Influence of zero-G on single-cell systems and zero-G fermenter design concepts

An analysis was made to identify potential gravity-sensitive mechanisms that may be present in the single-cell growth system. Natural convection (density gradients, induced sedimentation, and buoyancy) is important in microbial systems. The absence of natural convection in the space-flight environment could provide an opportunity for new approaches for developments in industrial fermentation and agriculture. Some of the potential influences of gravity (i.e., convection, sedimentation, etc.) on the cell were discussed to provide insight into what experimental areas may be pursued in future space-flight research programs

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2

Receptor-mediated increase in cytosolic calcium in LLC-PK1 cells by platelet activating factor and thromboxane A2. Several studies indicate an important role of platelet activating factor (PAF) and thromboxane A2 (TXA2) in glomerular pathophysiology. However, the potential role of PAF or TXA2 in renal tubular pathophysiology has received little attention, and the presence of functional receptors for these autacoids in renal tubular epithelium has not been previously studied. We examined the effects of PAF and the TXA2 analogue, ONO11113, on the cytosolic free calcium concentration ([Ca2+]i) in cultured LLC-PK1, cell line using a fluorescent probe, fura-2. In these cells, the addition of PAF or ONO11113 caused a significant increment in [Ca2+]i in a dose-dependent manner: both agonists (10-7 M) increased [Ca2+]i from 148 ± 16 to 288 ± 39 nM and from 130 ± 8 to 240 ± 18 nM, with the values of EC50 for PAF and ONO11113 being 17 ± 4 and 17 ± 2 nM, respectively. These effects were both rapid and transient, returning to baseline in two minutes. The effect of PAF was selectively blocked by PAF receptor antagonist BN50730, but not by TXA2 receptor antagonist L657925. Similarly ONO1113 response was abolished by L657925, but not by BN50730. PAF- or ONO11113-challenged cells did not respond to a second addition of the same agent and showed heterologous desensitization to the other agonist. The initial peaks of [Ca2+]i as well as the sustained elevations in [Ca2+]i induced by PAF or ONO11113 were reduced following the chelation of extracellular Ca2+ by 10 mM ethylene glycol-bis(β-aminomethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). In the absence of extracellular Ca2+ 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (which has extensively been used to study the contribution of Ca2+ released from intracellular storage sites in the increase in [Ca2+]i), blocked the increment in [Ca2+]i induced by PAF or ONO1113. These results indicate that LLC-PKi cells express discrete receptors for PAF and TXA2 that are coupled to an increase in [Ca2+]i through mobilization of calcium from both intracellular storage sites and extracellular milieu, and suggest the possible importance of PAF and TXA2 in tubular pathophysiology

Calendar year 2003 : annual site enviromental report for Tonopah Test Range, Nevada and Kauai Test Facility, Hawaii.

Tonopah Test Range (TTR) in Nevada and Kauai Test Facility (KTF) in Hawaii are government-owned, contractor-operated facilities operated by Sandia Corporation, a subsidiary of Lockheed Martin Corporation. The U.S. Department of Energy (DOE), National Nuclear Security Administration (NNSA), through the Sandia Site Office (SSO), in Albuquerque, NM, manages TTR and KTF's operations. Sandia Corporation conducts operations at TTR in support of DOE/NNSA's Weapons Ordnance Program and has operated the site since 1957. Westinghouse Government Services subcontracts to Sandia Corporation in administering most of the environmental programs at TTR. Sandia Corporation operates KTF as a rocket preparation launching and tracking facility. This Annual Site Environmental Report (ASER) summarizes data and the compliance status of the environmental protection and monitoring program at TTR and KTF through Calendar Year (CY) 2003. The compliance status of environmental regulations applicable at these sites include state and federal regulations governing air emissions, wastewater effluent, waste management, terrestrial surveillance, and Environmental Restoration (ER) cleanup activities. Sandia Corporation is responsible only for those environmental program activities related to its operations. The DOE/NNSA, Nevada Site Office (NSO) retains responsibility for the cleanup and management of ER TTR sites. Currently, there are no ER Sites at KTF. Environmental monitoring and surveillance programs are required by DOE Order 450.1, Environmental Protection Program (DOE 2003) and DOE Order 231.1 Chg 2., Environment, Safety, and Health Reporting (DOE 1996)

Characterization of cytosolic proliferating cell nuclear antigen (PCNA) in neutrophils: antiapoptotic role of the monomer.

We have shown previously that PCNA, a nuclear factor involved in DNA replication and repair in proliferating cells, is localized exclusively in the cytoplasm of neutrophils, where it regulates their survival. Nuclear PCNA functions are tightly linked to its ring-shaped structure, which allows PCNA to bind to numerous partner proteins to orchestrate DNA-related processes. We have shown that only monomeric PCNA can expose its NES to be relocalized from nucleus to cytosol during granulocyte differentiation. This study tested the hypothesis that monomeric PCNA could have a biological role in neutrophils. With the use of a combination of cross-linking and gel-filtration experiments, trimeric and monomeric PCNAs were detected in neutrophil cytosol. The promyelocytic cell line PLB985 was next stably transfected to express the monomeric PCNAY114A mutant to examine its function compared with the WT trimeric PCNA. Monomeric PCNAY114A mutant potentiated DMF-induced differentiation, as evidenced by an increased percentage of CD11b- and gp91phox-positive PLB985PCNAY114A cells and by an increased, opsonized zymosan-triggered NADPH oxidase activity compared with PLB985PCNA or PLB985 cells overexpressing WT PCNA or the empty plasmid, respectively. Regarding antiapoptotic activity, DMF-differentiated PLB985 cells overexpressing WT or the monomeric PCNAY114A mutant displayed a similar antiapoptotic activity following treatment with gliotoxin or TRAIL compared with PLB985. The molecular basis through which cytoplasmic PCNA exerts its antiapoptotic activity in mature neutrophils may, at least in part, be independent of the trimeric conformation

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

Background: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material

Clinical and biomarker changes in dominantly inherited Alzheimer\u27s disease

BACKGROUND: The order and magnitude of pathologic processes in Alzheimer\u27s disease are not well understood, partly because the disease develops over many years. Autosomal dominant Alzheimer\u27s disease has a predictable age at onset and provides an opportunity to determine the sequence and magnitude of pathologic changes that culminate in symptomatic disease. METHODS: In this prospective, longitudinal study, we analyzed data from 128 participants who underwent baseline clinical and cognitive assessments, brain imaging, and cerebrospinal fluid (CSF) and blood tests. We used the participant\u27s age at baseline assessment and the parent\u27s age at the onset of symptoms of Alzheimer\u27s disease to calculate the estimated years from expected symptom onset (age of the participant minus parent\u27s age at symptom onset). We conducted cross-sectional analyses of baseline data in relation to estimated years from expected symptom onset in order to determine the relative order and magnitude of pathophysiological changes. RESULTS: Concentrations of amyloid-beta (Aβ) 42 in the CSF appeared to decline 25 years before expected symptom onset. Aβ deposition, as measured by positron-emission tomography with the use of Pittsburgh compound B, was detected 15 years before expected symptom onset. Increased concentrations of tau protein in the CSF and an increase in brain atrophy were detected 15 years before expected symptom onset. Cerebral hypometabolism and impaired episodic memory were observed 10 years before expected symptom onset. Global cognitive impairment, as measured by the Mini-Mental State Examination and the Clinical Dementia Rating scale, was detected 5 years before expected symptom onset, and patients met diagnostic criteria for dementia at an average of 3 years after expected symptom onset. CONCLUSIONS: We found that autosomal dominant Alzheimer\u27s disease was associated with a series of pathophysiological changes over decades in CSF biochemical markers of Alzheimer\u27s disease, brain amyloid deposition, and brain metabolism as well as progressive cognitive impairment. Our results require confirmation with the use of longitudinal data and may not apply to patients with sporadic Alzheimer\u27s disease. (Funded by the National Institute on Aging and others; DIAN ClinicalTrials.gov number, NCT00869817.

Screening for Toxic Amyloid in Yeast Exemplifies the Role of Alternative Pathway Responsible for Cytotoxicity

The relationship between amyloid and toxic species is a central problem since the discovery of amyloid structures in different diseases. Despite intensive efforts in the field, the deleterious species remains unknown at the molecular level. This may reflect the lack of any structure-toxicity study based on a genetic approach. Here we show that a structure-toxicity study without any biochemical prerequisite can be successfully achieved in yeast. A PCR mutagenesis of the amyloid domain of HET-s leads to the identification of a mutant that might impair cellular viability. Cellular and biochemical analyses demonstrate that this toxic mutant forms GFP-amyloid aggregates that differ from the wild-type aggregates in their shape, size and molecular organization. The chaperone Hsp104 that helps to disassemble protein aggregates is strictly required for the cellular toxicity. Our structure-toxicity study suggests that the smallest aggregates are the most toxic, and opens a new way to analyze the relationship between structure and toxicity of amyloid species

S100A7, a Novel Alzheimer's Disease Biomarker with Non-Amyloidogenic α-Secretase Activity Acts via Selective Promotion of ADAM-10

Alzheimer's disease (AD) is the most common cause of dementia among older people. At present, there is no cure for the disease and as of now there are no early diagnostic tests for AD. There is an urgency to develop a novel promising biomarker for early diagnosis of AD. Using surface-enhanced laser desorption ionization-mass spectrometry SELDI-(MS) proteomic technology, we identified and purified a novel 11.7-kDa metal- binding protein biomarker whose content is increased in the cerebrospinal fluid (CSF) and in the brain of AD dementia subjects as a function of clinical dementia. Following purification and protein-sequence analysis, we identified and classified this biomarker as S100A7, a protein known to be involved in immune responses. Using an adenoviral-S100A7 expression system, we continued to examine the potential role of S100A7 in AD amyloid neuropathology in in vitro model of AD. We found that the expression of exogenous S100A7 in primary cortico-hippocampal neuron cultures derived from Tg2576 transgenic embryos inhibits the generation of β-amyloid (Aβ)1–42 and Aβ1–40 peptides, coincidental with a selective promotion of “non- amyloidogenic” α-secretase activity via promotion of ADAM (a disintegrin and metalloproteinase)-10. Finally, a selective expression of human S100A7 in the brain of transgenic mice results in significant promotion of α-secretase activity. Our study for the first time suggests that S100A7 may be a novel biomarker of AD dementia and supports the hypothesis that promotion of S100A7 expression in the brain may selectively promote α-secretase activity in the brain of AD precluding the generation of amyloidogenic peptides. If in the future we find that S1000A7 protein content in CSF is sensitive to drug intervention experimentally and eventually in the clinical setting, S100A7 might be developed as novel surrogate index (biomarker) of therapeutic efficacy in the characterization of novel drug agents for the treatment of AD