Cell cycle re-entry mediated neurodegeneration and its treatment role in the pathogenesis of Alzheimer's disease.

As one of the earliest pathologic changes, the aberrant re-expression of many cell cycle-related proteins and inappropriate cell cycle control in specific vulnerable neuronal populations in Alzheimer's disease (AD) is emerging as an important component in the pathogenesis leading to AD and other neurodegenerative diseases. These events are clearly representative of a true cell cycle, rather than epiphenomena of other processes since, in AD and other neurodegenerative diseases, there is a true mitotic alteration that leads to DNA replication. While the exact role of cell cycle re-entry is unclear, recent studies using cell culture and animal models strongly support the notion that the dysregulation of cell cycle in neurons leads to the development of AD-related pathology such as hyperphosphorylation of tau and amyloid-beta deposition and ultimately causes neuronal cell death. Importantly, cell cycle re-entry is also evident in mutant amyloid-beta precursor protein and tau transgenic mice and, as in human disease, occurs prior to the development of the pathological hallmarks, neurofibrillary tangles and amyloid-beta plaques. Therefore, the study of aberrant cell cycle regulation in model systems, both cellular and animal, may provide extremely important insights into the pathogenesis of AD and also serve as a means to test novel therapeutic approaches.

Neuroprotective effect of cocoa flavonoids on in vitro oxidative stress.

BACKGROUND: Cocoa is a rich source of flavonoids that, among other functions, can act as antioxidants. In living systems, the production of reactive oxygen species (ROS) activate an array of intracellular cascades, including mitogen-activated protein kinases (MAPK), that are closely associated with cell death or survival pathways. AIM OF THE STUDY: To ascertain the role of a cocoa extract and its main flavonoid, (-)-epicatechin, in an in vitro model of oxidative stress induced in a neuronal cell line. METHODS: We analyzed ROS production by fluorometry (dichlorofluorescein assay), and activation of MAPK pathways including extracellular signal-regulated kinases 1/2 (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38, by Western blot analysis. RESULTS: Cells incubated with cocoa extract or (-)-epicatechin, reduced ROS production in a dose-dependent manner, reaching 35% inhibition. pJNK and p38, involved in apoptosis, were down-modulated by cocoa extract and (-)-epicatechin with p38 inhibition reaching up to 70%. CONCLUSIONS: Our results show that cocoa extract and (-)-epicatechin may exert a neuroprotective action by reducing ROS production and modulating MAPK activation.

Development of a GC x GC-TOFMS method using SPME to determine volatile compounds in cacao beans.

A method to analyze volatile compounds from cacao beans has been developed and evaluated. The method utilizes solid phase micro extraction (SPME) sampling followed by comprehensive 2-D (GC x GC) coupled with TOFMS. For the SPME procedure, a polydimethyl siloxane/divinyl benzene (PDMS/DVB) fiber was implemented. Cacao beans from four geographical origins were studied under two storage conditions, either dry or high moisture. A given cacao bean sample was sealed in a SPME vial and heated for 15 min. Extraction temperatures of 45, 60, 80, and 100 degrees C were analyzed and an optimal extraction temperature of 60 degrees C was determined. Many peaks were found to change as a function of storage conditions with Fisher Ratio analysis. Four representative compounds were identified and quantified (on a relative basis): acetic acid, nonanal, tetramethyl pyrazine, and trimethyl pyrazine. Acetic acid and nonanal were elevated in samples without evident mold on the bean surface, while the two pyrazines were elevated when mold was evident on the bean surface. The results for these comparisons, indicate that metabolism at the bean surface plays a role in the concentration of analytes, and can be readily determined using this analytical technology and methodology.

Neuronal cell cycle re-entry mediates Alzheimer disease-type changes.

Evidence showing the ectopic re-expression of cell cycle-related proteins in specific vulnerable neuronal populations in Alzheimer disease led us to formulate the hypothesis that neurodegeneration, like cancer, is a disease of inappropriate cell cycle control. To test this notion, we used adenoviral-mediated expression of c-myc and ras oncogenes to drive postmitotic primary cortical neurons into the cell cycle. Cell cycle re-entry in neurons was associated with increased DNA content, as determined using BrdU and DAPI, and the re-expression of cyclin B1, a marker for the G2/M phase of the cell cycle. Importantly, we also found that cell cycle re-entry in primary neurons leads to tau phosphorylation and conformational changes similar to that seen in Alzheimer disease. This study establishes that the cell cycle can be instigated in normally quiescent neuronal cells and results in a phenotype that shares features of degenerative neurons in Alzheimer disease. As such, our neuronal cell model may be extremely valuable for the development of novel therapeutic strategies.

The application of microarray technology to neuropathology: cutting edge tool with clinical diagnostics potential or too much information?

Microarray technology is a tremendously powerful method for simultaneously monitoring the expression of thousands of species of nucleic acids, usually cellular mRNA, producing a high-resolution representation of the genes encoded or expressed in a cell. As such, microarray technology has great potential for impacting research and clinical approaches to treatment. However, this complex technology has been challenging to apply as a result of difficulties discerning biologic variation from technologic issues, therefore slowing the application of the technology to human diagnostics. Nevertheless, significant advances in microarray technology, improvements that avoid potential pitfalls, and a wider spectrum of application are making this technology easier to apply. Indeed, microarray technology has provided valuable insights into mechanisms involving gene regulation and expression in Alzheimer disease, and it remains a powerful tool to identify biomarkers for disease diagnosis. Ultimately, the most robust markers will enable the application of more specific treatments particular to disease stages or subcategories. Currently, no widely applicable molecular test is available to identify those at risk for developing Alzheimer disease or those who have early markers of pathology but show discernible cognitive impairment. The progression of this technology will lead to earlier detection of the disease through enhanced understanding of disease onset and progression.

Quantitative assessment of moisture damage for cacao bean quality using two-dimensional gas chromatography combined with time-of-flight mass spectrometry and chemometrics.

Quality control of cacao beans is a significant issue in the chocolate industry. In this report, we describe how moisture damage to cacao beans alters the volatile chemical signature of the beans in a way that can be tracked quantitatively over time. The chemical signature of the beans is monitored via sampling the headspace of the vapor above a given bean sample. Headspace vapor sampled with solid-phase micro-extraction (SPME) was detected and analyzed with comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS). Cacao beans from six geographical origins (Costa Rica, Ghana, Ivory Coast, Venezuela, Ecuador, and Panama) were analyzed. Twenty-nine analytes that change in concentration levels via the time-dependent moisture damage process were measured using chemometric software. Biomarker analytes that were independent of geographical origin were found. Furthermore, prediction algorithms were used to demonstrate that moisture damage could be verified before there were visible signs of mold by analyzing subsets of the 29 analytes. Thus, a quantitative approach to quality screening related to the identification of moisture damage in the absence of visible mold is presented.

Clinical benefit and preservation of flavonols in dark chocolate manufacturing.

The consumption of high-cacao-content chocolate has been associated with positive health benefits ascribed to flavanol [corrected] antioxidants derived from the ground, fermented cocoa seeds of Theobroma cacao. However, flavanols [corrected] impart a bitter, astringent flavor to foodstuffs, frequently masked in chocolates and confections by aggressive processing and adulteration with other flavors. Recent reports have implied that not all varieties of dark chocolate are created equally, and significant caveats exist regarding its potential health benefits. It is perhaps not surprising that extensive processing, dilution, and the addition of flavor modifiers may improve the palatability of chocolate, but could have negative nutritional and clinical benefits. This article examines the chemical composition of chocolate and the clinical data associated with the consumption of flavonoid-rich cocoa. We review the steps in chocolate manufacturing that directly affect the antioxidant levels in chocolate products, and the caveats associated with claims of health benefits from the consumption of dark chocolate.

Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

Use of semiconductor-based oligonucleotide microarrays for influenza a virus subtype identification and sequencing.

In the face of concerns over an influenza pandemic, identification of virulent influenza A virus isolates must be obtained quickly for effective responses. Rapid subtype identification, however, is difficult even in well-equipped virology laboratories or is unobtainable in the field under more austere conditions. Here we describe a genome assay and microarray design that can be used to rapidly identify influenza A virus hemagglutinin subtypes 1 through 15 and neuraminidase subtypes 1 through 9. Also described is an array-based enzymatic assay that can be used to sequence portions of both genes or any other sequence of interest.

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.