Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU): Trial Satisfaction and Attitudes towards Future Clinical Trials.

BACKGROUND: Clinical trial satisfaction is increasingly important for future trial designs and is associated with treatment adherence and willingness to enroll in future research studies or to recommend trial participation. In this post-trial survey, we examined participant satisfaction and attitudes toward future clinical trials in the Dominantly Inherited Alzheimer Network Trials Unit (DIAN-TU). METHODS: We developed an anonymous, participant satisfaction survey tailored to participants enrolled in the DIAN-TU-001 double-blind clinical trial of solanezumab or gantenerumab and requested that all study sites share the survey with their trial participants. A total of 194 participants enrolled in the trial at 24 study sites. We utilized regression analysis to explore the link between participants' clinical trial experiences, their satisfaction, and their willingness to participate in upcoming trials. RESULTS: Survey responses were received over a sixteen-month window during 2020-2021 from 58 participants representing 15 study sites. Notably, 96.5% of the survey respondents expressed high levels of satisfaction with the trial, 91.4% would recommend trial participation, and 96.5% were willing to enroll again. Age, gender, and education did not influence satisfaction levels. Participants reported enhanced medical care (70.7%) and pride in contributing to the DIAN-TU trial (84.5%). Satisfaction with personnel and procedures was high (98.3%). Respondents had a mean age of 48.7 years, with most being from North America and Western Europe, matching the trial's demographic distribution. Participants' decisions to learn their genetic status increased during the trial, and most participants endorsed considering future trial participation regardless of the DIAN-TU-001 trial outcome. CONCLUSION: Results suggest that DIAN-TU-001 participants who responded to the survey exhibited high motivation to participate in research, overall satisfaction with the clinical trial, and willingness to participate in research in the future, despite a long trial duration of 4-7 years with detailed annual clinical, cognitive, PET, MRI, and lumbar puncture assessments. Implementation of features that alleviate barriers and challenges to trial participation is like to have a high impact on trial satisfaction and reduce participant burden.

Multiomics Blood-Based Biomarkers Predict Alzheimer's Predementia with High Specificity in a Multicentric Cohort Study.

BACKGROUND: The primary criteria for diagnosing mild cognitive impairment (MCI) due to Alzheimer's Disease (AD) or probable mild AD dementia rely partly on cognitive assessments and the presence of amyloid plaques. Although these criteria exhibit high sensitivity in predicting AD among cognitively impaired patients, their specificity remains limited. Notably, up to 25% of non-demented patients with amyloid plaques may be misdiagnosed with MCI due to AD, when in fact they suffer from a different brain disorder. The introduction of anti-amyloid antibodies complicates this scenario. Physicians must prioritize which amyloid-positive MCI patients receive these treatments, as not all are suitable candidates. Specifically, those with non-AD amyloid pathologies are not primary targets for amyloid-modifying therapies. Consequently, there is an escalating medical necessity for highly specific blood biomarkers that can accurately detect pre-dementia AD, thus optimizing amyloid antibody prescription. OBJECTIVES: The objective of this study was to evaluate a predictive model based on peripheral biomarkers to identify MCI and mild dementia patients who will develop AD dementia symptoms in cognitively impaired population with high specificity. DESIGN: Peripheral biomarkers were identified in a gene transfer-based animal model of AD and then validated during a retrospective multi-center clinical study. SETTING: Participants from 7 retrospective cohorts (US, EU and Australia). PARTICIPANTS: This study followed 345 cognitively impaired individuals over up to 13 years, including 193 with MCI and 152 with mild dementia, starting from their initial visits. The final diagnoses, established during their last assessments, classified 249 participants as AD patients and 96 as having non-AD brain disorders, based on the specific diagnostic criteria for each disorder subtype. Amyloid status, assessed at baseline, was available for 82.9% of the participants, with 61.9% testing positive for amyloid. Both amyloid-positive and negative individuals were represented in each clinical group. Some of the AD patients had co-morbidities such as metabolic disorders, chronic diseases, or cardiovascular pathologies. MEASUREMENTS: We developed targeted mass spectrometry assays for 81 blood-based biomarkers, encompassing 45 proteins and 36 metabolites previously identified in AAV-AD rats. METHODS: We analyzed blood samples from study participants for the 81 biomarkers. The B-HEALED test, a machine learning-based diagnostic tool, was developed to differentiate AD patients, including 123 with Prodromal AD and 126 with mild AD dementia, from 96 individuals with non-AD brain disorders. The model was trained using 70% of the data, selecting relevant biomarkers, calibrating the algorithm, and establishing cutoff values. The remaining 30% served as an external test dataset for blind validation of the predictive accuracy. RESULTS: Integrating a combination of 19 blood biomarkers and participant age, the B-HEALED model successfully distinguished participants that will develop AD dementia symptoms (82 with Prodromal AD and 83 with AD dementia) from non-AD subjects (71 individuals) with a specificity of 93.0% and sensitivity of 65.4% (AUROC=81.9%, p<0.001) during internal validation. When the amyloid status (derived from CSF or PET scans) and the B-HEALED model were applied in association, with individuals being categorized as AD if they tested positive in both tests, we achieved 100% specificity and 52.8% sensitivity. This performance was consistent in blind external validation, underscoring the model's reliability on independent datasets. CONCLUSIONS: The B-HEALED test, utilizing multiomics blood-based biomarkers, demonstrates high predictive specificity in identifying AD patients within the cognitively impaired population, minimizing false positives. When used alongside amyloid screening, it effectively identifies a nearly pure prodromal AD cohort. These results bear significant implications for refining clinical trial inclusion criteria, facilitating drug development and validation, and accurately identifying patients who will benefit the most from disease-modifying AD treatments.

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles.

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV-vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

Raman Imaging: An Impending Approach Towards Cancer Diagnosis.

In accordance with the recent studies, Raman spectroscopy is well experimented as a highly sensitive analytical and imaging technique in biomedical research, mainly for various disease diagnosis including cancer. In comparison with other imaging modalities, Raman spectroscopy facilitate numerous assistances owing to its low background signal, immense spatial resolution, high chemical specificity, multiplexing capability, excellent photo stability and non-invasive detection capability. In cancer diagnosis Raman imaging intervened as a promising investigative tool to provide molecular level information to differentiate the cancerous vs non-cancerous cells, tissues and even in body fluids. Anciently, spontaneous Raman scattering is very feeble due to its low signal intensity and long acquisition time but new advanced techniques like coherent Raman scattering (CRS) and surface enhanced Raman scattering (SERS) gradually superseded these issues. So, the present review focuses on the recent developments and applications of Raman spectroscopy-based imaging techniques for cancer diagnosis.

Elucidating a Thermoresponsive Multimodal Photo-Chemotherapeutic Nanodelivery Vehicle to Overcome the Barriers of Doxorubicin Therapy.

In an attempt to circumvent the major pitfalls associated with conventional chemotherapy including drug resistance and off-target toxicity, we have adopted a strategy to simultaneously target both mitochondrial DNA (Mt-DNA) and nuclear DNA (n-DNA) with the aid of a targeted theranostic nanodelivery vehicle (TTNDV). Herein, folic acid-anchored p-sulfo-calix[4]arene (SC4)-capped hollow gold nanoparticles (HGNPs) were meticulously loaded with antineoplastic doxorubicin (Dox) and its mitochondrion-targeted analogue, Mt-Dox, in a pretuned ratio (1:100) for sustained thermoresponsive release of cargo. This therapeutic strategy was enabled to eradicate both n-DNA and Mt-DNA leaving no space to develop drug resistance. The SC4-capped HGNPs (HGNPSC4) were experimented for the first time as a photothermal (PTT) agent with 61.6% photothermal conversion efficiency, and they generated tunable localized heat more efficiently than bare HGNPs. Moreover, the cavity of SC4 facilitated the formation of an inclusion complex with folic acid to target the folate receptor expressing cancer cells and imparted enhanced biocompatibility. The as-synthesized TTNDV was demonstrated to be an ideal substrate for surface-enhanced Raman scattering (SERS) to monitor the molecular-level therapeutic progression in cells and a spheroidal model. A significant reduction in the tumor mass with a marked survival benefit was achieved in syngraft murine models through this synergistic photo-chemotherapy. Collectively, this multifunctional nanoplatform offers a robust approach to treat cancer without any scope of generating Dox resistance and off-target toxicity.

Exploring Mitochondria-Mediated Intrinsic Apoptosis by New Phytochemical Entities: An Explicit Observation of Cytochrome c Dynamics on Lung and Melanoma Cancer Cells.

Hydnocarpin (Hy) is a flavonoid isolated and purified from the seeds of Hydnocarpus wightiana Blume. Herein, we have developed a built-in semi-synthetic modification on Hy by one-pot multi-component reaction and a [3 + 2] cycloaddition strategy to append five membered isoxazole and isoxazolone as new phytochemical entities (NPCEs). Two selected NPCEs viz Hy-ISO-VIII and Hy-ISO-G from the library of 20 newly synthesized derivatives after in vitro screening unveiled promising cytotoxicity and induced caspase-mediated apoptosis against the human lung and melanoma cancer cells which were well supported by virtual screening based on ligand binding affinity and molecular dynamic simulations. As a new insight, we introduced surface-enhanced Raman spectroscopy to identify the chemo-marker molecular fingerprint to confirm the cellular uptake, cytochrome c release, and DNA fragmentation in a label-free manner. The present findings throw up a surfeit of seminal reasons behind the semi-synthetic modification of Hy, stepping forward to cancer chemotherapy.

Curcuma raktakanda Induces Apoptosis and Suppresses Migration in Cancer Cells: Role of Reactive Oxygen Species.

Although over 100 species of Curcuma are reported, only Curcuma longa is extensively studied. Curcuma raktakanda, a poorly studied species, is most commonly distributed in the Kerala state of India. For the first time, we examined the efficacy of different fractions (acetone, hexane, and ethyl acetate) of C. raktakanda against glioma, cervical, and breast cancer cell lines. As determined by mitochondrial reductase activity assay, the viability of cancer cells was decreased in a concentration-dependent manner by the three fractions. The half maximal inhibitory concentration (IC-50) values after the treatment of C-6 glioma cells for 48 h was found to be 32.97 µg/mL (acetone extract), 40.63 µg/mL (hexane extract), and 51.65 µg/mL (ethyl acetate extract). Of the three fractions, the acetone fraction was more effective. The long-term colony formation of cancer cells was significantly suppressed by the acetone fraction. Analyses using DAPI (4',6-diamidino-2-phenylindole) staining, AO/PI (acridine orange/propidium iodide) staining, DNA laddering, and sub-G1 population revealed that the acetone extract induced apoptosis in glioma cells. The extract induced reactive oxygen species generation and suppressed the expression of cell survival proteins. The migration of cancer cells was also suppressed by the acetone extract. The gas chromatography-mass spectrometry (GC-MS) analysis indicated that tetracontane, dotriacontane, hexatriacontane, pentacosane, hexacosane, and eicosane are the major components in the acetone extract. Collectively, the extract from C. raktakanda exhibited anti-carcinogenic activities in cancer cells. We are exploring whether the phytoconstituents, individually, or collectively contribute to the anti-cancer activities of C. raktakanda.

Exploring the margins of SERS in practical domain: An emerging diagnostic modality for modern biomedical applications.

Excellent multiplexing capability, molecular specificity, high sensitivity and the potential of resolving complex molecular level biological compositions augmented the diagnostic modality of surface-enhanced Raman scattering (SERS) in biology and medicine. While maintaining all the merits of classical Raman spectroscopy, SERS provides a more sensitive and selective detection and quantification platform. Non-invasive, chemically specific and spatially resolved analysis facilitates the exploration of SERS-based nano probes in diagnostic and theranostic applications with improved clinical outcomes compared to the currently available so called state-of-art technologies. Adequate knowledge on the mechanism and properties of SERS based nano probes are inevitable in utilizing the full potential of this modality for biomedical applications. The safety and efficiency of metal nanoparticles and Raman reporters have to be critically evaluated for the successful translation of SERS in to clinics. In this context, the present review attempts to give a comprehensive overview about the selected medical, biomedical and allied applications of SERS while highlighting recent and relevant outcomes ranging from simple detection platforms to complicated clinical applications.

Flies blown disease--oral myiasis.

Myiasis, a term first introduced by Hope (1840), refers to the invasion of tissues and organs of animals and human wounds and certain body cavities by the dipteran larvae which manifests as subcutaneous furunculoid or boillike lesions. Oral myiasis is a rare pathology and a risk to the patient's life. Higher incidence is seen in rural areas affecting the tropical and sub-tropical zones of Africa and America. Myiasis affecting the oro-dental complex is rare. Here is a case report of oral myiasis in an 18-year-old male patient who is mentally challenged with anterior open bite, incompetent lips, and periodontal disease. The lesion was treated with turpentine oil, which forced larvae out and irrigated with normal saline solution. Follow-up examination revealed complete remission and healing of the lesion.

Effect of losartan and enalapril on cognitive deficit caused by Goldblatt induced hypertension.

The present study was designed to evaluate the learning and memory, in an altered physiological state associated with increased blood pressure and activated renin angiotensin system in Wistar rats. The role of angiotensin in cognitive function was assessed by treatment with angiotensin converting enzyme (ACE) inhibitor enalapril (2 mg/kg), angiotensin 1 receptor (AT(1)) antagonist losartan (5 mg/kg) and their combination. The experimental renal hypertension was induced by the method of Goldblatt. Learning and memory was assessed using the radial arm maze test. Acetylcholine esterase (AChE) levels in the pons medulla, hippocampus, striatum and frontal cortex were measured as a cholinergic marker of learning and memory. Results indicate that in comparison to normotensive rats, renal hypertensive rats committed significantly higher number of errors and took more trials and days to learn the radial arm maze learning and exhibited memory deficit in the radial arm maze retrieval after two weeks of retention interval, indicating impaired acquisition and memory. Treatment with enalapril, losartan and their combination attenuated the observed memory deficits indicating a possible role of renin angiotensin system in cognitive function. AChE level was reduced in hippocampus and frontal cortex of renal hypertensive rats which could be attributed to the observed memory deficit in hypertensive rats. It can be concluded that, renal hypertensive rats had a poor acquisition, retrieval of the learned behavior, perhaps a possible disturbance in memory consolidation process and that this state was reversed with ACE inhibitor enalapril and AT 1 receptor antagonist losartan.

Age at focal epilepsy onset varies by sex and hemispheric lateralization.

BACKGROUND: Previous studies have shown that interictal epileptiform discharges favor the left hemisphere in adults but the right side in children up until age 5. This may be due to sex-influenced asymmetric brain maturation. To clarify this relationship, the authors analyzed age at epilepsy onset by sex and by lateralization of epileptiform activity. METHODS: An adult epilepsy center long-term monitoring database was used to define patients with exclusively unilateral epileptiform findings. Three groups were studied: any epileptiform activity (n = 404), ictal activity (n = 287), and interictal activity (n = 265). The second and third groups were drawn from the first group and the second and third groups overlapped with each other. Side of lateralized finding and sex were analyzed via factorial two-way analysis of variance with the outcome variable being age at epilepsy onset. Comparison analysis included patients with generalized epilepsy (n = 114), nonepileptic seizures (NES, n = 232), and surgical mesial temporal sclerosis (MTS, n = 116). RESULTS: Patients with unilateral epileptiform activity displayed bimodal epilepsy onset ages with infant and adolescent peaks. For patients with a right-sided focus, epilepsy onset was earlier in men (14.4 years) than women (20.7 years). In contrast, among patients with a left-sided focus, epilepsy began earlier in women (18.2 years) than men (19.9 years, p < 0.01). Parallel results were found in unilateral ictal (p < 0.01) and unilateral interictal activity (p = 0.01). Patients with surgical MTS, NES, or generalized seizure showed no similar patterns. CONCLUSIONS: In adult patients with focal epilepsy, sex and lateralized epileptiform abnormalities may be related to age at epilepsy onset.

Assessment of cisplatin-induced nephrotoxicity by microarray technology.

Microarrays are a new technology used to study global gene expression and to decipher biological pathways. In the current study, microarrays were used to examine gene expression patterns associated with cisplatin-mediated nephrotoxicity. Sprague-Dawley rats received either single or seven daily ip doses of cisplatin (0.5 or 1 mg/kg/day) or the inactive isomer transplatin (1 or 3 mg/kg/day). Histopathological evaluation revealed renal proximal tubular necrosis in animals that received cisplatin for 7 days, but no hepatotoxic findings. Microarray analyses were performed using rat specific arrays containing 250 toxicity-related genes. Prominent gene expression changes were observed only in the kidneys of rats that received cisplatin for 7 days. Mechanistically, the gene expression pattern elicited by cisplatin (e.g., Bax upward arrow and SMP-30 downward arrow) suggested the occurrence of apoptosis and the perturbation of intracellular calcium homeostasis. The induction of multidrug resistance genes (MDR1 upward arrow, P-gp upward arrow) and tissue remodeling proteins (clusterin upward arrow, IGFBP-1 upward arrow, and TIMP-1 upward arrow) indicated the development of cisplatin resistance and tissue regeneration. Select gene expression changes were further confirmed by TaqMan analyses. Gene expression changes were not observed in the liver following cisplatin administration. In contrast to these in vivo findings, studies using NRK-52E kidney epithelial cells and clone-9 liver cells suggested that liver cells were more sensitive to cisplatin treatment. The discrepancies between the in vivo and in vitro results suggest that caution should be taken when extrapolating data from in vivo to in vitro systems. Nonetheless, the current study elucidates the biochemical pathways involved in cisplatin toxicity and demonstrates the utility of microarrays in toxicological studies.

Elevated ceramide is downstream of altered calcium homeostasis in low serum-induced apoptosis.

Two immortalized cell lines, sup (+) and sup (-), derived from mutagenized Syrian hamster embryo cells, were used to study the relationship and temporal order between calcium and ceramide signals during apoptosis. The early preneoplastic cells, termed sup (+), suppress tumorigenicity when hybridized with tumor cells, whereas later-stage sup (-) cells do not. In reduced serum conditions, sup (+) cells cease proliferating and undergo apoptosis; in contrast, sup (-) cells continue slow growth and undergo necrosis. In sup (+) cells, decreased endoplasmic reticulum (ER) calcium occurs 4 h after low serum treatment and precedes apoptosis. Significant elevations in ceramide are observed 16 h after reduced serum treatment of sup (+) cells but are not found in sup (-) cells. Inhibiting ER calcium depletion in low serum-treated sup (+) cells by treating with high levels of calcium prevents both ceramide generation and apoptosis. Conversely, inducing ER calcium depletion in sup (-) cells by treating with low serum plus thapsigargin results in elevated ceramide levels and apoptosis. Furthermore, C(6)-ceramide treatment induced apoptosis of sup (-) cells in low serum, a condition that does not normally cause apoptosis. C(6)-ceramide treatment did not induce apoptosis in either sup (+) or sup (-) cells in 10% serum but did cause G(2)/M arrest. These studies show that ceramide production is downstream of ER calcium release.

Homologous and heterologous phosphorylation of the AT(2) angiotensin receptor by protein kinase C.

The angiotensin AT(2) receptor is an atypical seven transmembrane domain receptor that is coupled to activation of tyrosine phosphatase and inhibition of MAP kinase, and does not undergo agonist-induced internalization. An investigation of the occurrence and nature of AT(2) receptor phosphorylation revealed that phorbol ester-induced activation of protein kinase C (PKC) in HA-AT(2) receptor-expressing COS-7 cells caused rapid and specific phosphorylation of a single residue (Ser(354)) located in the cytoplasmic tail of the receptor. Agonist activation of AT(2) receptors by angiotensin II (Ang II) also caused rapid PKC-dependent phosphorylation of Ser(354) that was prevented by the AT(2) antagonist, PD123177, and by inhibitors of PKC. In cells coexpressing AT(1) and AT(2) receptors, Ang II-induced phosphorylation of the AT(2) receptor was reduced by either PD123177 or the AT(1) receptor antagonist, DuP753, and was abolished by treatment with both antagonists or with PKC inhibitors. These findings indicate that the AT(2) receptor is rapidly phosphorylated via PKC during homologous activation by Ang II, and also undergoes heterologous PKC-dependent phosphorylation during activation of the AT(1) receptor. The latter process may regulate the counteracting effects of AT(2) receptors on growth responses to AT(1) receptor activation.

The oncostatic action of melatonin in an ovarian carcinoma cell line.

Melatonin is reported to reduce proliferation in many cell types, but the effect is small and the results are inconsistent. Information on the mechanism by which melatonin exerts its antiproliferative effects might provide insight into the variability of the response. In an ovarian adenocarcinoma cell line (BG-1), we find that melatonin at concentrations of 10(-9)-10(-7) M caused a 20-25% reduction in cell number. Melatonin also resulted in a similar reduction in [3H]-thymidine incorporation with no significant increase in cell death as measured by trypan blue incorporation. The Kd for melatonin reduction in cell number was approximately 5 x 10(-10) M. Melatonin ML2 receptors have a Kd for melatonin binding in the low nM range and are linked to the production of the calcium mobilizing agent inositol-1,4,5-trisphosphate (IP3). To investigate whether melatonin signaling involves an increase in cytosolic-free calcium. BG-1 cells were loaded with the calcium sensitive indicator, fura-2. Acute addition of melatonin (10(-5)-10(-9) M) did not alter cytosolic calcium. Addition of the putative nuclear receptor agonist CGP52608 caused a dose-dependent inhibition of cell number with a Kd of approximately 2 x 10(-9) M. Addition of CGP52608 caused a similar reduction in [3H]-thymidine incorporation. Neither melatonin (10(-8) M-10(-5) M) nor CGP52608 at concentrations below 10(-7) M induced cell death associated with the inhibition of cell proliferation; however, addition of CGP52608 at a high dose (10(-7) M) caused an increase in cell death, consistent with apoptosis. Growth inhibition by melatonin or CGP52608 did not alter the percentage of cells in G1 versus S/G2/M.

N-linked glycosylation is required for optimal AT1a angiotensin receptor expression in COS-7 cells.

The nature and role of glycosylation in AT1 angiotensin receptor (AT1-R) function were investigated by expressing glycosylation-deficient influenza hemagglutinin (HA) epitope-tagged rat AT1a-Rs (HA-AT1a-Rs) in COS-7 cells. All three asparagine residues (Asn4, Asn176, Asn188) contained within consensus sites for N-linked glycosylation could be glycosylated in Cos-7 cells and appeared to be glycosylated on the endogenous AT1-R in bovine adrenal glomerulosa cells. Heterogeneity of glycosylation at each site accounted for the broad migration pattern of the AT1-R in SDS-PAGE. Mutation at each glycosylation site, either alone or in combination, had little effect on ligand binding parameters (although the N4K mutant had higher affinity) or signaling activity. However, an increasing number of mutated glycosylation sites was associated with decreasing cell surface receptor expression, which was minimal for the unglycosylated N4K/N176Q/N188Q receptor. Decreased surface expression of mutant HA-AT1a-Rs was correlated with decreased total cell receptor content as revealed by immunoblotting with an anti-HA antibody. These findings suggest that glycosylation enhances receptor stability, possibly by protecting nascent receptors from proteolytic degradation.

Reduced capacitative calcium entry correlates with vesicle accumulation and apoptosis.

A preneoplastic variant of Syrian hamster embryo cells, sup(+), exhibits decreased endoplasmic reticulum calcium levels and subsequently undergoes apoptosis in low serum conditions (Preston, G. A., Barrett, J. C., Biermann, J. A., and Murphy, E. (1997) Cancer Res. 57, 537-542). This decrease in endoplasmic reticulum calcium appears to be due, at least in part, to reduced capacitative calcium entry at the plasma membrane. Thus we investigated whether inhibition of capacitative calcium entry per se could reduce endoplasmic reticulum calcium and induce apoptosis of cells. We find that treatment with either SKF96365 (30-100 microM) or cell-impermeant 1,2-bis(o-amino-5-bromophenoxy)ethane-N,N,N', N'-tetraacetic acid (5-10 mM) is able to induce apoptosis of cells in conditions where apoptosis does not normally occur. Because previous work has implicated vesicular trafficking as a mechanism of regulating capacitative calcium entry, we investigated whether disruption of vesicular trafficking could lead to decreased capacitative calcium entry and subsequent apoptosis of cells. Coincident with low serum-induced apoptosis, we observed an accumulation of vesicles within the cell, suggesting deregulated vesicle trafficking. Treatment of cells with bafilomycin (30-100 nM), an inhibitor of the endosomal proton ATPase, produced an accumulation of vesicles, decreased capacitative entry, and induced apoptosis. These data suggest that deregulation of vesicular transport results in reduced capacitative calcium entry which in turn results in apoptosis.

Agonist-induced phosphorylation of the angiotensin AT1a receptor is localized to a serine/threonine-rich region of its cytoplasmic tail.

The agonist-induced phosphorylation sites of the rat AT1a angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta342) but was abolished by truncation at Ser325 (Delta325). Truncation at Ser335 (Delta335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by approximately 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta325 < Delta335 < ST-AA < Delta342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta325 > Delta335 > ST-AA = Delta342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.