Role of cytochrome P450 for vitamin D metabolisms in patients with neurodegenerative disorders.

Introduction: We previously reported lower serum 25-hydroxyvitamin D concentrations in patients with Alzheimer's disease (AD), Parkinson's disease (PD) and Multiple system atrophy (MSA) compared to healthy controls (HC), whereas 1,25-di-hydroxyvitamin D levels were solely lower in MSA patients. We investigate serum concentrations of P450 involved in Vitamin D(VD) hydroxylation to clarify the responsible hydroxylase for the low serum concentrations of VD metabolites. Methods: A total of 79 individuals were enrolled including 20 HC, 20 AD, 19 PD and 20 MSA patients. The serum concentrations of P450 involved in VD hydroxylation were assayed by ELISA. The data were analyzed by the nonparametric Kruskal-Wallis test between groups. Results: Though CYP2R1 and CYP27A1 mediate 25-hydroxylation for VD, CYP2R1 is the main hydroxylase, and CYP27A1 is also involved in VD synthesis. CYP2R1 concentrations showed no differences among groups, while lower CYP27A1 concentrations were found in PD (p < 0.05) and MSA (p < 0.005) compared to HC and differences between AD and MSA (p < 0.05), however no differences between PD and MSA. CYP27B1 is the main 1α-hydroxylase for 25-hydroxyvitamin D and showed differences between HC and PD (p < 0.05), between HC and MSA (p < 0.005) and between PD and MSA (p = 0.055). CYP24A1, which inactivate 1,25-di-hydroxyvitamin D, showed no differences among groups. Conclusions: CYP27A1 might affect VD synthesis and cause low 25-hydroxyvitamin D levels in AD, PD and MSA patients. Low 1,25-di-hydroxyvitamin D levels in MSA patients might be caused by impaired feedback mediated by CYP27B1.

Circulatory 25(OH)D and 1,25(OH)2D as differential biomarkers between multiple system atrophy and Parkinson's disease patients.

BACKGROUND AND PURPOSE: There is sufficient evidence to support vitamin D's noncalcemic effects and the role of vitamin D deficiency in the development of a wide range of neurological disorders. This study aimed to evaluate whether serum 25(OH)D and 1,25(OH) 2 D could be used as biomarkers to differentiate between healthy subjects (HS), multiple system atrophy (MSA) and Parkinson's disease (PD) patients of both genders. METHODS: A total of 107 subjects were included in this study, divided into three groups: 1- HS (n = 61), 2- MSA patients (n = 19), and 3- PD patients (n = 27). The patients were assessed using UMSARS II, UPDRS III, H&Y, MMSE and MoCA rating scales. The levels of 25(OH)D and 1,25(OH) 2 D in serum were determined using the radioimmunoassay technique. RESULTS: The levels of 25(OH)D and 1,25(OH) 2 D in HS were 26.85 +/- 7.62 ng/mL and 53.63 +/- 13.66 pg/mL respectively. 25(OH)D levels were lower in both MSA and PD by 61% and 50%, respectively (P = 0.0001 vs. HS). 1,25(OH) 2 D levels were lower in MSA by 29%(P = 0.001 vs HS). There was a correlation between 25(OH)D and 1,25(OH) 2 D in MSA and PD, but not in HS. 1,25(OH) 2 D regressed with MMSE (ß = 0.476, P = 0.04, R 2 = 0.226) in MSA, and with UPDRS III (ß = -0.432, P = 0.024, R 2 = 0.187) and MoCA (ß = 0.582, P = 0.005,R 2 = 0.279) in PD. 25(OH)D displayed considerable differentiative strength between HS and MSA (Wald = 17.123, OR = 0.586, P = 0.0001; AUC = 0.982, sensitivity and Youden index = 0.882, P = 0.0001) and PD (Wald = 18.552, OR = 0.700, P = 0.0001; AUC = 0.943, sensitivity = 0.889, YI = 0.791, P = 0.0001). 1,25(OH) 2 D distinguished MSA from PD (Wald 16.178, OR = 1.117, P = 0.0001; AUC = 0.868, sensitivity = 0.926, Youden index =0.632, P = 0.0001). H&Y exhibited the highest sensitivity, AUC, and significant distinguishing power between MSA and PD. CONCLUSIONS: Serum 25(OH)D and 1,25(OH) 2 D could be useful biomarkers for MSA and PD. 25(OH)D and H&Y provided the highest sensitivity and group classification characteristics.

Table tennis for patients with Parkinson's disease: A single-center, prospective pilot study.

INTRODUCTION: Table tennis is a popular sport worldwide. However, no study has examined whether it is an effective exercise for patients with Parkinson's disease (PD). The efficacy and safety of table tennis exercise for PD patients was examined. METHODS: This 6-month prospective study investigated if our table tennis exercise program could improve parkinsonian motor symptoms, cognition and psychiatric symptoms. Twelve PD patients with Hoehn & Yahr stage ≤4 were recruited. Patients participated in a 6-hour exercise session once weekly. All patients were assessed with the Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) parts I-IV, Montreal Cognitive Assessment (MoCA), Frontal Assessment Battery (FAB), Self-Rating Depression Scale (SDS), and Starkstein Apathy Scale (SAS) at baseline, 3 months, and 6 months. RESULTS: Nine of 12 PD patients were analyzed, except for three patients for which data was missing. MDS-UPDRS parts II and III were improved at 3 months (median -4.0, p = 0.012 and median -10.0, p = 0.012) and 6 months (median -7.0, p = 0.015 and median -12.0, p = 0.008), whereas MDS-UPDRS total parts I scores and total IV scores, MoCA, FAB, SDS, and SAS were unchanged. Adverse events included fall and backache in one patient each. CONCLUSION: A table tennis exercise program is relatively safe and may improve activities of daily living and motor symptoms in patients with PD.

A novel diagnostic marker for progressive supranuclear palsy targeting atrophy of the subthalamic nucleus.

INTRODUCTION: Despite great progress in radiological diagnostic tools for neurodegenerative disorders, their diagnostic accuracy has been unsatisfactory. One of the pathological hallmarks of progressive supranuclear palsy (PSP) is atrophy of the subthalamic nucleus, which has not attracted much attention for imaging analysis. METHODS: The clinical data of patients with PSP, multiple system atrophy (MSA), Parkinson's disease (PD), and corticobasal syndrome (CBS) who underwent brain magnetic resonance imaging at our department between June 2019 and March 2020 were retrospectively reviewed. The volumes of the subthalamic nucleus and of the whole cerebrum were then analyzed and compared among the disorders. RESULTS: Fourteen PSP-Richardson syndrome (RS), 14 MSA, 14 PD, and 8 CBS patients were assessed. The mean volume of the bilateral subthalamic nuclei was smaller in PSP patients (0.148 ± 0.012 cm3) than in MSA (0.183 ± 0.026 cm3; p < 0.001), PD (0.209 ± 0.031 cm3; p < 0.001), and CBS (0.180 ± 0.056 cm3; p < 0.001) patients. The volume of the whole cerebrum was not significantly different among the disorders. Using an STN volume cut-off of 0.01925, the sensitivity and specificity for differential diagnosis between PSP and the other disorders were 0.846 and 0.972, respectively. CONCLUSION: Subthalamic nucleus volume may be a useful diagnostic marker for PSP; it may easily differentiate it from other neurodegenerative parkinsonian disorders.

Safety Assessment of Bangle (Zingiber purpureum Rosc.) Rhizome Extract: Acute and Chronic Studies in Rats and Clinical Studies in Human.

Bangle (Zingiber purpureum Rosc.) rhizome extract (BRE) contains phenylbutenoid dimers (banglenes), which exert neurotrophic effects and possess the potential capability to regenerate hippocampal neurons in mice. The acute and chronic oral toxicities of BRE powder were evaluated in Sprague-Dawley rats. A dose of BRE powder was estimated to be higher than 2000 mg/kg containing BRE 534 mg/kg as minimum lethal dose in a single-dose oral toxicity study. The no-observed-adverse-effect-level for the BRE powder was 1000 mg/kg/day (BRE 267 mg/kg) in the 90 day oral toxicity study. Four week clinical studies of BRE tablets in humans suggested that the ingestion of BRE tablets within 850 mg/man/day (BRE 227 mg/man/day) was safe for at least 1 month and in a usual manner. The C max, t max, and AUC of cis- and trans-(E)-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-enes (c- and t-banglenes) were calculated after the ingestion of BRE tablets (BRE 227 mg) and were 17.73 and 22.61 ng/mL, 1.8 and 1.8 h, and 71.47 and 95.53 ng/mL/h, respectively.

Serum vitamin D in patients with mild cognitive impairment and Alzheimer's disease.

Objectives: To determine the relevance of Mini-Mental State Examination (MMSE), serum 25-hydroxyvitamin D (25(OH)D3), and 1,25(OH)2D3 concentrations to mild cognitive impairment (MCI) and various stages of Alzheimer's disease (AD). Materials and Methods: The study included 230 participants (>74 years) allocated to three main groups: 1-healthy subjects (HS, n = 61), 2-patients with MCI (n = 61), and 3- patients with Alzheimer's disease (AD) subdivided into three stages: mild (n = 41), moderate (n = 35), and severe AD (n = 32). The cognitive status was evaluated using MMSE. Serum 25 (OH)D3 (ng/ml) and 1,25(OH)2D3 concentrations (pg/ml) were determined by competitive radioimmunoassay. Results: MMSE scores and 25(OH)D3 were decreased in MCI and all stages of the AD in both genders. MMSE variability was due to gender in HS (11%) and to 25(OH)D3 in MCI (15%) and AD (26%). ROC analysis revealed an outstanding property of MMSE in diagnosis of MCI (AUC, 0.906; CI 95%, 0.847-0.965; sensitivity 82%; specificity, 98%) and AD (AUC, 0.997; CI 95%, 0.992-1; sensitivity, 100%; specificity, 98%). 25(OH)D3 exhibited good property in MCI (AUC, 0.765; CI 95%, 0.681-0.849; sensitivity, 90%; specificity, 54%) and an excellent property in diagnosis of AD (AUC, 0.843; CI 95%, 0.782-0.904; sensitivity, 97%; specificity, 79%). Logistic analyses revealed that, in MCI, MMSE could predict (or classify correctly) with 97.6% accuracy (Wald, 15.22, ß, -0.162; SE, 0.554; OR = 0.115:0.039-0.341; p = .0001), whereas 25(OH)D3 with 80% accuracy (Wald, 41,013; ß, -0.213; SE, 0.033; OR = 0.808: 0.757-863; p = .0001). 25(OH)D3 was the only significant predictor for the severe AD and contributed to MMSE variability. Age and gender were significant predictors only in the moderate AD. In patients with MCI, 25(OH)D3 and 1,25(OH)2D3 were correlated men, but in case of the AD, they were correlated in women. Conclusions: MMSE and serum 25(OH)D3 concentrations could be useful biomarkers for prediction and diagnosis of MCI and various stages of the AD. The results support the utility of vitamin D supplementation in AD therapy regimen.

Monocytic elastase-mediated apolipoprotein-E degradation: Potential involvement of microglial elastase-like proteases in apolipoprotein-E proteolysis in brains with Alzheimers disease.

Impaired clearance of soluble Aß (amyloid-ß) promotes Aß aggregation in brains with Alzheimer's disease (AD), while apolipoprotein-E (ApoE) in microglia mediates Aß clearance. We studied the protease responsible for ApoE(4) degradation in human peripheral monocyte extracts, which are from the same lineage as microglia. We detected the hydrolytic activity for ApoE(4) in high-salt extracts with 2 M NaCl and found that the activity was inhibited by a serine protease inhibitor and an elastase-specific inhibitor, but not by other protease inhibitors. The extracts exhibited higher activity for the elastase substrate, and we followed the activity with ion-exchange and gel-filtration chromatography. Through silver staining, we partially purified a protein of 28 kDa, which was clarified as elastase by liquid chromatography-tandem mass spectrometry. These observations suggest that elastase is the key protease for ApoE(4) degradation. We also detected ApoE(4) hydrolytic activity in high-salt extracts in mouse microglial (BV-2) cell lysates, and showed that the ApoE(4) fragments by the BV-2 extracts differed from the fragments by the monocyte extracts. Though the ApoE(4) degradation by the extracts was not inhibited with elastase-specific inhibitors, it was inhibited by an elastase-specific monoclonal antibody, suggesting that elastase-like proteases in microglia differ from those of monocytes. Immunohistochemistry revealed that both elastase and ApoE were expressed in the senile plaques of brains with AD. In vitro studies also disclosed the localization of elastase in the microglial cell line, BV-2. Our results suggest that elastase-like proteases in the microglial cells surrounding Aß plaques are responsible for ApoE degradation in the brain.

Novel neurotrophic phenylbutenoids from Indonesian ginger Bangle, Zingiber purpureum.

Two new curcuminoids 1 and 2, and a new phenylbutenoid dimer 3, were isolated from Bangle (Zingiber purpureum). Their structures were determined on the basis of comprehensive spectroscopic data and their biogenetic pathway. Compounds 1 and 2 are the first example of curcumin coupled with phenylbutenoid. Compounds 1 and 2 promoted neurite outgrowth of NGF-mediated PC12 cells at concentrations ranging from 1 to 10 µM. In addition, compound 1 was found to accelerate the prevention of Aß42 aggregation.

Different effect of vitamin D2 and vitamin D3 on amyloid-ß40 aggregation in vitro.

The seeding of amyloid-ß 40 (Aß40) oligomers from monomers is the initial step of Aß aggregation, and many reports have suggested that cholesterol enhances this step. We studied the potential of secosteroid vitamin D derivatives for Aß40 aggregation in vitro. The quartz-crystal microbalance technique demonstrated that vitamin D3 does not show any effect on Aß40 aggregation while vitamin D2 promoted it and docking simulation but that vitamin D2 has high potential in this regard. Thus, stacking of the Phe19 benzene ring in Aß40 and the C22-C23 double bond in vitamin D2 may alter the energy of these molecules. Electron microscopy revealed the potential of vitamin D2 to increase Aß40 aggregation. Thioflavin-T assays indicated that Vitamin D2 induced increased fluorescence at 490 nm, as typically observed for amyloid fibrils but also for protofibrils; in both cases this reflects of the increase of ß-sheet contents. Aß40 aggregation was further confirmed in ELISA, SDS-PAGE and dot blot analysis which revealed changes in protease K resistance. These results suggest a possible mechanism, of how vitamin D2 could increase Aß40 aggregation and the docking simulation explains, why the same is not observed with vitamin D3.

Vizantin inhibits endotoxin-mediated immune responses via the TLR 4/MD-2 complex.

Vizantin has immunostimulating properties and anticancer activity. In this study, we investigated the molecular mechanism of immune activation by vizantin. THP-1 cells treated with small interfering RNA for TLR-4 abolished vizantin-induced macrophage activation processes such as chemokine release. In addition, compared with wild-type mice, the release of MIP-1ß induced by vizantin in vivo was significantly decreased in TLR-4 knockout mice, but not in TLR-2 knockout mice. Vizantin induced the release of IL-8 when HEK293T cells were transiently cotransfected with TLR-4 and MD-2, but not when they were transfected with TLR-4 or MD-2 alone or with TLR-2 or TLR-2/MD-2. A dipyrromethene boron difluoride-conjugated vizantin colocalized with TLR-4/MD-2, but not with TLR-4 or MD-2 alone. A pull-down assay with vizantin-coated magnetic beads showed that vizantin bound to TLR-4/MD-2 in extracts from HEK293T cells expressing both TLR-4 and MD-2. Furthermore, vizantin blocked the LPS-induced release of TNF-α and IL-1ß and inhibited death in mice. We also performed in silico docking simulation analysis of vizantin and MD-2 based on the structure of MD-2 complexed with the LPS antagonist E5564; the results suggested that vizantin could bind to the active pocket of MD-2. Our observations show that vizantin specifically binds to the TLR-4/MD-2 complex and that the vizantin receptor is identical to the LPS receptor. We conclude that vizantin could be an effective adjuvant and a therapeutic agent in the treatment of infectious diseases and the endotoxin shock caused by LPS.

Vitamin D 2 interacts with Human PrP(c) (90-231) and breaks PrP(c) oligomerization in vitro.

PrP(sc), the pathogenic isoform of PrP(c), can convert PrP(c) into PrP(sc) through direct interactions. PrP(c) oligomerization is a required processing step before PrP(sc) formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D 2 and human recombinant PrP(c) (90-231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109-112 of PrP(c), inhibited this interaction. An ELISA study using3F4 antibody showed that PrP(c) (101-130), corresponding sequence to human PrP, was affected by vitamin D 2, supporting the results of Biacore studies and suggesting that the PrP(c) sequence around the 3F4 epitope was responsible for the interaction with vitamin D 2. Furthermore, the effects of vitamin D 2 on disruption of PrP(c) (90-231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D 2, we propose that vitamin D 2 may be a suitable agent to target PrP(c) in the brain and therefore is a potential therapeutic candidate for prion disease.

Concise synthesis of a probe molecule enabling analysis and imaging of vizantin.

Trehalose 6,6'-dicorynomycolate (TDCM) was first characterized in 1963 as a cell surface glycolipid of Corynebacterium spp. by Ioneda and co-workers. TDCM shows potent anti-tumor activity due to its immunoadjuvant properties. Furthermore, the toxicity of TDCM in mice is much weaker than the related trehalose diester of mycolic acid; trehalose 6,6'-dimycolate (TDM, formerly known as cord factor). We have investigated the chemical modification of this class of compound to generate novel agents that display increased immunoadjuvant activity with minimal associated toxicity. During the course of this work we recently developed 6,6'-bis-O-(3-nonyldodecanoyl)-α,α'-trehalose (designated as vizantin). Our results show that vizantin exhibited a potent prophylactic effect on experimental lung metastasis of B16-F0 melanoma cells without a loss of body weight and death in mice. Furthermore, vizantin effectively stimulated human macrophages in an in vitro model, making it a promising candidate for a safe adjuvant in clinical applications. In order to elucidate the pharmacokinetics of vizantin, a probe molecule with similar activity was developed on the basis of a structure-activity relationship (SAR) study with vizantin. The distribution of the probe molecule after intravenous administration into a mouse was assessed by macro confocal microscopy, where it was found to accumulate in the lungs and liver.

Environmental factors preceding aß40 monomer to oligomers and the detection of oligomers in Alzheimer's disease patient serum.

We present here environmental factors including pH shifts, temperature, and metal ions surrounding Aß40 monomer to precede the oligomers. We also suggest a new idea to detect Aß40 oligomers with anti-Aß40 monoclonal antibody using enzyme-linked immunosorbent assay. This method involves the different sensitivity of the thermal shifts between Aß40 monomer and the oligomers. The idea is useful for the diagnostics of Alzheimer's disease to detect Aß40 oligomers in the serum from the patients.

L-tryptophan suppresses rise in blood glucose and preserves insulin secretion in type-2 diabetes mellitus rats.

Ample evidence indicates that a high-protein/low-carbohydrate diet increases glucose energy expenditure and is beneficial in patients with type-2 diabetes mellitus (T2DM). The present study was designed to investigate the effects of L-tryptophan in T2DM. Blood glucose was measured by the glucose dehydrogenase assay and serum insulin was measured with ELISA in both normal and hereditary T2DM rats after oral glucose administration with or without L-D-tryptophan and tryptamine. The effect of tryptophan on glucose absorption was examined in the small intestine of rats using the everted-sac method. Glucose incorporation in adipocytes was assayed with [(3)H]-2-deoxy-D-glucose using a liquid scintillation counter. Indirect computer-regulated respiratory gas-assay calorimetry was applied to assay energy expenditure in rats. L-Tryptophan suppressed both serum glucose and insulin levels after oral glucose administration and inhibited glucose absorption from the intestine. Tryptamine, but not L-tryptophan, enhanced insulin-stimulated [(3)H]-glucose incorporation into differentiated adipocytes. L-Tryptophan increased glucose-associated energy expenditure in rats in vivo. L-Tryptophan-rich chow consumed from a young age preserved the secretion of insulin and delayed the progression of T2DM in hereditary diabetic rats. The results suggested that L-tryptophan suppresses the elevation of blood glucose and lessens the burden associated with insulin secretion from ß-cells.

Reversal of temperature-induced conformational changes in the amyloid-beta peptide, Abeta40, by the beta-sheet breaker peptides 16-23 and 17-24.

BACKGROUND AND PURPOSE: Aggregates of the protein amyloid-beta (Abeta) play a crucial role in the pathogenesis of Alzheimer's disease (AD). Most therapeutic approaches to AD do not target Abeta, so determination of the factor(s) that facilitate aggregation and discovering agents that prevent aggregation have great potential therapeutic value. EXPERIMENTAL APPROACH: We investigated ex vivo the temperature-sensitive regions of Abeta1-40 (Abeta40) and their interactions with octapeptides derived from sequences within Abeta40 -beta-sheet breaker peptides (betaSBP) - using enzyme-linked immunosorbent assay, and dot blot and far-UV circular dichroism (CD) spectroscopy. We measured changes within the physiological limits of temperature, using antibodies targeting epitopes 1-7, 5-10, 9-14 and 17-21 within Abeta40. KEY RESULTS: Temperature-dependent conformational changes were observed in Abeta40 at epitopes 9-14 and 17-21 at 36-38 and 36-40 degrees C respectively. The betaSBPs 16-23 and 17-24, but not 15-22 and 18-25, could inhibit the changes. Moreover, betaSBPs 16-23 and 17-24 increased digestion of Abeta40 by protease K, indicating a decreased aggregation of Abeta40, whereas betaSBPs 15-22 and 18-25 did not increase this digestion. CD spectra revealed that beta-sheet formation in Abeta40 at 38 degrees C was reduced with betaSBPs 16-23 and 17-24. CONCLUSIONS AND IMPLICATIONS: The epitopes 9-14 and 17-21 are the temperature-sensitive regions within Abeta40. The betaSBPs, Abeta16-23 and 17-24 reversed temperature-induced beta-sheet formation, and decreased Abeta40 aggregation. The results suggest that the 17-23 epitope of Abeta40 is crucially involved in preventing Abeta40 aggregation and consequent deposition of Abeta40 in AD brain.

Amyloid-beta causes apoptosis of neuronal cells via caspase cascade, which can be prevented by amyloid-beta-derived short peptides.

Amyloid beta 1-42 (Abeta42) and Abeta17-42 are major constituents of diffuse plaque in brains with Alzheimer's disease (AD). We demonstrate the potent cytotoxicity of Abeta42 and Abeta17-42, lesser toxicity of Abeta1-40 (Abeta40) and lack of toxicity of Abeta1-16 (Abeta16) in neuronal cells as measured by inhibition of cell proliferative response using thymidine incorporation assay and that this cytotoxicity can be reduced with Abeta16 and eight-residue Abeta derivatives such as Abeta1-8 and Abeta9-16. FACS analysis also revealed that Abeta16 could dramatically protect against the apoptosis induced by Abeta17-42 with over 80% viable cells. We determined the caspases involved in the Abeta-mediated apoptotic pathway using caspase-specific inhibitors in MTT assays. For all Abetas, the executor was caspase 3, while the initiator was caspase 9 for Abeta42 and caspase 8 for Abeta40 and Abeta17-42. Microscopic observation of lucifer-yellow-labeled neuronal cells demonstrated the occurrence of lysosomal membrane injury of the cells, corresponding to the severe cytotoxic effects of Abeta42. Our findings suggest that the apoptosis of neuronal cells due to Abeta42, Abeta40 and Abeta17-42 is mediated by the different caspase pathways and that this apoptosis can be reduced with the eight-residue Abeta-derived fragments Abeta1-8, Abeta9-16 and Abeta16.

Eight-residue Abeta peptides inhibit the aggregation and enzymatic activity of Abeta42.

Insoluble Abeta1-42 is the main component of the amyloid plaque. We have previously demonstrated that exposure to low pH can confer the molten globule state on soluble Abeta1-42 in vitro [Biochem. J. 361 (2000) 547] and unfolding experiments with guadinine hydrochloride (GdnHCl) have now confirmed this observation. The molten globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimer's disease (AD). We therefore investigated the ability of a series of synthetic eight-residue peptides derived from Abeta1-42 to inhibit the acid-induced aggregation of Abeta1-42 and identified the potent peptides to be Abeta15-22, Abeta16-23 and Abeta17-24. A1-antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family is another major component of the amyloid plaque. In the present study, we investigated the proteolytic activity of Abeta1-42 against casein at different pHs. Chemical modification of amino acid residues in Abeta1-42 indicated that serine and histidine residues, but not aspartic acid, are necessary for enzymatic activity, suggesting that it is a serine proteinase. Amino acid substitution studies indicate that glutamic acids at positions 11 and 22 participate indirectly in proteolysis and we surmise that amino acid residues 29-42 are required to stabilize the conformer. A study of metal ions suggested that Cu2+ affected the enzymatic activity, but Zn2+ and Fe2+ did not. Interestingly, Abeta14-21 and Abeta15-22 were the only peptides that inhibited the proteolytic activity of Abeta42. Therefore, Abeta15-22 may control both aggregation of Abeta1-42 at acidic pH and its proteolytic activity at neutral pH. Consequently, we suggest that it may be of use in the therapy of Alzheimer's disease.

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors.

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.

Nitric oxide protects against chloroquine resistant Plasmodium yoelii nigeriensis parasites in vitro.

Malaria is a life-threatening disease of global concern. The role of nitric oxide in the clearance of malarial parasites is still under debate. Several reports suggest a possible role for nitric oxide in the protection during initial stages of malarial infection. In the present study, we demonstrate that the nitric oxide in combination with low concentrations of chloroquine controls the parasitaemia in vitro. Activated peritoneal macrophages co-cultured with lipopolysaccharide+interferon-gamma or extracts from Tenospora cordifolia as an immunomodulator promoted nitric oxide production by macrophages. The high concentration of nitric oxide in combination with sub-optimal chloroquine suppressed the parasitaemia in the chloroquine resistant malarial infection. Further, the nitric oxide synthase inhibitor, N(G)-mono-methyl-l-arginine, downregulated nitric oxide production by peritoneal macrophages and the resulting levels of parasitaemia were higher, similar to those of untreated controls. These findings support the proposition that nitric oxide has a crucial role in the control of parasitaemia at the initial periods of blood stage malarial infection.

Conformational changes preceding amyloid-fibril formation of amyloid-beta and stefin B; parallels in pH dependence.

Amyloid beta (A beta) protein is the key component of amyloid plaques in Alzheimer's disease brain whereas stefin B is an intracellular cysteine proteinase inhibitor, broadly distributed in different tissue and recently reported to form amyloid fibrils in vitro. By reducing the pH to 4.6, the native conformation of both polypeptides are changed into less ordered metastable intermediates that are stabilized by formation of the more stable fibrils. In A beta, the Glu at position 11 was found to be responsible for the conformational change at pH 4.6. Metal ions, including copper and zinc, could also induce conformational changes of A beta at neutral pH. The acid modified A beta conformer exhibited protease K resistance, preferential internalization and accumulation in the human glial cells. In stefin B, reducing the pH to pH 3.3 results in another intermediate of the molten-globule type which also leads to amyloid fibril formation. Multiple sequence alignment revealed distinct similarities of A beta (1-42) peptide, stefin B (13 to 61 residues) and prion fragment (90 to 144 residues).