Investigation of anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L): in vitro and in vivo studies / Investigación de la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L): estudios in vitro e in vivo

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Sever cognitive and memory impairments, huge increase in the prevalence of the disease, and lacking definite cure have absorbed worldwide efforts to develop therapeutic approaches. Since many drugs have failed in the clinical trials due to multifactorial nature of AD, symptomatic treatments are still in the center attention and now, nootropic medicinal plants have been found as versatile ameliorators to reverse memory disorders. In this work, anti-Alzheimer's activity of aqueous extract of areca nuts (Areca catechu L.) was investigated via in vitro and in vivo studies. It depicted good amyloid ß (Aß) aggregation inhibitory activity, 82% at 100 µg/mL. In addition, it inhibited beta-secretase 1 (BACE1) with IC50 value of 19.03 µg/mL. Evaluation of neuroprotectivity of the aqueous extract of the plant against H2O2-induced cell death in PC12 neurons revealed 84.5% protection at 1 µg/mL. It should be noted that according to our results obtained from Morris Water Maze (MWM) test, the extract reversed scopolamine-induced memory deficit in rats at concentrations of 1.5 and 3 mg/kg.

La enfermedad de Alzheimer (EA) es un trastorno neurodegenerativo relacionado con la edad. Los severos deterioros cognitivos y de la memoria, el enorme aumento de la prevalencia de la enfermedad y la falta de una cura definitiva han absorbido los esfuerzos mundiales para desarrollar enfoques terapéuticos. Dado que muchos fármacos han fallado en los ensayos clínicos debido a la naturaleza multifactorial de la EA, los tratamientos sintomáticos siguen siendo el centro de atención y ahora, las plantas medicinales nootrópicas se han encontrado como mejoradores versátiles para revertir los trastornos de la memoria. En este trabajo, se investigó la actividad anti-Alzheimer del extracto acuoso de nueces de areca (Areca catechu L.) mediante estudios in vitro e in vivo. Representaba una buena actividad inhibidora de la agregación de amiloide ß (Aß), 82% a 100 µg/mL. Además, inhibió la beta-secretasa 1 (BACE1) con un valor de CI50 de 19,03 µg/mL. La evaluación de la neuroprotección del extracto acuoso de la planta contra la muerte celular inducida por H2O2 en neuronas PC12 reveló una protección del 84,5% a 1 µg/mL. Cabe señalar que, de acuerdo con nuestros resultados obtenidos de la prueba Morris Water Maze (MWM), el extracto revirtió el déficit de memoria inducido por escopolamina en ratas a concentraciones de 1,5 y 3 mg/kg.

The role of GLP-1/GIP receptor agonists in Alzheimer's disease.

BACKGROUND: New glucagon-like peptide-1 (GLP-1) analogues developed in recent years have a long half-life and offer further prospects for clinical application. At present, the neuroprotection of GLP-1 analogues in Alzheimer's disease (AD) has just begun to be explored. OBJECTIVES: To investigate how glucagon-like peptide-1 (liraglutide) plays a protective role in AD by regulating tau activation and BACE1 expression. MATERIAL AND METHODS: Human neuroblastoma cell line SH-SY5Y cells were cultured in vitro and pretreated with different concentrations of liraglutide, and then treated with different concentrations of okadaic acid (OA) in order to observe the apoptosis of the SH-SY5Y cells. After liraglutide treatment, the apoptosis of neurons in AD rats was detected using flow cytometry, and tau activation and ß-site APP cleaving enzyme 1 (BACE1) expression were detected using western blot. RESULTS: Different concentrations of OA were able to induce apoptosis of SH-SY5Y cells in a dose-dependent manner. Different concentrations of liraglutide were used to pretreat SH-SY5Y cells, which were able to protect the SH-SY5Y cells from apoptosis induced by OA. Okadaic acid significantly increased tau activation and BACE1 expression in the SH-SY5Y cells, which was blocked with liraglutide pretreatment. The results of a water maze experiment showed that liraglutide had significant protective effects on memory and cognitive ability in AD rats induced with OA, inhibited apoptosis of neural cells in AD rats, and inhibited tau activation and BACE1 expression of neural cells in AD rats induced with OA. CONCLUSIONS: Liraglutide has a protective effect on AD in vivo and in vitro, which may be mediated by preventing neuronal apoptosis and inhibiting the activation of tau and the expression of BACE1.

Activation of peroxisome proliferator-activated receptor delta suppresses BACE1 expression by up-regulating SOCS1 in a JAK2/STAT1-dependent manner.

Neuronal expression of beta-secretase 1 (BACE1) has been implicated in the progression of Alzheimer's disease. However, the mechanisms that regulate BACE1 expression are unclear. Here, we show that peroxisome proliferator-activated receptor delta (PPARδ) decreases BACE1 expression by up-regulating suppressor of cytokine signaling 1 (SOCS1) in SH-SY5Y neuroblastoma cells. The activation of PPARδ by GW501516, a specific PPARδ agonist, inhibited expression of BACE1. This effect was abrogated by shRNA-mediated knockdown of PPARδ and by treatment with the PPARδ antagonist GSK0660, indicating that PPARδ is involved in GW501516-mediated suppression of BACE1 expression. On the other hand, GW501516-activated PPARδ induced expression of SOCS1, which is a negative regulator of cytokine signal transduction, at the transcriptional level by binding to a PPAR response element in its promoter. This GW501516-mediated induction of SOCS1 expression led to down-regulation of BACE1 expression via inactivation of signal transducer and activator of transcription 1. GW501516-activated PPARδ suppressed the generation of neurotoxic amyloid beta (Aß) in accordance with the decrease in BACE1 expression. Taken together, these results indicate that PPARδ attenuates BACE1 expression via SOCS1-mediated inhibition of signal transducer and activator of transcription 1 signaling, thereby suppressing BACE1-associated generation of neurotoxic Aß.

Impaired flow-induced arterial remodeling in DOCA-salt hypertensive rats.

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) µm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) µm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 µm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) µm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 µm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 µm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 µm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 µm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

Judicialização da saúde na garantia do acesso ao medicamento / The judicialisation of health as a means ensuring access to medicines / La Judicialización de la salud como garantía de acceso a medicamentos

Objetivo Visando conhecer o impacto das demandas judiciais sobre a organização dos serviços públicos de saúde, realizou-se uma revisáo sistemática com enfoque na "judicialização da saúde" para fornecimento de medicamentos. Métodos Foram analisados artigos originais publicados no período de 2007 a 2011, na literatura nacional e internacional, resultando no total de 49239 artigos disponíveis nas bases de dados Science Direct e BIREME. Resultados A pesquisa indicou predominância da bibliografia proveniente do Brasil, principalmente do sudeste, bem como de estudo realizado na Colômbia. Discursáo Dentre os pleitos, configuraram-se como principais agravos relatados as doenças crônicas, podendo-se citar: diabetes, hipertensáo, cânceres e artrite reumatóide. Por serem afecções parte de programas específicos do Sistema Único de Saúde, a dificuldade de acesso a esses fármacos e consequente judicialização da saúde demonstrou a fragilidade das políticas públicas existentes. Conclusão Por fim, conclui-se que a via judicial, apesar de ser uma estratégia para garantir o acesso ao medicamento, apresenta inabilidade para lidar com o julgamento das ações e gera, dessa forma, distorções no fluxo dos sistemas públicos.

Objective A systematic review, focusing on the judicialisation of health regarding gaining access to medicines, was aimed at understanding the impact of lawsuits on the organisation of public health services. Method Original articles published between 2007 and 2011 in the pertinent national and international literature were analysed, resulting in 49,239 articles being found in Science Direct and BIREME databases. Results The survey indicated a predominance of literature from Brazil, mainly the southeast, as well as a study from Colombia. Discussion The aforementioned chronic disease-related claims involved diabetes, high blood pressure, cancer and rheumatoid arthritis. Forming part of specific Unified Healthcare System programmes highlighted the difficulty in gaining access to the appropriate medicine and consequent health judicialisation demonstrated the fragility of existing public policy. Conclusion It was concluded that the courts (despite being a strategy for ensuring access to medicine) were unable to deal with the current spate of lawsuits, thereby leading to disruption regarding the flow of public systems.

Objetivo El estudio tiene como objetivo evaluar el impacto de las demandas judiciales sobre la organización de los servicios públicos de salud, mediante la realización de una revisión sistemática centrada en el uso de los tribunales para el suministro de medicamentos. Método Fueron identificados 49239 artículos en las bases de datos Science Direct e BIREME. Resultado El estudio indicó que la mayor parte de la bibliografía es de Brasil, con uno estudio en Colombia. Discusión Aparecen como los principales trastornos de salud relatados a las enfermedades crónicas, se pueden citar: la diabetes, la hipertensión, el cáncer y la artritis reumatoide. Debido a que son parte de los programas específicos de lo sistema de salud, la dificultad de acceso a estos fármacos y la consiguiente judicialización de la salud de manifiesto la fragilidad de las políticas públicas existentes. Conclusiones Por último, está la conclusión de que los tribunales, a pesar de ser una estrategia para garantizar el acceso a la medicina, presenta incapacidad para hacer frente al juicio de las acciones y por lo tanto genera distorsiones en el flujo de los sistemas públicos.

Oleic acid ameliorates amyloidosis in cellular and mouse models of Alzheimer's disease.

Several lines of evidence support protective as well as deleterious effects of oleic acid (OA) on Alzheimer's disease (AD) and other neurological disorders; however, the bases of these effects are unclear. Our investigation demonstrates that amyloid precursor protein (APP) 695 transfected Cos-7 cells supplemented with OA have reduced secreted amyloid-beta (Aß) levels. An early-onset AD transgenic mouse model expressing the double-mutant form of human APP, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings when they were fed a high-protein, low-fat (18% reduction), cholesterol-free diet enriched with OA. These mice exhibited an increase in Aß40/Aß42 ratio, reduced levels of beta-site APP cleaving enzyme (BACE) and reduced presenilin levels along with reduced amyloid plaques in the brain. The decrease in BACE levels was accompanied by increased levels of a non-amyloidogenic soluble form of APP (sAPPα). Furthermore, the low-fat/+OA diet resulted in an augmentation of insulin-degrading enzyme and insulin-like growth factor-II. These results suggest that OA supplementation and cholesterol intake restriction in a mouse model of AD reduce AD-type neuropathology.

Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases.

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

Trailing end-point phenotype antibiotic-sensitive strains of Candida albicans produce different amounts of aspartyl peptidases

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

The peptidase inhibitor CGS-26303 increases endothelin converting enzyme-1 expression in endothelial cells through accumulation of big endothelin-1.

BACKGROUND AND PURPOSE: CGS-26303 inhibits endothelin converting enzyme (ECE)-1 more specifically than phosphoramidon. We have studied the effect of CGS-26303 on ECE-1 expression in bovine aortic endothelial cells. METHODS: ECE-1 activity and big endothelin (ET)-1 levels were measured by ELISA, ECE-1 expression using western and northern blot and promoter activity using transfection assays. KEY RESULTS: ECE-1 activity was completely inhibited by CGS-26303 25 microM and phosphoramidon 100 microM. CGS-26303 and phosphoramidon, though not thiorphan, a neutral endopeptidase (NEP) inhibitor, stimulated ECE-1 expression in cells (maximal effect at 16 h, 25 microM). Cycloheximide abolished that effect. CGS-26303 induced ECE-1 mRNA expression and ECE-1 promoter activity. CGS-35066, a selective ECE-1 inhibitor, mimicked the effects of CGS-26303, suggesting that the effect was specific to ECE-1 inhibition. Big ET-1 accumulated in the cells and in the supernatants after CGS-26303 treatment. Neither exogenously added ET-1 nor the blockade of their receptors with bosentan modified ECE-1 protein. When big ET-1 was added to cells, significant increases in ECE-1 protein content and ECE-1 promoter activity were found. Bosentan did not block those effects. CGS-26303 did not modify prepro-ET-1 expression. CGS-26303 and big ET-1 induced the same effects in human endothelial cells, at lower doses. CONCLUSIONS: These results suggest that the accumulation of big ET-1 is responsible for the effects of CGS-26303 on ECE-1 and they did not depend on NEP blockade. Changes in ECE-1 protein after the administration of CGS-26303 could lead to a decreased response in long-term treatments.

The inhalation anesthetic isoflurane induces a vicious cycle of apoptosis and amyloid beta-protein accumulation.

The anesthetic isoflurane has been reported to induce apoptosis and increase Abeta generation and aggregation. However, the molecular mechanism underlying these effects remains unknown. We therefore set out to assess whether the effects of isoflurane on apoptosis are linked to amyloid beta-protein (Abeta) generation and aggregation. For this purpose, we assessed the effects of isoflurane on beta-site amyloid beta precursor protein (APP)-cleaving enzyme (BACE) and gamma-secretase, the proteases responsible for Abeta generation. We also tested the effects of inhibitors of Abeta aggregation (iAbeta5, a beta-sheet breaker peptide; clioquinol, a copper-zinc chelator) on the ability of isoflurane to induce apoptosis. All of these studies were performed on naive human H4 neuroglioma cells as well as those overexpressing APP (H4-APP cells). Isoflurane increased the levels of BACE and gamma-secretase and secreted Abeta in the H4-APP cells. Isoflurane-induced Abeta generation could be blocked by the broad-based caspase inhibitor Z-VAD. The Abeta aggregation inhibitors, iAbeta5 and clioquinol, selectively attenuated caspase-3 activation induced by isoflurane. However, isoflurane was able to induce caspase-3 activation in the absence of any detectable alterations of Abeta generation in naive H4 cells. Finally, Abeta potentiated the isoflurane-induced caspase-3 activation in naive H4 cells. Collectively, these findings suggest that isoflurane can induce apoptosis, which, in turn, increases BACE and gamma-secretase levels and Abeta secretion. Isoflurane also promotes Abeta aggregation. Accumulation of aggregated Abeta in the media can then promote apoptosis. The result is a vicious cycle of isoflurane-induced apoptosis, Abeta generation and aggregation, and additional rounds of apoptosis, leading to cell death.

IFN-gamma-induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytes.

Beta-site APP cleaving enzyme 1 (BACE1) is an essential enzyme for the production of beta amyloid. Since we found that injection of interferon-gamma (IFN-gamma) into young mouse brains increased BACE1 expression in astrocytes, we investigated molecular mechanisms underlying this process by cloning a putative BACE1 promoter. BACE1 promoter activity was differentially regulated by IFN-gamma in a region specific manner and down-regulated by an inhibitor of Janus kinase 2 (JAK2). A dominant negative mutant of signal transducer and activator of transcription 1 (STAT1) expression suppressed BACE1 promoter activity, and this was rescued by transfecting wild type STAT1. Electrophoretic mobility shift assay and promoter activity assays indicated that STAT1 binds directly to the putative STAT1 binding sequence of BACE1 promoter. Because IFN-gamma treatment induced STAT1 phosphorylation, we examined whether the expression of a suppressor of cytokine signaling (SOCS), negative regulator of JAK2, suppresses BACE1 promoter activity. The results show that SOCS1 or SOCS3 expression suppressed BACE1 promoter by blocking phosphorylation of Tyr701 residue in STAT1. Also, because IFN-gamma treatment specifically potentiated extracellular signal regulated MAP kinase (ERK) 1/2 activation, pretreatment of mitogen-activated or extracellular signal-regulated protein kinase (MEK) inhibitor, PD98059, significantly attenuated IFN-gamma-induced BACE1 promoter activity and protein expression through blocking phosphorylation of Ser727 residue in STAT1, suggesting that ERK1/2 is associated with IFN-gamma-induced STAT1 signaling cascade. Taken together, our results suggest that IFN-gamma activates JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region to modulate BACE1 protein expression in astrocytes.

Up-regulation in endothelin-1 by Helicobacter pylori lipopolysaccharide interferes with gastric mucin synthesis via epidermal growth factor receptor transactivation.

OBJECTIVE: Endothelin-1 (ET-1), a key mediator of inflammatory processes associated with bacterial infection, is a 21-amino acid peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1) that acts through G protein-coupled ET(A) and ET(B) receptors. Here we report on the role of ET-1 in the mediation of the detrimental influence of Helicobacter pylori on the synthesis of gastric mucin. MATERIAL AND METHODS: Rat gastric mucosal cells were exposed to H. pylori key virulence factor, lipopolysaccharide (LPS). RESULTS: The LPS inhibitory effect on gastric mucin synthesis was accompanied by a marked increase in ET-1 generation and enhancement in ECE-1 activity. Inhibition of ECE-1 with phosphoramidon not only led to the impedance of LPS-induced ET-1 generation, but also countered the detrimental effect of LPS on mucin synthesis. Moreover, the LPS inhibitory effect on mucin synthesis was blocked by ET(A) receptor antagonist BQ610, but not by ET(B) receptor antagonist BQ788. Furthermore, the LPS-induced suppression in gastric mucin synthesis was countered in a concentration-dependent fashion by PD153035 (81.7%), a specific inhibitor of epidermal growth factor receptor (EGFR) kinase as well as PP2 (69.8%), a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR transactivation. CONCLUSIONS: Our findings are the first to show that the detrimental effect of H. pylori on gastric mucin synthesis is intimately linked to the events associated with ECE-1 up-regulation, enhancement in ET-1 production, and G protein-coupled ET(A) receptor activation that triggers the EGFR transactivation.

Altered intracellular signaling and reduced viability of Alzheimer's disease neuronal cybrids is reproduced by beta-amyloid peptide acting through receptor for advanced glycation end products (RAGE).

Activation of apoptosis by increased production of amyloid beta peptides (Abeta) has been implicated in neuronal cell death of Alzheimer's disease (AD). We used mitochondrial transgenic cybrid models of sporadic AD (SAD), which overproduce Abeta compared to control (CTL) cybrids, to investigate the effects of endogenously generated Abeta on intracellular signaling pathways and viability. Reducing SAD Abeta production with gamma-secretase inhibition altered the total phosphorylation profile of SAD cybrid to one similar to CTL cybrids and enhanced viability to approximately CTL cybrid levels. Treating CTL cybrids with exogenous Abeta or conditioned media (CM) from SAD cybrids activated the signaling pathways active in SAD cybrids under basal condition and decreased viability. Antibodies against receptor for advanced glycation end products (RAGE) blocked Abeta-induced activation of the p38, JNK pathways, and NF-kappaB in CTL cybrids and offered protection against the neurotoxic effects of Abeta. Expression of SAD mitochondrial genes in cybrids activates stress-related signaling pathways and reduces viability. This SAD phenotype is produced by endogenously generated Abeta and can be replicated by exogenous Abeta acting through RAGE.

Amyloid precursor protein compartmentalization restricts beta-amyloid production: therapeutic targets based on BACE compartmentalization.

Alzheimer's disease (AD) is defined by deposits of the 42-residue amyloid-beta peptide (Abeta42) in the brain. Abeta42 is a minor metabolite of the amyloid precursor protein (APP), but its relative levels are increased by mutations on APP and presenilins 1 and 2 linked to familial AD. beta-secretase (BACE-1), an aspartyl protease, cleaves approx 10% of the APP in neuronal cells on the N-terminal side of Abeta to produce the C-terminal fragment (CTFbeta), which is cleaved by gamma-secretase to produce mostly Abeta of 40 residues (90%) and approx10% Abeta42. A third enzyme, alpha-secretase, cleaves APP after Abeta16 to secrete sAPPalpha and CTFalpha, the major metabolites of APP. Moreover, previous studies have demonstrated that phorbol esters stimulate processing of APP by alpha-secretase. Because alpha-secretase and BACE-1 cleave APP within the secretory pathway, it is likely that the two enzymes compete for the APP substrate. This type of competition can explain the failure to saturate the minor BACE-1 pathway by overexpressing APP in the cell. In this study, we demonstrate that inhibition of constitutive alpha-secretase processing in a human neuroblastoma cell line does not increase the yield of Abeta, suggesting that the APP substrate targeted for alpha-secretase processing is not diverted to the BACE-1 pathway. However, when phorbol ester-induced alpha-secretase was similarly inhibited, we detected an increase in BACE-1 processing and AB yield. We explain these results compartmentalization of BACE-1 and alpha-secretase with processing depending on sorting of APP to the two compartments. The simplest explanation for the detection of competition between the two pathways upon phorbol ester stimulation is the partial failure of this compartmentalization by phorbol ester-induced release of secretory vesicles.

Salt-stress induced alterations in protein profile and protease activity in the mangrove Bruguiera parviflora.

Two-month-old seedlings of Bruguiera parvifora were treated with varying levels of NaCl (100, 200 and 400 mM) under hydroponic culture. Total proteins were extracted from leaves of control and NaCl treated plants after 7, 14, 30 and 45 d of treatment and analysed by SDS-PAGE. As visualized from SDS-PAGE, the intensity of several protein bands of molecular weight 17, 23, 32, 33 and 34 kDa decreased as a result of NaCl treatment. The degree of decrease of these protein bands seemed to be roughly proportional to the external NaCl concentration. The most obvious change concerned a 23 kDa-polypeptide (SSP-23), which disappeared after 45 d treatment in 400 mM NaCl. Moreover, the SSP-23 protein, which disappeared in B. parviflora under salinity stress, reappeared when these salinized seedlings were desalinized. These observations suggest the possible involvement of these polypeptides for osmotic adjustment under salt stress. NaCl stress also caused an increase in the activity of both acid and alkaline protease. The increasing activity of proteases functions as a signal of salt stress in B. parviflora, which induces the reduction of protein level.

Nonsteroidal anti-inflammatory drugs and peroxisome proliferator-activated receptor-gamma agonists modulate immunostimulated processing of amyloid precursor protein through regulation of beta-secretase.

Long-term treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk for Alzheimer's disease (AD). To determine the mechanisms by which inflammation affects AD and how NSAIDs protect against it, we stimulated neuroblastoma cells stably transfected with amyloid precursor protein (APP) with proinflammatory cytokines, which increased the secretion of amyloid-beta and APP ectodomain. Addition of ibuprofen, indomethacin, peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists, or cotransfection with PPARgamma cDNA reversed this effect. The inhibitory action of ibuprofen and indomethacin was suppressed by PPARgamma antagonists. Finally, we observed that the mRNA levels, expression, and enzymatic activity of beta-secretase were increased by immunostimulation and normalized by NSAIDs. In conclusion, proinflammatory cytokines activate beta-secretase, and NSAIDs inhibit this effect through PPARgamma.

Effect of mifepristone on pregnancy, pregnancy-specific protein B (PSPB), progesterone, estradiol-17beta, prostaglandin F2alpha (PGF2alpha) and prostaglandin E (PGE) in ovariectomized 90-day pregnant ewes.

The objective of this experiment was to determine the effect of mifepristone, a progesterone receptor antagonist, on pregnancy and secretion of steroids, pregnancy-specific protein B (PSPB) and prostaglandins at mid-pregnancy in ewes. Ninety-day pregnant ewes were ovariectomized (OVX) and treatments were initiated 72 h post-OVX. Ewes received (1) vehicle, (2) prostaglandin F2alpha (PGF2alpha, 8 mg/58 kg/bw, i.m.) 84 h post-OVX, (3) mifepristone (50 mg intrajugular at 72, 84, 96, and 108 h post-OVX), (4) mifepristone (50mg) + PGF2alpha, (5) mifepristone (100 mg intrajugular at 72, 84, 96, and 108 h), and (6) mifepristone (100 mg) + PGF2alpha. Ewes treated with vehicle or PGF2alpha alone did not abort (P > or = 0.05). But, 60, 80, 60, and 100% of ewes treated with mifepristone (50 mg), mifepristone (50 mg) + PGF2alpha, mifepristone (100 mg), and mifepristone (100 mg) + PGF2alpha, respectively, aborted (P < or = 0.05). Profiles of progesterone, estradiol-17beta, prostaglandin E (PGE), or PSPB did not differ (P > or = 0.05) among treatment groups. Profiles of PGF2alpha of treatment groups receiving mifepristone with or without PGF2alpha differed (P < 0.05) from vehicle or PGF2alpha alone-treated ewes. It is concluded that progesterone actions are necessary to suppress uterine/placental secretion of PGF2alpha and that maintenance of critical progesterone: estradiol-17beta and PGE:PGF2alpha ratios are necessary for maintenance of pregnancy.

HTLV-I protease cleavage of P19/24 substrates is not dependent on NaCl concentration.

Understanding the factors that affect the activity of Human T-cell Leukemia Virus type I (HTLV-I) protease is essential for the discovery of inhibitors to be used for the treatment of HTLV-I infection, but little has been reported on the protease to date. Here we report the production of HTLV-I protease in purified yields greater than 150 mg/L, determination of its extinction coefficient, and determination of the optimum conditions for cleavage of the p19/24 substrates (DABCYL)-(GABA)-PQVL-Nph-VMH-(EDANS), (DABSYL)-(GABA)-PQVL-Nph-VMH-(EDANS), and (DABSYL)-(GABA)-PQVLPVMH-(EDANS). The highest activity was found at pH 5.2-5.3 and 37 degrees C. There was no effect on activity upon change in sodium chloride concentration from 0 to 1500 mM. The values of K(m) and k(cat) for cleavage of these substrates by the protease with and without the histidine tag were determined.

HIV-1 Vif-derived peptide inhibits drug-resistant HIV proteases.

Vif, one of the six accessory genes expressed by HIV-1, is essential for the productive infection of natural target cells. Previously we suggested that Vif acts as a regulator of the viral protease (PR): It prevents the autoprocessing of Gag and Gag-Pol precursors until virus assembly, and it may control the PR activity in the preintegration complex at the early stage of infection. It was demonstrated before that Vif, and specifically the 98 amino acid stretch residing at the N'-terminal part of Vif (N'-Vif), inhibits both the autoprocessing of truncated Gag-Pol polyproteins in bacterial cells and the hydrolysis of synthetic peptides by PR in cell-free systems. Linear synthetic peptides derived from N'-Vif specifically inhibit and bind HIV-1 PR in vitro, and arrest virus production in tissue culture. Peptide mapping of N'-Vif revealed that Vif88-98 is the most potent PR inhibitor. Here we report that this peptide inhibits both HIV-1 and HIV-2, but not ASLV proteases in vitro. Vif88-98 retains its inhibitory effect against drug-resistant HIV-1 PR variants, isolated from patients undergoing long-term treatment with anti-PR drugs. Variants of HIV protease bearing the mutation G48V are resistant to inhibition by this Vif-derived peptide, as shown by in vitro assays. In agreement with the in vitro experiments, Vif88-98 has no effect on the production of infectious particles in cells infected with a G48V mutated virus.

Increase of Candida cell virulence by anticancer drugs and irradiation.

The influence of anticancer drugs and irradiation on Candida cell proliferation, adherence to HeLa cells and susceptibility to antifungal drugs (amphotericin B and miconazole) and neutrophils were examined using two Candida albicans strains. After treatment with 5-fluorouracil (25 microg/ml to 250 microg/ml), cis-diammine-dichloroplatinum (10 microg/ml to 100 microg/ml), peplomycin (0.5 microg/ml to 5 microg/ml) or 137Cs (20 Gy to 40 Gy) for 3 days or more, surviving Candida cells proliferated more rapidly than did untreated control cells. Anticancer agent-pretreated Candida cells revealed an increased adhesion to HeLa cells corresponding to an increase of binding to the lectins. The concentration of half limited colony formation (IC50) of amphotericin B and miconazole was increased to near two-fold that of the control by pretreatment of Candida cells with the anticancer agents, except peplomycin, which only weakly increased IC50. In addition, the enolase and Candida acid proteinase activities in the culture supernatants were increased by pretreatment with the drugs and irradiation. Correspondingly, surviving Candida cells after these treatments were resistant to neutrophils, with a reduction to half of the killing. These results indicate that anti-cancer drugs and irradiation potentiate the virulence of Candida cells, or they eliminate Candida cells with low virulence, thereby enhancing the risk of oral and systemic candidiasis.