Simultaneous quantification of losartan potassium and its active metabolite, EXP3174, in rabbit plasma by validated HPLC-PDA.

Herein, we report a novel, accurate and cost-effective validated analytical method for the quantification of losartan potassium and its active metabolite, EXP 3174, in rabbit plasma by reversed-phase high-performance liquid chromatography. Valsartan was used as an internal standard. The method was validated as per International Conference on Harmonization guidelines. The analytes were extracted in rabbit plasma using liquid-liquid extraction technique and analyzed at 247 nm after separation through a reverse-phase C18 column. The isocratic mobile phase used is a mixture of acetonitrile, water and glacial acetic acid in the ratio of 60:40:1 v/v/v maintained at pH 3.4. All calibration curves showed a good linear relationship (r > 0.995) within the test range. Precision was evaluated by intra- and interday tests with RSDs <1.91% and accuracy showed validated recoveries of 86.20-101.11%. Based on our results, the developed method features good quantification parameters and can serve as an effective quality control method for the standardization of drugs.

Development, method validation and simultaneous quantification of eleven bioactive natural products from high-altitude medicinal plant by high-performance liquid chromatography.

Herein, a novel, rapid, reliable, simple method validation and simultaneous quantification of 11 bioactive compounds (mostly xanthones) have been described. International Conference on Harmonization guidelines were used for the analytical method validation. Good linearity, repeatability, intra- and inter-day precision, accuracy and reliability were well-illuminated in the method validation procedure. The calibration curves showed a good linear relationship (r > 0.999) within test range. Precision was evaluated by intra- and inter-day tests with relative standard deviation <2.79% and accuracy validation recovery of 74.16%-91.84%. On quantification study, the validated method described the high content of bioactive xanthone derivatives, including 1-hydroxy-3, 5-dimethoxyxanthone (7), 2-(allyloxy)-8-hydroxy-1, 6-dimethoxyxanthone (6) 1, 7, 8-trihydroxy-3-methoxyxanthone (9) and Coxanthone E (5) in Codonopsis ovata, which is advantageous given the numerous pharmacological and biological effects associated with these compounds, which mostly exhibit anti-cancerous, antioxidant, anti-inflammatory, anti-mutagenic and anti-obesity effects. The bulk abundance of these compounds can also be used for further modification to produce better lead molecules for drug discovery with low toxicity and high potency. The proposed method makes it possible to simultaneously determine all bioactive compounds in one run and can be extended to marker-based standardization of herbal formulations in medicinal and pharmaceutical industries.

Synthesis and biological evaluation of novel bavachinin analogs as anticancer agents.

A library of 28 analogs of bavachinin including aliphatic and aromatic ethers, epoxide, chalcone, oxime, semicarbazide, oxime ether and triazole derivatives have been synthesized and evaluated for cytotoxicity against four different human cancer cell lines. Bio-evaluation studies exhibited better cytotoxic profile for many analogs compare to bavachinin. Best results were observed for a 1,2,3-triazole analog (17i) with IC50 values 7.72, 16.08, 7.13 and 11.67 µM against lung (A549), prostate (PC-3), colon (HCT-116) and breast (MCF-7) cancer cell lines respectively. This analog showed three and four fold improvement in cytotoxicity against HCT-116 and A549 cell lines than parent molecule (1). Structure activity relationship (SAR) study for all synthesized analogs was carried out. Further, mechanistic study of the lead molecule (17i) revealed that it inhibits colony formation and in vitro migration of human colon cancer cells (HCT-116). Also, it induced the morphological changes and mediated the apoptotic cell death of HCT-116 cells with perturbance in mitochondrial membrane potential (MMP) and PARP cleavage.

Biologically active xanthones from Codonopsis ovata.

Five new xanthones, named coxanthones A-E (1-5), together with 21 known secondary metabolites (6-26) that include seven xanthones, five flavonoids, two steroids and seven triterpenoids were isolated from the chemically unexplored whole plant Codonopsis ovata. The structures of new metabolites were elucidated by HRMS, interpretation of NMR spectra and other spectroscopic techniques. The absolute configuration of the stereogenic centre of coxanthone B (2) was determined by electronic circular dichroism (ECD) spectroscopy. This is the first report of xanthones from the genus Codonopsis. All isolated metabolites were evaluated for cytotoxic activity by SRB assay against six human cancer cell lines A549 (lung), PC-3 (prostate), HCT-116 (colon), MCF-7 (breast), SF-295 (CNS), and MDAMB-435 (melanoma). Among the new compounds, coxanthone B (2) exhibited significant inhibitory activity against SF-295 and MDAMB-435 with IC50 values of 7.0 and 15.0 µM, respectively. Coxanthone A (1) displayed cytotoxicity against A549 cell line at IC50 value of 22.5 µM. Cytotoxic activity of 1-hydroxy-3,5-dimethoxyxanthone (7), swertiperenine (9) and 1,7,8-trihydroxy-3-methoxyxanthone (10) are reported here first time that exhibited the IC50 values of 3.0, 5.0 and 21.0 µM against A549, MDAMB-435, and A549 cell lines, respectively. Kaempferol (13) showed most potent cytotoxic activity with an IC50 values in the 1.0-2.3 µM range against all tested cancer cell lines.

A phase-field model for fracture in biological tissues.

This work presents a recently developed phase-field model of fracture equipped with anisotropic crack driving force to model failure phenomena in soft biological tissues at finite deformations. The phase-field models present a promising and innovative approach to thermodynamically consistent modeling of fracture, applicable to both rate-dependent or rate-independent brittle and ductile failure modes. One key advantage of diffusive crack modeling lies in predicting the complex crack topologies where methods with sharp crack discontinuities are known to suffer. The starting point is the derivation of a regularized crack surface functional that [Formula: see text]-converges to a sharp crack topology for vanishing length-scale parameter. A constitutive balance equation of this functional governs the crack phase-field evolution in a modular format in terms of a crack driving state function. This allows flexibility to introduce alternative stress-based failure criteria, e.g., isotropic or anisotropic, whose maximum value from the deformation history drives the irreversible crack phase field. The resulting multi-field problem is solved by a robust operator split scheme that successively updates a history field, the crack phase field and finally the displacement field in a typical time step. For the representative numerical simulations, a hyperelastic anisotropic free energy, typical to incompressible soft biological tissues, is used which degrades with evolving phase field as a result of coupled constitutive setup. A quantitative comparison with experimental data is provided for verification of the proposed methodology.

Retinoblastoma protein phosphorylation at multiple sites is associated with neurofibrillary pathology in Alzheimer disease.

The re-expression of multiple cell cycle markers representing various cell cycle phases in postmitotic pyramidal neurons suggests that neurons in Alzheimer disease (AD) attempt to re-enter the cell cycle. Entry into the cell cycle requires activation of G1 to S phase cell cycle proteins, among which retinoblastoma protein (pRb) is a key regulator. pRb inhibits the transcription of cell cycle proteins in the nucleus of healthy cells by interaction and consequent blocking of the active site of E2F, dependent upon the phosphate stoichiometry and combination of the locations of their 16 potential phosphorylation sites on pRb. Therefore, to determine whether pRb is involved in the aberrant cell cycle phenotype in AD neurons, a systematic immunocytochemical evaluation of the phosphorylation status of pRb protein using antibodies specific for multiple phosphorylation sites (i.e., pSpT249/252, pS612, pS795, pS807, pS811 and pT821) was carried out in the hippocampal regions of brains from AD patients. Increased levels of phospho-pRb (ppRb) for all these phosphorylation sites were noted in the brains of AD patients as compared to control cases. More importantly, redistribution of ppRb from the nucleus to the cytoplasm of susceptible neurons, with significant localization in neurofibrillary tangles and neuritic plaques, was observed. Additional studies revealed extensive co-localization between phospho-p38 and ppRb, implicating that p38 activation may contribute to cell cycle abnormalities through pRb phosphorylation. Taken together, these data supports the concept of neuronal cell cycle re-entry in AD and indicates a crucial role for pRb in this process.

BRCA1 may modulate neuronal cell cycle re-entry in Alzheimer disease.

In Alzheimer disease, neuronal degeneration and the presence of neurofibrillary tangles correlate with the severity of cognitive decline. Neurofibrillary tangles contain the antigenic profile of many cell cycle markers, reflecting a re-entry into the cell cycle by affected neurons. However, while such a cell cycle re-entry phenotype is an early and consistent feature of Alzheimer disease, the mechanisms responsible for neuronal cell cycle are unclear. In this regard, given that a dysregulated cell cycle is a characteristic of cancer, we speculated that alterations in oncogenic proteins may play a role in neurodegeneration. To this end, in this study, we examined brain tissue from cases of Alzheimer disease for the presence of BRCA1, a known regulator of cell cycle, and found intense and specific localization of BRCA1 to neurofibrillary tangles, a hallmark lesion of the disease. Analysis of clinically normal aged brain tissue revealed systematically less BRCA1, and surprisingly in many cases with apparent phosphorylated tau-positive neurofibrillary tangles, BRCA1 was absent, yet BRCA1 was present in all cases of Alzheimer disease. These findings not only further define the cell cycle reentry phenotype in Alzheimer disease but also indicate that the neurofibrillary tangles which define Alzheimer disease may have a different genesis from the neurofibrillary tangles of normal aging.

Apoptosis in Alzheimer disease: a mathematical improbability.

Neuronal cell dysfunction and death are cardinal features of Alzheimer disease and a great deal of effort is being expended not only to understand factors involved in the cause and progression of disease (i.e., disease initiators and propagators) but, ultimately, the precise mechanism by which neurons die (for want of a better word, the terminators). Understanding each and every component of the complex pathway that ultimately leads to disease (a clinical phenotype) is clearly of paramount importance for the development of effective therapeutic strategies. Of particular intrigue for many scientists, perhaps the more macabre among us, has been to decipher the final event - namely cell death. Broadly speaking, cell death falls into two categories, apoptotic and necrotic. The former, apoptosis, by definition, is a controlled event; thereby offering the potential for intervention, whereas necrosis is a more stochastic process. Since many of the propagators and exacerbators involved in Alzheimer disease are pro-apoptotic, it is not surprising that certain aspects of apoptosis are evident. However, it would be a mistake to call this apoptosis. In fact, as reviewed herein, the chronic course of disease together with the necessarily slow rate of neuronal death makes apoptotic cell death in Alzheimer disease a mathematical improbability. The numbers simply do not add up.

The cell cycle in Alzheimer disease: a unique target for neuropharmacology.

Several hypotheses have been proposed attempting to explain the pathogenesis of Alzheimer disease including, among others, theories involving amyloid deposition, tau phosphorylation, oxidative stress, metal ion dysregulation and inflammation. While there is strong evidence suggesting that each one of these proposed mechanisms contributes to disease pathogenesis, none of these mechanisms are able to account for all the physiological changes that occur during the course of the disease. For this reason, we and others have begun the search for a causative factor that predates known features found in Alzheimer disease, and that might therefore be a fundamental initiator of the pathophysiological cascade. We propose that the dysregulation of the cell cycle that occurs in neurons susceptible to degeneration in the hippocampus during Alzheimer disease is a potential causative factor that, together with oxidative stress, would initiate all known pathological events. Neuronal changes supporting alterations in cell cycle control in the etiology of Alzheimer disease include the ectopic expression of markers of the cell cycle, organelle kinesis and cytoskeletal alterations including tau phosphorylation. Such mitotic alterations are not only one of the earliest neuronal abnormalities in the disease, but as discussed herein, are also intimately linked to all of the other pathological hallmarks of Alzheimer disease including tau protein, amyloid beta protein precursor and oxidative stress, and even risk factors such as mutations in the presenilin genes. Therefore, therapeutic interventions targeted toward ameliorating mitotic changes would be predicted to have a profound and positive impact on Alzheimer disease progression.

The role of metabotropic glutamate receptors in Alzheimer's disease.

While glutamatergic transmission is severely altered by early degeneration of cortico-cortical connections and hippocampal projections in Alzheimer's disease (AD), the role of glutamate receptors in the pathogenesis of AD is not yet defined clearly. Nonetheless, as reviewed here, the topographical distribution of different types of receptors likely contributes to the regional selective nature of neuronal degeneration. In particular, metabotropic glutamate receptors (mGluR) may contribute the pathogenesis of many neurological conditions and also regulate neuronal vulnerability against cytotoxic stress. Thus, we here discuss the possible role of mGluR in the pathogenesis of AD based on the results from other neurodegenerative diseases that may give us clues to solve the mysterious selective neurodegeneration evident in AD.

Alzheimer's disease and the cell cycle.

Current views associate the reappearance of cell cycle markers with early events in Alzheimer's disease. Even though, the cell cycle was implicated early in the study of this disease, only recently has it been associated with selective early vulnerability of neurons. The pathological hallmarks of Alzheimer's disease namely tau and amyloid have been associated with having effects on or being affected by cell cycle progression. Indeed the mitogenic component looms large early in the onset of Alzheimer's disease. Although quite a number of markers of reentry have been catalogued, the common denominator is abortosis, the unalterable march towards neuronal dysfunction, stasis and eventually death. We feel that complete understanding of the mechanisms, acting either positively by stimulation or through removal of inhibitory signals will provide promising molecular targets for pharmacological interventions which have been static for a number of years by being relegated to inhibition of the enzyme cholinesterase. In our opinion, investigating more proximal mechanisms will provide answers to changing the natural course of this illness.

Neuroprotective properties of Bcl-w in Alzheimer disease.

While there is a host of pro-apoptotic stimuli that target neurons in Alzheimer disease (AD), given the chronicity of the disease and the survival of many neurons, those neurons must either avoid or, at minimum, delay apoptotic death signaling. In this study, we investigated Bcl-w, a novel member of the Bcl-2 family that promotes cell survival. In AD, we found increased levels of Bcl-w associated with neurofibrillary pathology and punctate intracytoplasmic structures whereas, in marked contrast, there are only low diffuse levels of Bcl-w in the neuronal cytoplasm of age-matched control cases. Immunoblot analysis confirmed that Bcl-w levels were significantly increased in AD. By electron microscopy, we determined that the increased Bcl-w expression in AD was ultrastructurally localized to mitochondria and neurofibrillary pathology. To investigate the cause and consequence of Bcl-w up-regulation in neurons, we found that fibrillized amyloid-beta led to increased Bcl-w protein levels in M17 human neuroblastoma cells, and that overexpression of Bcl-w significantly protected neurons against staurosporine- and amyloid-beta-induced apoptosis. Taken together, these series of results suggest that Bcl-w may play an important protective role in neurons in the diseased brain and that this aspect could be therapeutically harnessed to afford neuroprotection.

Homocysteine and Alzheimer's disease: a modifiable risk?

A hypothesis is proposed that reconciles the epidemiological observation of elevated homocysteine in Alzheimer's disease (AD) with clinical features of the disease, particularly evidence of increased oxidative stress. We propose homocysteine is involved in an iron dysregulation/oxidative stress cycle that has a central role in the pathogenesis of AD. The implications of the hypothesis and some strategies for testing it are discussed.

Oxidative stress signalling in Alzheimer's disease.

Multiple lines of evidence demonstrate that oxidative stress is an early event in Alzheimer's disease (AD), occurring prior to cytopathology, and therefore may play a key pathogenic role in the disease. Indeed, that oxidative mechanisms are involved in the cell loss and other neuropathology associated with AD is evidenced by the large number of metabolic signs of oxidative stress as well as by markers of oxidative damage. However, what is intriguing is that oxidative damage decreases with disease progression, such that levels of markers of rapidly formed oxidative damage, which are initially elevated, decrease as the disease progresses to advanced AD. This finding, along with the compensatory upregulation of antioxidant enzymes found in vulnerable neurons in AD, indicates that reactive oxygen species (ROS) not only cause damage to cellular structures but also provoke cellular responses. Mammalian cells respond to extracellular stimuli by transmitting intracellular instructions by signal transduction cascades to coordinate appropriate responses. Therefore, not surprisingly stress-activated protein kinase (SAPK) pathways, pathways that are activated by oxidative stress, are extensively activated during AD. In this paper, we review the evidence of oxidative stress and compensatory responses that occur in AD with a particular focus on the roles and mechanism of activation of SAPK pathways.

Alzheimer's disease: the two-hit hypothesis.

There are many lines of evidence showing that oxidative stress and aberrant mitogenic changes have important roles in the pathogenesis of Alzheimer's disease (AD). However, although both oxidative stress and cell cycle-related abnormalities are early events, occurring before any cytopathology, the relation between these two events, and their role in pathophysiology was, until recently, unclear. However, on the basis of studies of mitogenic and oxidative stress signalling pathways in AD, we proposed a "two-hit hypothesis" which states that although either oxidative stress or abnormalities in mitotic signalling can independently serve as initiators, both processes are necessary to propagate disease pathogenesis. In this paper, we summarise evidence for oxidative stress and abnormal mitotic alterations in AD and explain the two-hit hypothesis by describing how both mechanisms are necessary and invariant features of disease.

Elevated expression of a regulator of the G2/M phase of the cell cycle, neuronal CIP-1-associated regulator of cyclin B, in Alzheimer's disease.

Adult neurons are generally accepted to be in a quiescent, nonproliferative state. However, it is becoming increasingly apparent that, in Alzheimer's disease (AD), alterations in cell cycle machinery, suggesting an attempt to reenter cell cycle, relate temporally and topographically to degenerating neurons. These findings, together with the fact that neurons lack the necessary components for completion of mitosis, have led to the notion that an ill-regulated attempt to reenter the cell cycle is associated with disease pathogenesis and, ultimately, neuronal degeneration. To understand better the role of such cell cycle abnormalities in AD, we undertook a study of CIP-1-associated regulator of cyclin B (CARB), a protein that associates with two key proteins, p21 and cyclin B, involved in cellular checkpoints in the cell cycle. Our results show that there are increases in CARB localized to intraneuronal neurofibrillary tangles and granulovacuolar degeneration in susceptible hippocampal and cortical neurons in AD. By marked contrast, CARB is found only at background levels in these neuronal populations in nondiseased age-matched controls. Our data not only provide another line of evidence indicative of cell cycle abnormalities in neurons in AD but also lend further credence to the hypothesis that susceptible neurons may be arrested at the G2/M phase of the cell cycle before they die. Therefore, therapeutics targeted toward initiators of the cell cycle are likely to prove of great efficacy for the treatment of AD.

A metabolic basis for Alzheimer disease.

Most studies of Alzheimer's disease (AD) have focused on a single precipitating alteration as the etiological event rather than global changes closely linked to aging. Recent evidence suggests that the most significant of these global changes are metabolic. Here we present data indicating that metabolic rate, nutrition, and neuronal size are all early indicators of AD. Understanding the cellular and molecular basis for these changes may open a new dimension to understanding AD.

Oxidative stress and neuronal adaptation in Alzheimer disease: the role of SAPK pathways.

Recent evidence indicates that oxidative stress occurs early in the progression of Alzheimer disease, significantly before the development of the hallmark pathologies, namely neurofibrillary tangles and senile plaques. The interaction of abnormal mitochondria, redox transition metals, and oxidative stress response elements contributes to the generation of reactive oxygen species in diseased neurons. Oxidative damage to major cellular molecules is seen in a number of disease states that are either acute or chronic and it is apparent that without eliciting compensations that restore redox balance, cells will rapidly succumb to death. Indeed, although oxidative stress is a prominent feature in Alzheimer disease, few vulnerable neurons show clear signs of apoptosis, suggesting that the level of oxidative stress does not significantly exceed neuronal oxidative defenses. In light of this observation, we propose that neurons in Alzheimer disease are exposed to low, but chronic, levels of oxidative stress that lead neurons to elicit adaptive responses such as the activation of stress-activated protein kinase pathways.

Hydroxynonenal, toxic carbonyls, and Alzheimer disease.

Cytoskeletal disruption is one of the distinguishing characteristics of the vulnerable neurons in Alzheimer disease (AD). It has been suggested that these cytoskeletal changes occur secondarily to covalent modifications of the protein components. Despite the abundance and probable importance of these changes, there has been very little data regarding the identity of the modified proteins or the precise chemistry of the modifications. Here we review a specific type of modification, namely carbonylation of proteins, which has been shown to be a common result of cellular oxidative stress. Hopefully, the following discussion will help elucidate the relationship between oxidative stress, protein modification and the pathogenesis of AD.

Increased p27, an essential component of cell cycle control, in Alzheimer's disease.

A number of recent findings have demonstrated re-expression of cell cycle-related proteins in vulnerable neurones in Alzheimer's disease. We hypothesize that this attempt by neurones to re-enter mitosis is a response to external growth stimuli that leads to an abortive re-entry into the cell cycle and, ultimately, neuronal degeneration. In this study, to further delineate the role of mitotic processes in the pathogenesis of Alzheimer's disease, we investigated p27, a cyclin-dependent kinase inhibitor that plays a negatively regulatory role in cell cycle progression that, once phosphorylated at Thr187, is degraded via an ubiquitin-proteasome pathway. Here we report that both p27 and phosphorylated p27 (Thr187) show increases in the cytoplasm of vulnerable neuronal populations in Alzheimer's disease vs. age-matched control subjects. Importantly, phosphorylated p27 (Thr187) shows considerable overlap with tau-positive neurofibrillary pathology, including neurofibrillary tangles, dystrophic neurites and neuropil threads. The findings presented here suggest that dysregulation of the cell cycle plays a crucial role in the pathogenesis of Alzheimer's disease that may provide a novel mechanistic basis for therapeutic intervention.