Genetic association of the very low density lipoprotein (VLDL) receptor gene with sporadic Alzheimer's disease.

A specific isoform of apolipoprotein E has been associated with the accelerated rate of disease expression of sporadic Alzheimer's disease (AD) and late-onset familial AD (FAD). An earlier age at onset has also been demonstrated in familial AD patients with mutations in the amyloid precursor protein (APP) gene (APP717 and APP670/671)13 carrying the APOE epsilon-4 allele compared to those who do not, but not in familial AD patients with APP692 or 693 mutations, or in chromosome 14-linked familial AD patients. Hypothesizing that receptors for apoE-containing lipoproteins act as a potential risk factor for AD, we performed an association study using a polymorphic triplet (CGG) repeat in the gene for the VLDL receptor (VLDL-R), a receptor for apoE-containing lipoproteins. The frequency of the 5-repeat allele was significantly higher in all of the Japanese sporadic AD patients (P < 0.02) than in the Japanese controls. Moreover, the odds ratio was significantly increased in the AD patients homozygous for the 5-repeat allele (OR = 2.1, 95% CI = [1.1-4.2]). Multiple logistic regression analysis reveals that the relative risk conferred by the presence of two copies of the 5-repeat allele and at least one copy of the APOE epsilon-4 allele is 8.7 (95% CI = [2.9-25.8]). Our results suggest that the VLDL-R gene is a susceptibility gene for AD.

An increased dose of insulin detemir improves glycaemic control and reduces body weight of Japanese patients with diabetes.

AIMS: The aim of study was to evaluate the safety and efficacy of insulin detemir as a basal insulin switching from neutral protamine Hagedorn insulin (NPH) and insulin glargine in patients with diabetes on an intensive insulin therapy regimen. METHODS: This 6-month multicentre, prospective, treat-to-target [glycosylated haemoglobin (HbA(1c) ) less than 6.5%] trial included 92 people with diabetes (61 type 1, 29 type 2 and two unknown diabetes types). Detemir was administered first with fixed dose and injection times and then adapted to optimal dose after 3 months. RESULTS: Mean HbA(1c) (%) of all the subjects at months 4 to 6 of the study was improved compared with month 0 (7.34 ± 0.87, 7.28 ± 0.88, 7.25 ± 0.93 vs. 7.55 ± 1.18; p < 0.05 paired t-test). However, significant improvement was seen only among the patients who had previously used NPH as a basal insulin. Twice-daily injection of basal insulin increased among people in the type 1 previously injected insulin glargine. Total insulin dose increased in the type 1 glargine group. The mean body weight change in the highest quartile body mass index (BMI) group was from 70.7 to 69.3 kg over the 6 months. Quality of life (QoL) relating to the patients' glycaemic control tended to improve without a change in frequency of hypoglycaemia. CONCLUSIONS: The results suggest that insulin detemir has a greater effect on glycaemic control in subjects with poor glycaemic control using NPH; can reduce or maintain body weight in obese patients; and obtains perceptive stability for patients with unstable glycaemic control.

Interleukin 3 as a trophic factor for central cholinergic neurons in vitro and in vivo.

We have found that interleukin 3 (IL-3), a growth factor for hematopoietic cells, is a novel trophic factor for mouse and rat central cholinergic neurons. It enhanced neurite outgrowth and elevated choline acetyltransferase activity. The effect seems to be specific for cholinergic neurons, since somatostatin release and glutamic acid decarboxylase and 2',3'-cyclic nucleotide 3'-phosphodiesterase activities were not significantly influenced by IL-3. In vivo, IL-3 was infused into the lateral ventricles of rats after unilateral axotomy of the septohippocampal pathways. Two weeks later, the IL-3-treated animals showed significant numbers of acetylcholinesterase-positive neurons remaining in the septal region.

Estrogen receptors in human myeloma cells.

It has recently been reported that the human myeloma cell line U266 proceeds to undergo apoptosis after cultivation with the antiestrogen tamoxifen, thus raising the possibility that antiestrogens may be candidates for use in myeloma therapy. To obtain basic information on the effects of antiestrogens on myeloma cells, we investigated the mRNA expression levels of estrogen receptor (ER)-alpha, ER-beta, and coactivators and corepressors in nine human myeloma cell lines and compared them with those of seven human breast cancer cell lines including four ER-positive and three ER-negative lines. The alterations in cell growth and mRNA expression of the target genes of ER or those of cytokines in the myeloma lines by estradiol or antiestrogens (tamoxifen and toremifene) were also investigated. In addition, effects on membrane Fas expression, appearance of apoptosis, and cell cycle perturbation were analyzed. It was revealed that ER-beta and corepressors were dominantly expressed in myeloma cells, and antiestrogens induced growth inhibition through apoptosis mediated by a Fas-related pathway and G1 arrest of the cell cycle in myeloma cell lines.

Combined action of paraquat and superoxide on the peroxidation of detergent-dispersed linolenic acid.

We investigated the peroxidative effect of paraquat and active oxygens on detergent-dispersed linolenic acid in phosphate buffer (pH 7.5) from the malondialdehyde (MDA) level. Our complete system and further inclusion of catalase were effective in stimulating MDA formation. On the other hand, xanthine oxidase (XOD) or paraquat omission, superoxide dismutase (SOD) inclusion or anaerobic incubation inhibited the formation of MDA. Ferrous ion was weakly associated with phosphate of the buffer, forming a complex, and the release of ferrous ion from the complex intensified the MDA levels with the complete and catalase inclusion systems. The electron paramagnetic resonance (EPR) spectra using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that superoxide, produced immediately after the addition of XOD, played a crucial role. We could obtain a DMPO-OOH signal at the starting stage whenever MDA stimulation was observed. The omission of paraquat, however, produced no increase in MDA level in spite of an appearance of DMPO-OOH signal, indicating that paraquat also plays an important role. On the other hand, Desferal, a ferric chelator, showed a concentration-dependent inhibition effect. There was an immediate strong intensity of DMPO-OOH and paraquat signals. We did not, however, observe MDA stimulation at 250 microM Desferal, which confirms that ferrous ion plays an essential role in the lipid peroxidation. These results indicate a combined action of paraquat (or its radical) and superoxide on the accessibility of ferrous ion, including its release from the complex with phosphate, which may be an endogenous chelator. The possibility of ternary complex participation is also discussed.

In vitro excess ammonia production in human myeloma cell lines.

It is well known that cases with multiple myeloma reveal various clinical manifestations such as pancytopenia, hyperproteinemia, renal dysfunction, bone lesions, hypercalcemia and immunodeficiency. Recently, a few more clinical features associated with myeloma, such as salivary type hyperamylasemia and elevated serum C-reactive protein (CRP) concentration, have been reported. The elevation of CRP is thought to be related to interleukin-6 (IL-6) production by myeloma cells, because of identification of IL-6 as an autocrine and/or paracrine growth factor for myeloma cells. More recently, there have been several reports of cases with myeloma associated with hyperammonemia. This hyperammonemia is not considered to be due to liver dysfunction, because in most of these cases tests revealed normal hepatic function, and some cases showed different patterns of serum amino acid distribution than that associated with hepatic failure. However, there have been no apparent observations of ammonia production by myeloma cells. In this study, we used six human myeloma cell lines including KMS-18, which was recently established from a myeloma case associated with hyperammonemia. These lines were treated with MRA (mycoplasma removal agent) to observe ammonia production in vitro. They produced and released significantly higher levels of ammonia into culture medium than non-myeloma hematological cell lines or the HepG2 human hepatic carcinoma cell line. Although attempts to analyze the relative expression levels of the enzymes related to ammonia biosynthesis using the reverse transcriptase-polymerase chain reaction assay failed to detect any differences between these myeloma lines and other cell lines, in vitro excess ammonia production by the myeloma cells was confirmed and the relevance to clinical manifestations is discussed.

Further treatment with MPTP does not produce parkinsonism in marmosets showing behavioural recovery from motor deficits induced by an earlier exposure to the toxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or 0.9% saline, was administered to a group of common marmosets initially treated with the toxin 12-18 months previously. Initial treatment had consisted of a cumulative dose of 6-22 mg/kg (i.p.) which caused marked parkinsonism. Subsequently, the animals gradually recovered normal motor function. Further treatment consisted of a cumulative dose of MPTP of 78-83 mg/kg (i.p.) but this produced only modest akinesia. At 12-18 months after the initial treatment with MPTP, the content of dopamine, HVA and DOPAC in the caudate and putamen was markedly reduced. However, levels of dopamine, HVA and DOPAC in the nucleus accumbens were normal. Three months after the second treatment with MPTP there was no further decrease in the content of dopamine in the caudate-putamen. However, in the nucleus accumbens the content of dopamine, HVA and DOPAC was now reduced. The initial treatment with MPTP substantially decreased the binding of [3H]mazindol in the caudate-putamen but less so in the nucleus accumbens. Only a small additional decrease occurred upon further treatment with MPTP. The density of tyrosine hydroxylase (TH) immunoreactive cells in substantia nigra was reduced after the initial treatment with MPTP. However, the cell loss was far less marked than the decrease in terminal density, assessed by the binding of [3H]mazindol. Subsequent treatment with MPTP caused a small further loss of tyrosine hydroxylase-positive cells. Initial treatment with MPTP may kill the majority of MPTP-sensitive dopamine cells in the nigra. Compensation by the remaining nigrostriatal neurones may account for the behavioural recovery observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of alternatively spliced variant messages of Fas gene and mutational screening of Fas and Fas ligand coding regions in peripheral blood mononuclear cells derived from silicosis patients.

Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities such as the appearance of autoantibodies and complications of autoimmune diseases. Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, has been considered to play a role in the pathogenesis of autoimmune diseases. It has been found that serum soluble Fas (sFas) levels are elevated in silicosis patients (SIL) and the sFas message is dominantly expressed in peripheral blood mononuclear cells (PBMC) derived from these individuals. In the present study, one tried to detect alternatively spliced variant messages including typical sFas message and found four that were highly and frequently expressed, and which possess a signal peptide domain, but not transmembrane and signal transducing domains, in PBMC derived from SIL. Functional mutations were not detected in Fas and FasL genes in silicosis PBMC. Still, alternative spliced variants of the Fas gene including typical sFas message appear to play an important role in the immunological dysregulation in SIL.

Crocidolite asbestos suppresses the differentiation of HL-60 cells induced by DMSO.

This study was undertaken to determine if the biological function of inducers for cell differentiation is affected by asbestos fibers, which are sometimes deposited in human tissues. Protein kinase C activity, c-myc protein expression and cell surface CR3 expression were used as the markers of cell differentiation. The function of dimethylsulfoxide (DMSO), an inducer of cell differentiation, was suppressed by the co-culturing of crocidolite asbestos, because DMSO reacted with the hydroxyl radical released after the stimulation with crocidolite and spent itself. Superoxide dismutase (SOD) inhibited the effect of crocidolite, reacting rapidly with .O2- before the secondary release of .OH. Asbestos fibers deposited in tissues may inhibit the function of inducers which stimulate immature cells to differentiate, because such inducers frequently are also radical scavengers.

Activation of human CD4+CD45RA+ T cells by chrysotile asbestos in vitro.

Chrysotile asbestos stimulates T lymphocyte subsets. Cell surface CD4 or CD45RA expression in peripheral blood mononuclear cells (PBMC) was downregulated after incubation with chrysotile asbestos in vitro temporarily. The percentage of CD4+CD45RA+ cells and mean fluorescence intensity in CD4 or CD45RA decreased after incubation with asbestos and returned to the original level after 24 h of incubation, which suggests that chrysotile asbestos activates CD4+CD45RA+ cells. No change was observed in CD29 expression. An increased percentage of IL-2R positive cells and an elevated intracellular Ca++ level were also indicative of the activation of PBMC by chrysotile asbestos.

TNF-alpha stimulates the biosynthesis of complement C3 and factor B by human umbilical cord vein endothelial cells.

The human umbilical cord vein (HUVEC) endothelial cells synthesize and secrete complement components. We analyzed the regulation of biosynthesis of the third component of complement (C3) and factor B (Bf) by cytokines in endothelial cells. Production of C3 increased after stimulation with TNF-alpha or IL-1beta and that of Bf with TNF-alpha, TNF-beta and IFN-gamma. TNF-alpha was the most effective in stimulating the secretion of C3 and Bf or the expression of mRNA of C3 or Bf. Preincubation with TNF-alpha for at least 36 or 24 h was needed for the stimulation, respectively. TNF-alpha, together with IFN-gamma had synergistic effects and the release of C3 or Bf increased to about 40- and 26-fold higher than those in control cells after incubation with both cytokines.

Down regulation of protein kinase C during growth enhancement induced by interleukin-6 on a human myeloma cell line, KMS-11.

Interleukin-6 (IL-6) is one of the most important growth factors for myeloma cells. We examined the effect of recombinant IL-6 on the proliferation of five human myeloma cell lines, which were independently established AT Kawasaki Medical School. Only the KMS-11 cell line among these five lines showed growth enhancement induced by IL-6. Based on the results, a possible contribution of Ca(2+)-phospholipid-dependent protein kinase C (PKC) to the signal transduction in KMS-11 cells during growth enhancement was studied, since PKC may play an important role in malignant transformation or cell proliferation induced by some growth factors, such as IL-6. When exogenous IL-6 was added to KMS-11 culture, we observed (1) reduction of total PKC activity, and (2) translocation of PKC activity from its cytosol fraction to the membrane fraction. These findings may indicate that down regulation of PKC occurred during the myeloma cell proliferation induced by IL-6. However, IL-6 does not appear to be involved in cell proliferation and differentiation in the other cell lines studied.

5-HT(2A/2C) receptor agonist-induced increase in urinary isatin excretion in rats: reversal by both diazepam and dexamethasone.

Isatin, a stress-related biological substance, increases in rat urine in association with elevated catecholamine biosynthesis during stress. The goal of this study was to unravel how the biosynthetic pathway of isatin is related to stress response. The importance of the serotonergic compounds in anxiety, which is the major emotional process of stress response, has emerged. m-Chlorophenylpiperazine (m-CPP), a 5-HT(1A/1B/2A/2C) receptor agonist, and (+/-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane hydrochloride [(+/-)-DOI], a 5-HT(2A/2C) agonist, both of which have anxiogenic properties, induced a marked increase in 24-hr urinary isatin excretion, whereas neither 1-(m-chlorophenyl)-biguanide (m-CPBG), a 5-HT3 agonist, nor 2-methyl-5-HT, a 5-HT(3,4) agonist, affected urinary isatin excretion. 5-HT(2A/2C) receptor antagonists such as ketanserin and ritanserin prevented the increase in urinary isatin excretion induced by the 5-HT(2A/2C) receptor agonist m-CPP. These findings are the first to provide evidence that pharmacological substances cause increases in urinary isatin excretion via specific 5-HT receptors, probably 5-HT(2A/2C) receptors. In addition, both the synthetic glucocorticoid dexamethasone and diazepam prevented the m-CPP-induced increase in urinary isatin excretion. These observations suggest that the mechanism by which m-CPP elicits enhancing effects on urinary isatin excretion has something in common with stress response involving activation of hypothalamic CRF cells and the sympathetic nervous system.

Stress-induced increase in urinary isatin excretion in rats: reversal by both dexamethasone and alpha-methyl-P-tyrosine.

The effects of acute food deprivation and acute cold exposure on 24-hr urinary isatin excretion in rats and a mechanism responsible for changes in urinary isatin excretion during stress were investigated. This is the first study to demonstrate by HPLC that urinary isatin excretion is increased by stress. Both types of stress induced a marked increase in urinary isatin excretion during the 24 hr following the initiation of stress. Dexamethasone administration prevented the increase in urinary isatin excretion induced by both of the different types of stress. Furthermore, administration of either the benzodiazepine receptor agonist diazepam or the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine prevented the increase in urinary isatin excretion induced by acute food deprivation, whereas the dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate proved ineffective. These observations suggest that during stress, activated catecholamine-synthesizing cells and corticotropin-releasing factor cells, both of which play central roles in stress responses, may be involved in total isatin production. Isatin may serve as an endogenously generated marker for some types of stress.

Association study of structural mutations of the tyrosine hydroxylase gene with schizophrenia and Parkinson's disease.

Tyrosine hydroxylase (TH) gene is the rate-limiting enzyme in the synthesis of catecholamines. Functional polymorphisms of the TH gene may be involved in the pathogenesis of neuropsychiatric diseases such as schizophrenia, affective disorders, and Parkinsonism. This study examined a possible association of two polymorphisms, both of which result in an amino acid change of the TH protein, with schizophrenia and Parkinson's disease (PD). The Val81Met polymorphism is a common variation, although its effect on the enzyme expression is unclear. Leu205Pro polymorphism is a rare mutation that is reported to cause Parkinsonism in infancy for individuals who are homozygous for the mutated type. We genotyped a Japanese sample of 194 schizophrenics, 99 patients with PD, and 161 controls for the Val81Met polymorphism by using mis-match PCR and digestion by the restriction enzyme BalI. There was no significant allelic or genotypic association of the Val81Met polymorphism with schizophrenia or PD. The Leu205Pro polymorphism was examined by using PCR and digestion by AluI; however, there was no individual who carried the mutated type of Pro205 among 50 schizophrenics or 50 patients with PD. Thus we obtained no evidence for the involvement of the two structural mutations of the TH gene in the pathogenesis of schizophrenia or PD.

Alzheimer's disease and 5-HTTLPR polymorphism of the serotonin transporter gene: no evidence for an association.

Recently two independent research groups consistently reported a significant association between the serotonin transporter (5-HTT) gene and late-onset sporadic Alzheimer's disease (AD). They found that the "short" allele of the 5-HTT gene-linked polymorphic region (5-HTTLPR), which is associated with reduced transcriptional activity of the gene, increases the risk of developing late-onset AD. The present study tried to replicate this finding in a Japanese sample. We genotyped 41 patients with early-onset AD (<65 years), 82 with late-onset AD, and 336 controls. There was no significant difference in genotype or allele distribution between either patient group and controls in our sample, suggesting that the 5-HTTLPR does not play a major role in the pathogenesis of AD in Japanese.

No allelic association between Parkinson's disease and dopamine D2, D3, and D4 receptor gene polymorphisms.

Parkinson's disease is thought to be caused by a combination of unknown environmental, genetic, and degenerative factors. Evidence from necropsy brain samples and pharmacokinetics suggests involvement of dopamine receptors in the pathogenesis or pathophysiology of Parkinson's disease. Genetic association studies between Parkinson's disease and dopamine D2, D3 and D4 receptor gene polymorphisms were conducted. The polymorphism was examined in 71 patients with Parkinson's disease and 90 controls. There were no significant differences between two groups in allele frequencies at the D2, D3, and D4 dopamine receptor loci. Our findings do not support the hypothesis that susceptibility to Parkinson's disease is associated with the dopamine receptor polymorphisms examined.

Human cd4+ cd45ra+ lymphocytes-T can be stimulated by crocidolite, anthophyllite and amosite asbestos invitro.

Crocidolite, anthophyllite and amosite asbestos stimulate T lymphocyte subsets. Cell surface CD4 or CD45RA expression in peripheral blood mononuclear cells (PBMC) was downregulated temporarily after incubation with asbestos in vitro. The percentage of CD4+ CD45RA+ cells significantly decreased 12 h after incubation with asbestos, suggesting activation of the cells. An early increase in the intracellular Ca++ level was also indicative of activation. An elevated Ca++ level was observed after incubation in both PBMC and purified T cell fractions.

Establishment of a new human myeloma cell line, KMS-18, having t(4;14)(p16.3;q32.3) derived from a case phenotypically transformed from Ig A-lambda to BJP-lambda, and associated with hyperammonemia.

A new human myeloma cell line, KMS-18, was established from a 58-year-old male with multiple myeloma associated with hyperammonemia. The original leukemic cells and established KMS-18 cells possessed several of the same chromosomal abnormalities, including add(1)(q32), add(10) (q24) and add(17)(p11). In addition, the KMS-18 cells showed novel t(4;14)(p16.3;q32.3) masked translocation which was determined by the FISH method. Moreover, we compared the ammonia production in culture medium of the KMS-18 cell line with that of non-myeloma hematological malignant cell lines and a hepatocellular carcinoma cell line. KMS-18 produced higher levels of ammonia in medium than the other cell lines examined. This new cell line may prove helpful in analyzing the role and biological mechanisms of the t(4;14)(p16.3;q32.3) translocation in myeloma and also in investigating hyperammonemia in cases with myeloma.

Activation-induced cell death in human peripheral blood lymphocytes after stimulation with silicate in vitro.

Silica and related substances such as silicate have been proven to possess "adjuvant effects". We have previously reported a finding of polyclonal human T cell activation induced by silicate as a superantigen in vitro. In this study, we observed activation-induced cell death in human lymphocytes after stimulation with chrysotile, a kind of silicate. Apoptotic cells were detected flow cytometrically using the TUNEL assay, and the maximum appearance of TUNEL positive cells occurred on day 4 of incubation. Simultaneously the manifestation of small-sized cells in the specimens increased implying apoptosis. Fas expression on lymphocytes increased to day 3 of incubation with chrysotile, and then spontaneously decreased on day 4 when remarkable apoptosis could be detected. Based on these results it is conceivable that activation-induced cell death occurred through Fas-Fas ligand interaction in lymphocytes after stimulation with silicate in a concentration with which no acute cytotoxicity has been detected. Whether and how the repeated apoptosis in definite clones of lymphocytes causes the induction of sFas synthesis need clarification.