Results: In KHYG-1 cytolytic granules are constitutively polarized, normally a consequence of tumor target contact. Demecolcine treatment (10 μM) dispersed granules reversibly in about 90% cells. Cytotoxicity was significantly reduced at doses mentioned above, although, not completely abrogated. This was likely due to re-polarization of granules during assay duration as assessed in parallel imaging experiments. About 20-40% KHYG-1 were positive for Annexin V and about 10-20% positive for 7AAD at 0.1 to 30 μM doses. Degranulation after target contact was inhibited, expression of important activation receptors, NKG2D and NKp44, down-modulated to about 60%, and ERK phosphorylation to about 45%. In all experiments, a plateau effect at doses ≥1 μM was noted (Suck, G. et al. Int Immunol 2006, 18:1347-54).
Conclusions: Demecolcine significantly inhibited KHYG-1 functions and could be envisaged as a treatment for NK neoplasms, with the potential to induce apoptosis, interfere with cytotoxicity and proliferation as a cytostatic drug. A potential drawback is the reversibility of its effects. Combination therapy with the proteasome inhibitor Bortezomib, which has recently been shown to induce apoptosis in malignant NK cell lines, followed by hematopoietic stem cell transplant, is envisaged to improve this approach.
Recombinant therapeutic proteins - - - from the viewpoint of a biotechnologist
SUGIURA T
Daiichi-Sankyo Co., Ltd.
Recombinant proteins are possible magic bullets for pharmacological interventions of the various diseases. However, it is challenging to find a protein with a desirable effect. Moreover, it is also tough to produce a recombinant protein of interest in a form ahieving full activity. We have explored pharmacologically relevant proteins using recombinant technologies.
Protein C is an anti-coagulant protein requiring gamma-carboxylation of the Gla domain for its activity. To ensure its sufficient modification we needed to produce recombinant protein C in mammalian cells. The culture conditions must be optimized to accomplish the satisfactory level of its productivity with enough gamma-carboxylation. Now protein C is on the market as an anti-septic agent from Eli Lilly.
Periostin was first identified as an ostoeblast specific factor-2 from cultured osteoblastic cells with expectation that it should exhibit the activity to stimulate osteoblast cell growth. In order to seek its pharmacological activity toward bone we produced periostin in a high yield by the use of a baculovirus expression vector system. Recently periostin gains much attention due to its potential to treat patients with heart diseases despite the expectation at its discovery.
Thus, the multidisciplinary expertise is required for the development of recombinant magic bullets.
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Plasmodium Heme Crystallization: Methylene Blue to the Quinolines
SULLIVAN DJ
Dept Molecular Microbiology and Immunology and Infectious Diseases
Johns Hopkins Bloomberg School of Public Health
Background: Attempting to make quinine, WH Perkins created the aniline dye mauve, which provided the rational basis of staining microorganisms for inhibition. Ehrlich started from the aniline dye, methylene blue, to discover the first synthetic antimalarial proven effective in humans. Ehrlich later launched the first drug screen to identify compound 606 for syphilis. Methylene blue and the quinolines like chloroquine both bind heme and the heme crystal, hemozoin, to effectively kill Plasmodium parasites. Aims: 1) to show the mechanism of intracellular heme crystal formation and inhibition by methylene blue and the quinolines and 2) to compare inhibition of both P. falciparum and heme crystal inhibition by more than 2,000 existing drugs.
Methods: This study utilized subcellular fractionation, electron microscopy, mass spectrometry and heme crystallization assays to demonstrate heme crystal formation in Plasmodium. Different heme crystallizaton inhibition assays were compared to P. falciparum culture inhibition by the Johns Hopkins Clinical Compound Library, comprised of over 2000 approved drug molecules.
Results: P. falciparuim intracellular heme crystals were observed to grow within neutral lipid naonpheres by electron microscopy. Subcellular fractionation followed by mass spectrometry identified principally neutral lipids closely associated to heme crystals. The neutral lipids promoted efficient heme crystallization, inhibitable by the quinolines and methylene blue. The clinical compound drug screen inhibition of parasite and heme crystallization identified novel antimalarial activity of the antihistamine, astemizole, and the epidermal growth factor inhibitor, gefitinib.
Conclusions: Dye or quinoline binding to heme and heme crystals effectively kills malaria parasites. Screening an approved drug library for malaria or other diseases is potentially an efficient means to discover novel activities of existing drugs which can rapidly be translated into human clinical trials.
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Some Uses of Metal-Based Complexes as Anti-Tumor Agents
SUN RWY, CHE CM*
Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, The University of Hong Kong, Hong Kong, China
Background: Gold(III) complexes have long been known to exhibit anti-cancer properties. However, their medicinal application has been hampered by their poor stability in solutions. In this study, we aim at 1) preparing a variety type of physiologically stable gold(III) complexes; 2) studying the unprecedented structure-activity relationship for stable gold(III) complexes; 3) identifying potential anti-cancer drug hit(s) according to their in vitro/ in vivo anti-cancer and anti-angiogenic activities; and 4) illustrating their anti-cancer mechanism(s).
Methods: By using strong σ-donor dianionic porphyrinato ligands, we developed a family of gold(III) porphyrin complexes having different hydrophilic and hydrophobic substituents. Their IC50 values toward different panels of cancer cell lines were determined by MTT assay, and some of their in vivo anti-cancer activities were studied by using Buffalo rats and nude mice models. To elucidate the potent anti-cancer mechanism(s), DNA array, proteomics, western blotting and computational docking analyses have been employed.
Results: The IC50 values of the gold(III) porphyrin complexes were found to correlate with their lipophilicity and their cellular uptake. Two gold(III) porphyrins, namely gold-1a and gold-2a, exhibited tremendous in vitro cytotoxicity with IC50 ~ 50 nM toward some nasopharyngeal carcinoma cell lines. In vivo studies showed that these two complexes could prolong survival of the HCC-bearing rats and/or significantly inhibit tumor growth in nude mice models. Western blotting and computational docking analyses revealed that some anti-apoptotic proteins such as Bcl-2 and Mcl-1 are their important cellular targets. A gold(III) porphyrin with saccharide conjugation (gold-3a) was found to be relatively not cytotoxic, but exhibits significant cytotstatic and anti-angiogenic activities on MS1 cells.
Conclusions: The enhanced stabilization of the gold(III) ion and the ease of its structural modification render porphyrin ligands to be advantages in the development of physiologically stable bioactive gold(III) porphyrin complexes with potent anti-cancer and anti-angiogenic activities.
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Personalized Medication with Estramustine Phosphate for Advanced Prostate Cancer after Screening of the CYP1A1 gene polymorphisms
MOTOFUMI SUZUKI, TAKAYUKI KUROSAKI, MIAO LIU, YUTAKA ENOMOTO, HIROAKI NISHIMATSU, YUKIO HOMMA, TADAICHI KITAMURA
Department of Urology, The University of Tokyo, Tokyo, Japan
Background: Estramustine phosphate (EP) is a chemoendocrine agent applied for the treatment of advanced and/or hormone refractory prostate cancer. Gastrointestinal (GI) toxicity is observed frequently during EP therapy in prostate cancer patients. Formerly, we reported that polymorphisms in the CYP1A1 gene were significantly associated with GI toxicity during EP therapy. Aim of this study is to determine whether screening of the CYP1A1 gene polymorphisms is a useful method to achieve personalized medication with EP for advanced prostate cancer.
Methods: After screening of the CYP1A1 gene polymorphisms, a total of 39 patients with advanced or hormone refractory prostate cancer was regarded as a low-risk group for GI toxicity with EP. The methods of genotyping assay were a TaqMan assay or a direct-sequencing method. All of patients were administered EP 280 mg/day orally and assessed their toxicities monthly. Three of 39 patients withdrew EP therapy within 8 weeks because of other toxicities (liver dysfunction, lung edema, and gynecomastia), and they were excluded from the analysis. Formerly, we experienced EP therapy with same regimen for 55 patients of advanced prostate cancer without screening of the CYP1A1 gene polymorphisms. We compared the incidence of GI toxicity according to the NIH Common Terminology Criteria for Adverse Events v3.0 between former study and present one, and evaluated the efficacy of the gene screening. The Ethics Committee of the University of Tokyo approved this study.
Results: Follow-up period of present study was 557 ± 254.6 (mean± SD) days. Incidence of GI toxicity of former study and present one was 40.0% (22/55) and 19.4% (9/36), respectively. The odds ratio was 0.36 (95% CI, 0.13 – 0.94; P = 0.036). In the multivariate analysis, the hazard ratio was 0.21 (95% CI, 0.06 – 0.65; P = 0.006). The withdrawal of EP therapy because of GI toxicity in former study and present one was 18.2% (10/55) and 2.8% (1/36), respectively. Conclusions: Screening of the CYP1A1 gene polymorphisms prior to EP therapy could achieve a personalized medication with EP for the patients with advanced prostate cancer.
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L-type calcium channels in non-excitable cells: critical roles in cell survival
SUZUKI Y, YOSHIMARU T, INOUE T, RA C
Nihon University Graduate School of Medical Science, Tokyo, Japan
Background: Ca2+ is a highly versatile intracellular second messenger that regulates many complicated cellular processes, including cell functions, cell proliferation, and death. It has long been believed that in nonexcitable cells, including hematopoietic cells, store-operated Ca2+ entry is the principal route of Ca2+ influx. Increasing evidence suggests the existence of founctional L-type Ca2+ channels (LTCCs) in various immune cells, including T, B and natural killer cells, but their molecular entity and functions are poorly understood. We have previously reported that in mast cells IgE plus antigen (IgE/antigen) stimulates a dihydropyridine (DHP)-sensitive Ca2+ channel that is immunologically and pharmacologically related to LTCCs. In the present study we attempted to reveal the molecular entity of this Ca2+ channel. Moreover, since mast cells do not undergo apoptosis upon IgE receptor activation, while the Ca2+-ATPase inhibitor thapsigargin (Tg), which cannot activate the LTCC-related Ca2+ channel, causes sizable apoptosis, we elucidated the potential role of the Ca2+ channel in mast cell survival.
Methods: The expression of LTCCs mRNA was evaluated using RT-PCR analysis. The expression of LTCCs on the cell surface was determined by flow cytometry. Apoptotic cell death and/or overall cell death were evaluated by annexin V/ propidium iodide double-staining, cytochrome c leakage and the membrane potential collapse.
Results: The mast cell line rat basophilic leukemia (RBL-2H3) expressed the 1C subunit mRNA and express the 1C protein on their surface. The cells underwent to apoptosis when extracellular Ca2+ was absent. The LTCC antagonists such as nifedipine and diltiazem remakably augmented IgE-mediated apoptosis, while the LTCC agonist (S)-BayK8644 rescued the cells from Tg-induced apoptosis. The augmentation and reduction of apoptosis were accompanied by the enhancement and suppression, respectively of mitochondrial Ca2+ efflux, depolarization, cytochrome c leakage, and caspase-3/7 activation. Finally, when the expression of the 1c type of LTCC was knocked down using small interfering RNA, IgE/antigen alone caused substantial mitochondrial integrity disruption and apoptosis, as observed with IgE/antigen plus nifedipine or Tg stimulation, and (S)-BayK 8644 no longer protected cells from apoptosis.
Conclusions: LTCCs play a critical role in the survival of mast cells and potentially other immune cell types which express them through maintaning mitochondrial Ca2+ homeostasis and preventing mitochondrial integrity disruption.
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Chemical chaperone therapy: magic bullet to the brain in GM1-ggngliosidosis
SUZUKI Y1, NANBA E2, HIGAKI K2, SAKAKIBARA Y3, OGAWA S3, IIDA M4
1UInternat Univ Health Welfare, Otawara, Japan; Tottori Univ, Yonago, Japan; Keio Univ, Yokohama, Japan; Seikagaku Corp, Higashi Yamato, Japan
Background: Chemical chaperone therapy has been tried as a new molecular approach to brain damage in lysosomal diseases. Low molecular weight substrate analogues bind to mutant enzymes at neutral pH and dissociation occurs at acidic pH in the lysosome. Aims: 1) molecular analysis of chaperone-protein interaction in enzyme deficient cells. 2) assessment of chaperone effect in mice and humans.
Methods: Assays of in vitro enzyme inhibition and in situ chaperone effect of N-octyl-4-epi--valienamine (NOEV), using genetically engineered model mice expressing mutant human -galactosdase. Computational modeling of the human -galactosidase structure and theoretical calculation of NOEV-enzyme interaction, leading to the chaperone effect. DNA microarray on secondary gene expression. Analysis of neurotrophin receptor Trk using specific antibodies. NOEV effect on intracellular signal transduction, autophagy, and gene expression.
Results: NOEV was clinically effective within 3 month when oral administration was started at 2 month of age (p<0.05 at 5-7 months; n=11-15 for non-treated and n=7-16 for treated mice). The effect was not evident till 6 month of treatment (11 month of age) when treatment was started at 5 month of age (p<0.05 at 11 months; n=5 for non-treated and n=6 for treated mice). Computational prediction showed that human -galactosidase took the TIM barrel structure, and the NOEV-enzyme binding was less strong in the lysosomal environment at low pH, resulting in dissociation of the complex. DNA microarray revealed an abnormal pattern of gene expression in enzyme-deficient human and mouse fibroblasts, and mouse brain. Phosphorylated Trk and Bip/GRP-78 abnormally increased at the late clinical stage of GM1-gangliosidosis in the mouse brain. These pathological changes were corrected by NOEV treatment.
Conclusions: Early chaperone thrapy is mandatory for better neurological effect in model mice. Computational modeling is useful for analysis of molecular pathology in GM1-gangliosidosis. Multiphasic intracellular abnormalities were corrected by NOEV threrapy in association with the clinical effect. These new approaches are important for understanding chemical pathology and developing new therapeutic trials in lysosomal diseases.
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Antimalarial drug resistance in Indonesia: A molecular analysis
SYAFRUDDIN D1, ASIH PBS1, TAUFIK AS2, MULYANTO2,3
1Eijkman Institute for Molecular Biology, Jakarta, Indonesia, 2Immunobiology Laboratory, School of Medicine, University of Mataram, Mataram, Indonesia, 3
West Nusa Tenggara Hepatitis Laboratory, Mataram, Indonesia
Background: Malaria remains a major public health problem in Indonesia, causing approximately 30 million clinical cases and several thousands deaths annually. Since the first report of P. falciparum resistance to chloroquine (CQ), the first-line antimalarial, in the mid of 1970s in East Kalimantan and West Papua Provinces, respectively, resistance to this drug has rapidly spread throughout the archipelago. Resistance to CQ have also been reported in P. vivax and P. malaria in 1996 and 2003 respectively. Sulphadoxine-Pyrimethamine (SP) resistance was first reported in the early 1980s in West Papua and then sporadically found in other islands of Indonesia. The Ministry of Health has adopted the policy to use artemisinin-based combination therapy (ACT) as the first line antimalarial drug in the year 2004. To determine the extent of antimalarial drug resistance among the field isolates throughout the archipelago, a molecular epidemiologic survey has been conducted since 1998
Methods: Molecular assays of the parasite DNAs isolated from blood blots on filter paper, collected from several sentinel sites, were performed. The assays employed Polymerase Chain Reaction (PCR), restriction fragment length polymorphism (RFLP) to detect the allelic forms of the pfcrt and pfmdr1 genes, and pfdhfr and pfdhps genes and their orthologues in P. vivax, that were associated with CQ and SP resistance, respectively.
Results: The results revealed that the pfcrt 76-Thr and pfmdr1 86-Tyr alleles have been fixed in many of (VH1) P. falciparum isolates examined. Analysis of dhfr gene revealed three mutant alleles 16Val, 59Arg and 108Asn/Thr. The dhfr 108Asn mutations appeared either as a single mutation or paired with 59-Arg in the frequency of 20-90% among the isolates examined. Mutant alleles of the dhps gene appeared in much less frequency, mostly in the form of 437G and 540K alleles. Analysis of the P. vivax isolates did not find any mutations in the pvcg10 and pvmdr1 genes, but found several isolates that carried mutant alleles of the pvdhfr and pvdhps genes. The findings strongly indicate resistance to chloroquine in P. falciparum isolates and a growing resistance status to sulfadoxine-pyrimethamine combination in both P.falciparum and P. vivax.
Conclusions: molecular evidence indicates that the antimalarial drug resistance in Indonesia poses a continuing challenge to the malaria control programme and
highlights the need to the proper antimalarial deployment.
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Anti-apoptotic and apoptotic action of (-)deprenyl (Selegiline) and its metabolites
SZENDE B (1), MAGYAR K(2)
1'st Department of Pathology and Experimental Cancer Research
(1) and Department of Pharmacodynamics
(2) Semmelweis University, Budapest, Hungary
Background: The antiapoptotic effect of (-)deprenyl on human phaeochromocytoma cells after serum deprivation has been reported earlier.
Material and Methods: In our experiments the mode of cytoprotective action of (-) deprenyl was studied using A2058 human melanoma cells in culture. Serum deprivation for five days resulted in excessive number of apoptosis in the cell ciltures.
Results: Very low doses of (-) deprenyl (10(-7)-10(-13)M) caused an approximately 2 days delay in the onset if apoptosis. The known metabolites of (-)deprenyl, (-)desmethyl-deprenyl, (-) and (+) methylamphetamine and also (+) deprenyl failed to exert the same effect. The anti-apoptotic action of (-)deprenyl was prevented by the simultaneous application of the microsomal drug-metabolizing enzyme inhibitor SKF-525A, showing that (-) deprenyl needs metabolic conversion in order to be anti-apoptotic, but the effective metabolite is still unknown. On the other hand, higher dose (10(-3Ml) of (-) deprenyl, (-) desmethyl-deprenyl, (-) and (+) methylamphetamine induced apoptosis in the non-serum deprived A-2058 cell culture. SKF 525A did not prevent the apoptosis-inducing effect of (-) deprenyl which means that no metabolic changes are needed for this activity. High dose (10(-3) Ml) (-)deprenyl induced very high Caspase 3 activity in non-serum-deprived A-2058 cell culture, low doses (10(-9)-10(-13)M maintained Caspase 3 activity on control level in case of serum deprivation.
Conclusion: The neuroprotective effect of (-) deprenyl (Selegiline) may be due to the anti-apoptotic activity of this compound.
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Advent of Pharmacology of Nociceptors Initiated by Capsaicin Has Opened Also a New World in Neurohumoral Regulation
SZOLCSÁNYI J, PETHŐ G, PINTÉR E, HELYES ZS
Department of Pharmacology, University of Pécs, Hungary
Background: Capsaicin, the hot principle of chilli is the first magic bullet for nociceptors. Its receptor cloned in 1997 is a thermosensor ion channel gated by noxious stimuli, named Transient Receptor Potential Vanilloid 1 (TRPV1). Furthermore, substance P and other neuropeptides released from the activated capsaicin-sensitive nerve endings elicit – without axon reflexes – neurogenic inflammation and other efferent tissue responses. We also described that released somatostatin inhibits inflammation at distant sites.
Methods: 1. Mechanical, noxious heat thresholds of rat paw was measured in hyperalgesia (plantar incision, heat injury etc.) with and without stimulation of the acutely denervated contralateral paw. 2. Neurogenic inflammation evoked by electrical stimulation (dorsal roots, nerves) or by irritants was similarly tested after stimulating other parts of the body (paw, eye, vagal nerve). 3. Sensory neuropeptides were measured by radioimmunoassay. 4. Gene deleted mice (TRPV1, sst4R) were also used.
Results: 1. Contralateral counter-irritation (capsaicin, mustard oil) diminished the hyperalgesia by more than 50%. Antagonists of somatostatin (cyclosomatostatin) or cannabinoid CB1 receptor counteracted these effects. 2. Antidromic stimulation (dorsal root, sciatic nerve) enhanced the plasma somatostatin level over 3-fold and inhibited by around 50% inflammation evoked by capsaicin, carrageenin or contralateral dorsal root stimulation. The inhibition was absent in rats pretreated with somatostatin antibody or after perineural capsaicin pretreatment. 3. 0.1 Hz electrical stimulation did not evoke neurogenic inflammation but still produced systemic inhibitory effects.
Conclusions: Capsaicin-sensitive major subsets of sensory neurons subserve unorthodox local effector and systemic antinociceptive, antiinflammatory „sensocrine” functions. Drug candidates of TRPV1 antagonists or somatostatin receptor agonists are in Phase II clinical trials.
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Treating the Untreatable: Alpha 1-Adrenoreceptor Antagonist Prazosin for PTSD, Disruptive Agitation in AD and Alcohol Dependence
SZOT P, SIMPSON T, PESKIND E, RASKIND M
Mental Illness, Research Education and Clinical Center (MIRECC), VA Puget Sound Health Care System, 1660 S. Columbian Way, Seattle WA 98108
Background: Although the CNS noradrenergic system has been implicated in multiple neuropsychiatric disorders, the postsynaptic alpha 1-adrenoreceptor (alpha1-AR) has been addressed as a pharmacotherapeutic target only in cardiovascular and urologic disorders. Clinical and neuropathologic studies suggest that increased responsiveness to norepineprhine (NE) at the alpha1-AR contributes to the pathophysiology of post-traumatic stress disorder (PTSD) trauma nightmares, disruptive agitation in Alzheimer’s disease (AD) and alcohol dependence. Prazosin, a CNS active alpha1-AR antagonist, was evaluated for efficacy and tolerability in these three difficult to treat disorders.
Methods: Study 1: 40 Vietnam war veterans with PTSD and intractable nightmares were randomized to prazosin (achieved mean bedtime dose = 13 mg) or placebo. 3 weeks of titration and 8 weeks of mean dose. Study 2: 22 elderly AD patients with treatment resistant disruptive agitation were randomized to titrated prazosin (achieved daily dose 2 mg id and 3 mg bedtime) or placebo. 2 weeks of titration and 4 weeks of mean dose. Study 3: 17 men seeking abstinence for alcohol dependence were randomized to titrated prazosin (achieved dose 4 mg bid and 8 mg bedtime). 2 weeks of titration and 4 weeks of mean dose.
Results: Prazosin was well tolerated in all studies. Prazosin was significantly and substantially superior to placebo for: (study 1) reducing nightmares and improving sleep and overall clinical status in PTSD; (study 2) reducing disruptive agitation by Neuropsychiatric Index (NPI) and Brief Psychiatric Rating Scale (BPRS) scores in AD; and (study 3) reducing alcohol ingestion and days drinking in persons seeking abstinence.
Conclusion: Prazosin appears effective for three different neuropsychiatric disorders that have been very difficult to treat. Larger and longer placebo controlled trials of prazosin are underway for all three conditions. This data introduces the novel use of prazosin, an alpha1-AR antagonist, for several different neuropsychiatric disorders that have been very difficult to treat.
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Role of estradiol and testosterone in the regulation of bone metabolism in men and women
SZULC P
INSERM 831 Research Unit, Hôpital Edouard Herriot, University of Lyon, Lyon, France
Sex steroids (testosterone, 17-estradiol) are major regulators of bone metabolism in men and women. After menopause, dramatic decrease in the secretion of 17-estradiol results in an increase in bone resorption followed (but not matched!!) by increased bone formation. This imbalance between bone formation and bone resorption results in a decrease in bone mineral density (BMD) and an increase in the risk of fracture. Structural mechanisms underlying postmenopausal bone loss have been partly elucidated (trabecular perforation and loss, loss of trabecular connectivity, cortical thinning due to, mainly but not only, higher endocortical resorption and trabecularization of subendocortical bone).
In men, the major sex steroid is testosterone, however, 17-estradiol is probably the principal regulator of bone metabolism in men. During growth, 17-estradiol is necessary for closure of growth cartilages and growth arrest. Data are discordant as concerns the role of sex steroids in the regulation of the radial bone growth in boys. During ageing, lower concentration of 17-estradiol, especially of its bioavailable fraction, is associated with higher levels of biochemical bone turnover markers, lower BMD, slightly faster bone loss. Direct effect of testosterone on bone formation and bone resorption in men is probably weak. Less data concern the role of both sex steroids in the regulation of metabolism of trabecular bone and cortical bone in male and they are based largely on experimental studies. Trabecular bone seems to be more sensitive to lower 17-estradiol concentration, because its metabolically available area is higher. It is not clear if the sex steroids play a significant role in the regulation of periosteal apposition in older men. Deficits of both hormones increase the risk of fracture in older men; 17-estradiol deficit acts probably mainly through the effect on bone (BMD, microarchitecture) and testosterone deficit acts at least partly through its effect on muscle mass and risk of falls.
In both sexes, the 17-estradiol deficit may interact with other factors influencing bone metabolism. Smoking may aggravate consequences of 17-estradiol deficit through the influence on its synthesis and catabolism. Conversely, obesity can partly counteract the effect of 17-estradiol deficit, partly by the peripheral aromatization and partly by the mechanical effect.
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Effects of neurotoxic and neuroprotective compounds on cholinergic neurons are mediated by alterations in acetyl-CoA metabolism
SZUTOWICZ A1, BIELARCZYK H, DYS A, RONOWSKA A, KLIMASZEWSKA J, JANKOWSKA-KULAWY A
Department of Laboratory Medicine, Medical University of Gdansk, Gdansk, Poland
Background: The preferential loss of septal cholinergic neurons is a main cause of cognitive deficits in course of Alzheimer’s disease (AD) and other encephalopathies of advanced age. High susceptibility of cholinergic neurons to neurodegeneration might result from the fact that they utilize acetyl-CoA, not only for energy production but also for acetylcholine (ACh) synthesis. Therefore, acetyl-CoA metabolism is a likely target for both cholinotoxic insults as well as for therapeutic approaches.
Methods: Cholinergic septum-derived SN56 neuroblastoma cells were cultured in Dulbecco-Eagle’s medium for 2 days with subsequent treatment with neurotoxic or/and cytoprotective compounds.
Results: The differentiation of SN56 cells by retinoids, NGF or cAMP-mediated signals, caused the increase of choline acetyltransferase (ChAT) activity, ACh synthesis/quantal release and cytoplasmic acetyl-CoA content along with decrease in acetyl-CoA synthesis and its levels in neuronal mitochondria. The shortage of acetyl-CoA in mitochondria of differentiated cells caused their greater than nondifferentiated ones susceptibility to recognized AD pathogens such as amyloid-beta, NO, Al and Zn. These compounds caused dose-dependent increase of nonviable cell fraction and cytoplasmic cytochrome c levels, decreases in mitochondrial enzyme and ChAT activities, intramitochondrial and cytoplasmic acetyl-CoA and ACh levels, with loss of morphologic differentiation. Number of nonviable cells inversely correlated with pyruvate dehydrogenase activity (r=-0.79, p=0.002) and content of mitochondrial acetyl-CoA (r=-0.92, p=0.0002). Neuroprotective capacity of lipoates correlated with their ability to maintain high level of acetyl-CoA in mitochondria. On the other hand, the expression of cholinergcic phenotype positively correlated with alterations in cytoplasmic acetyl-CoA levels (r=0.90, p=0.002).
Conclusions: These data indicate the existence in cholinergic neurons two functionally independent pools of mitochondrial and cytoplasmic acetyl-CoA, that under pathologic conditions affect their viability and expression of cholinergic phenotype, respectively.
Authors’ disclosure statement: Supported by MNISW projects 2P05A 110 30.
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