL- 1 beta, but uPA mRNA increase by LPS began at 9 h after stimulation and the increase was maintained until the experiment ended at 24 h. These responses were independent of de novo synthesis, rather amplified in the presence of a protein synthesis inhibitor. The effects by IL-1 alpha and Il-1 beta were prevented by addition of anti-IL-1 alpha and anti-IL-1 beta neutralizing antibodies, respectively. In contrast, both antibodies did not prevent LPS-induced uPA gene expression. Therefore, it is unlikely that the effect by LPS is through induction of IL-1. Both IL-1 alpha and IL- 1 beta rapidly activated uPA gene transcription, but not increased stability of uPA mRNA. These results suggest that both IL-1 alpha and IL-1 beta cause a rapid activation of uPA gene transcription in which de novo protein synthesis is not required and that LPS induces uPA gene expression independently of the IL-1 pathway. These modulations of uPA production by inflammatory mediators may be implicated in tumor growth and metastasis.

Transcriptional regulation of tissue- and urokinase-type plasminogen activator genes by thrombin in human fetal lung fibroblasts.

The mechanism of thrombin induction of tissue- and urokinase-type plasminogen activator (t-PA and u-PA) biosynthesis was investigated in cultured human fetal lung fibroblast cells, IMR-90. Northern blot analysis of total RNA from thrombin-treated cells showed marked accumulations of both t-PA and u-PA mRNA during 24 h. Nuclear run-on experiments showed that the transcription rates of both genes were increased in the thrombin-treated cells. These thrombin effects were inhibited by cycloheximide (CHX), an inhibitor of protein biosynthesis. Treatment of IMR-90 cells with CHX alone caused an increase in u-PA mRNA but not in t-PA mRNA. CHX, however, did not affect the transcription rates of both genes in the cells. Thus, on-going protein synthesis is required for increased accumulations of both t-PA and u-PA mRNA by thrombin but not for the constitutive expression of u-PA gene in IMR-90 cells. Therefore, we conclude that the accumulations of t-PA and u-PA mRNA due to thrombin result mainly from increased rates of their gene transcriptions, and that this influence is exerted in part by proteins synthesized by thrombin stimulation. Thrombin also increased plasminogen activator inhibitor type-1 (PAI-1) in the levels of both antigen and mRNA more rapidly than it increased t-PA in IMR-90 cells. In conditioned medium, most of the secreted PAI-1 seemed to form a complex with t-PA. Northern blot analysis using a PAI-2 cDNA probe showed that the levels of PAI-2 mRNA were markedly increased in response to thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Transcriptional regulation of urokinase-type plasminogen activator receptor by cyclic AMP in PL-21 human myeloid leukemia cells: comparison with the regulation by phorbol myristate acetate.

We investigated the effect of dibutyryl cyclic AMP (Bt2-cAMP) on urokinase-type plasminogen activator receptor (uPAR) expression in human PL-21 myeloid leukemia cells and compared it with the effect of phorbol myristate acetate (PMA). Flow cytometric analysis clearly demonstrated that Bt2-cAMP and PMA both induced the cell surface expression of uPAR. Northern analysis and nuclear run-on assay revealed that cAMP and PMA activated the uPAR gene transcription and both additively increased the uPAR mRNA level. However, actinomycin-D decay experiment showed that PMA, but not cAMP, prolonged the uPAR mRNA half-life. Furthermore, inhibition of the ongoing protein synthesis with cycloheximide abrogated completely the PMA-induced uPAR mRNA accumulation but only partially the induction by PMA plus cAMP, whereas the induction by cAMP alone was rather amplified, indicating that the de novo protein synthesis is necessary in the induction by PMA but not in the induction by cAMP and that the cAMP pathway may be dominant in uPAR gene expression in the PL-21 cells as compared to the PMA pathway. These results suggest that cAMP induces the uPAR expression exclusively through activating the gene transcription in which a preexisting transcriptional factor may be involved, whereas PMA transcriptionally and posttranscriptionally regulates the uPAR gene expression.