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At least 685 records · Page 38Linked to original sources
PubMed2005

Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides.

We report four-color DNA sequencing by synthesis (SBS) on a chip, using four photocleavable fluorescent nucleotide analogues (dGTP-PC-Bodipy-FL-510, dUTP-PC-R6G, dATP-PC-ROX, and dCTP-PC-Bodipy-650) (PC, photocleavable; Bodipy, 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solution-phase polymerase reaction and the fluorophore could be cleaved using laser irradiation ( approximately 355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azido-labeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length.

Base Sequence↗
PubMed2006

Designing a nano-interface in a microfluidic chip to probe living cells: challenges and perspectives.

Nanotechnology-based materials are beginning to emerge as promising platforms for biomedical analysis, but measurement and control at the cell-chip interface remain challenging. This idea served as the basis for discussion in a focus group at the recent National Academies Keck Futures Initiative. In this Perspective, we first outline recent advances and limitations in measuring nanoscale mechanical, biochemical, and electrical interactions at the interface between biomaterials and living cells. Second, we present emerging experimental and conceptual platforms for probing living cells with nanotechnology-based tools in a microfluidic chip. Finally, we explore future directions and critical needs for engineering the cell-chip interface to create an integrated system capable of high-resolution analysis and control of cellular physiology.

Biomedical Engineering↗
PubMed1999

Chip-based genotyping by mass spectrometry.

Silicon chips with immobilized target DNAs were used for accurate genotyping by mass spectrometry. Genomic DNAs were amplified with PCR, and the amplified products were covalently attached to chip wells via N-succinimidyl (4-iodoacetyl)aminobenzoate (SIAB) chemistry. Primer annealing, extension, and termination were performed on a 1-microl scale directly in the chip wells in parallel. Diagnostic products thus generated were detected in situ by using matrix-assisted laser desorption ionization mass spectrometry. This miniaturized method has the potential for accurate, high-throughput, low-cost identification of genetic variations.

Base Sequence↗
PubMed2003

CHIP-Hsc70 complex ubiquitinates phosphorylated tau and enhances cell survival.

The microtubule-binding protein tau has been implicated in the neurofibrillary pathology of Alzheimer's disease. Within affected cells, ubiquitinated and hyperphosphorylated tau assembles into massive filamentous polymers. Eventually these tangle-bearing neurons die. The formation of neurofibrillary tangles closely parallels the progression and anatomic distribution of neuronal loss in Alzheimer's disease, suggesting that these lesions play a role in the disease pathogenesis. Mutations in the human tau gene cause autosomal dominant neurodegenerative disorders. These and other neurodegenerative conditions are also characterized by extensive neurofibrillary pathology. The mechanisms underlying tau-mediated neurotoxicity remain unclear; however, phosphorylated tau is a strong candidate for a toxic molecule, particularly those isoforms phosphorylated by the kinases glycogen synthase kinase 3beta and Cdk5. Here we show that Alzheimer tau binds to Hsc70, and its phosphorylation is a recognition requirement for the addition of ubiquitin (Ub) by the E3 Ub ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) and the E2 conjugating enzyme UbcH5B. Other E3 Ub ligases including parkin and Cbl failed to ubiquitinate phosphorylated tau. CHIP could rescue phosphorylated tau-induced cell death, and therefore the CHIP-Hsc70 complex may provide a new therapeutic target for the tauopathies.

Animals↗
PubMed2004

An androgen receptor NH2-terminal conserved motif interacts with the COOH terminus of the Hsp70-interacting protein (CHIP).

The NH2-terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species. In this study, a primary sequence homology comparison identified a 14-amino acid NH2-terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates. The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species. The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution. The function of the AR NH2-terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner. We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation. In support of this, two mutations in the AR NH2-terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR. Our results suggest that the AR NH2-terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity. Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH2-terminal domains and other intrinsically unstructured transcriptional regulatory proteins.

Amino Acid Motifs↗
PubMed2004

A foldable CFTR{Delta}F508 biogenic intermediate accumulates upon inhibition of the Hsc70-CHIP E3 ubiquitin ligase.

CFTRDeltaF508 exhibits a correctable protein-folding defect that leads to its misfolding and premature degradation, which is the cause of cystic fibrosis (CF). Herein we report on the characterization of the CFTRDeltaF508 biogenic intermediate that is selected for proteasomal degradation and identification of cellular components that polyubiquitinate CFTRDeltaF508. Nonubiquitinated CFTRDeltaF508 accumulates in a kinetically trapped, but folding competent conformation, that is maintained in a soluble state by cytosolic Hsc70. Ubiquitination of Hsc70-bound CFTRDeltaF508 requires CHIP, a U box containing cytosolic cochaperone. CHIP is demonstrated to function as a scaffold that nucleates the formation of a multisubunit E3 ubiquitin ligase whose reconstituted activity toward CFTR is dependent upon Hdj2, Hsc70, and the E2 UbcH5a. Inactivation of the Hsc70-CHIP E3 leads CFTRDeltaF508 to accumulate in a nonaggregated state, which upon lowering of cell growth temperatures, can fold and reach the cell surface. Inhibition of CFTRDeltaF508 ubiquitination can increase its cell surface expression and may provide an approach to treat CF.

Animals↗
PubMed2003

High quality ion channel analysis on a chip with the NPC technology.

In evaluating ion channel function, electrophysiology, e.g., patch clamping, provides the highest information content. For the analysis of ion channel-modulating compounds, one variant of the patch-clamp technique, the whole-cell configuration, is particularly useful. We present here patch-clamp recordings in the whole-cell configuration and single channel recordings performed with planar patch-clamp chips, which are microstructured from borosilicate glass substrate. The chips are used in the Port-a-Patch, an ion channel research/screening instrument that enables automated patch-clamp experiments on a single cell. A software runs the experiment by executing user-determined protocols for cell positioning, as well as for electrical stimulation and current readout. In various electrophysiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated. Quantitative pharmacological experiments are performed on sodium channels expressed in HEK cells using solution volumes in the low microliter range. The exceptionally low volume consumption in the experiments make the system attractive for work on rare or expensive compounds. Due to the low volumes necessary, a rapid solution exchange is facilitated, which is shown on RBL cells. The patch-clamp chip enables a rapid and precise perfusion, allowing sophisticated investigations on ion channel function with the Port-a-Patch.

Algorithms↗
PubMed2003

CHIP activates HSF1 and confers protection against apoptosis and cellular stress.

Induction of molecular chaperones is the characteristic protective response to environmental stress, and is regulated by a transcriptional program that depends on heat shock factor 1 (HSF1), which is normally under negative regulatory control by molecular chaperones Hsp70 and Hsp90. In metazoan species, the chaperone system also provides protection against apoptosis. We demonstrate that the dual function co-chaperone/ubiquitin ligase CHIP (C-terminus of Hsp70-interacting protein) regulates activation of the stress-chaperone response through induced trimerization and transcriptional activation of HSF1, and is required for protection against stress-induced apoptosis in murine fibroblasts. The consequences of this function are demonstrated by the phenotype of mice lacking CHIP, which develop normally but are temperature-sensitive and develop apoptosis in multiple organs after environmental challenge. CHIP exerts a central and unique role in tuning the response to stress at multiple levels by regulation of protein quality control and transcriptional activation of stress response signaling.

Amino Acid Substitution↗
PubMed2004

CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation.

Molecular chaperones, ubiquitin ligases and proteasome impairment have been implicated in several neurodegenerative diseases, including Alzheimer's and Parkinson's disease, which are characterized by accumulation of abnormal protein aggregates (e.g. tau and alpha-synuclein respectively). Here we report that CHIP, an ubiquitin ligase that interacts directly with Hsp70/90, induces ubiquitination of the microtubule associated protein, tau. CHIP also increases tau aggregation. Consistent with this observation, diverse of tau lesions in human postmortem tissue were found to be immunopositive for CHIP. Conversely, induction of Hsp70 through treatment with either geldanamycin or heat shock factor 1 leads to a decrease in tau steady-state levels and a selective reduction in detergent insoluble tau. Furthermore, 30-month-old mice overexpressing inducible Hsp70 show a significant reduction in tau levels. Together these data demonstrate that the Hsp70/CHIP chaperone system plays an important role in the regulation of tau turnover and the selective elimination of abnormal tau species. Hsp70/CHIP may therefore play an important role in the pathogenesis of tauopathies and also represents a potential therapeutic target.

Animals↗
PubMed2002

Mongolian gerbils can utilize provitamin-A carotenoids in deep-fried carrot chips.

Deep-fried carrot chips, containing provitamin-A carotenes, were developed as an alternative mode of dietary intervention to combat vitamin A deficiency. The biological use of carotenoids in this product as vitamin A precursors was evaluated in Mongolian gerbils. Male 4-wk-old gerbils were fed a diet containing all essential nutrients for 1 wk. Then six gerbils were killed, and the remaining gerbils were fed the diet without vitamin A for 6 wk to produce marginal vitamin A deficiency. After depletion, six gerbils were killed and the remainder divided into four groups of 12 gerbils each and fed vitamin A-containing diet (+VA), beta-carotene-containing diet (BC), carrot chip-containing diet (CC), or diet containing no vitamin A/provitamin-A carotenes (-VA). The first three diets contained approximately 6 microg RE/g. Six gerbils from each group were killed after 2 wk of consuming these diets, and 6 after 4 wk. Final body weight and weekly food consumption did not differ among groups after 2 or 4 wk of repletion. Total liver vitamin A stores of BC and CC gerbils killed after 4 wk of repletion were not different from those of gerbils killed before depletion, but those of -VA gerbils were significantly lower (P < 0.05) and those of +VA gerbils were significantly higher (P < 0.05). Plasma retinol levels of gerbils killed after 4 wk of repletion, including the -VA group, did not differ. Total liver alpha- and beta-carotenes and 9-cis beta-carotene contents of the CC group were significantly higher (P < 0.05) than in the BC group after 4 wk of repletion. This carrot chip product effectively reversed vitamin A deficiency in gerbils.

Animals↗
PubMed2002

Rapid parallel mutation scanning of gene fragments using a microelectronic protein-DNA chip format.

We have developed a method for the de novo discovery of genetic variations, including single nucleotide polymorphisms and mutations, on microelectronic chip devices. The method combines the features of electronically controlled DNA hybridisation on open-format microarrays, with mutation detection by a fluorescence-labelled mismatch- binding protein. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals, as demonstrated for mutations in different exons of the p53 gene. This microelectronic chip-based mutation discovery assay may substitute for time-consuming sequencing studies and will complement existing technologies in genomic research.

Adenosine Triphosphatases↗
PubMed1989

Phase II study of iproplatin (CHIP) in previously treated small-cell lung cancer.

Eighteen patients with previously treated extensive small-cell carcinoma of the lung were entered into a Phase II study employing iproplatin (CHIP), a cis-platin analog. Patients had received a mean of two prior chemotherapeutic regimens. Fifty-five percent had received prior cis-platinum and 33% had received prior radiation therapy. CHIP (225 mg/m2) was administered by intermittent intravenous infusion over 30 min for 5 days of a 28-day cycle without prehydration. Sixteen patients with Zubrod performance scores of less than or equal to 3 received 27 courses of therapy (mean 1.7, range 1-6). One partial response of 167 days duration was observed, with complete regression of involved lymph nodes and stabilization of nonevaluable disease in the chest. Six patients had stable disease following one cycle of CHIP, but all progressed following a second cycle of drug. The main toxicity was myelosuppression with prominent thrombocytopenia. Median survival was 75 days from initiation of therapy. In this group of heavily pretreated patients with advanced disease, iproplatin has only minimal activity as a single agent and does not show non-cross-resistance with cis-platinum.

Aged↗
PubMed1995

Phase II trial of CHIP for the treatment of advanced, hormonally refractory carcinoma of the prostate. A Southwest Oncology Group Study.

CHIP, a second generation analogue of cisplatin, was subjected to a Phase II trial for the treatment of advanced, hormonally refractory carcinoma of the prostate. Forty-six patients were treated with CHIP 300 mg/m2 intravenously every 4 weeks. Evaluations for tumor response were done after three cycles of therapy. Among 40 evaluable patients there were no complete responses, but there were 6 partial responses for an overall response rate of 15% (95% confidence interval of 5.7 to 29.8%). The median time to response was 2.4 months and the median progression-free survival was 4.1 months. Median survival was 8.4 months. The most common toxicities were hematologic and gastrointestinal. While CHIP can be administered on an outpatient basis, its response rate for prostatic carcinoma appears to be similar to that of cisplatin.

Adenocarcinoma↗
PubMed2006

A surgical treatment of severe late posttraumatic enophthalmos using sliced costal cartilage chip grafts.

The efficacy of sliced costal cartilage chip grafts for the treatment of late posttraumatic enophthalmos was investigated. Surgery was conducted based on the method reported by Matsuo et al. in 1989. After making an incision in the lower eyelid, dissecting the subperiosteum of the medial orbital wall, orbital floor and lateral orbital wall was performed to the posterior of the orbit, and then costal cartilage chips were gradually grafted in a step-like configuration to the subperiosteum from a location posterior to the equatorial plane of the eyeball. At this time, as well as to the area of concave depression in the orbital bone caused by the fracture, grafts were made to the subperiosteum of the non-deformed medial and lateral orbital wall, to move all of the orbital tissue, including the eyeball, forward. This was performed for five cases of severe late posttraumatic enophthalmos. Among the five cases, there were four cases of severe orbital fracture and one case for which malignant orbital tumor extirpation and radiation therapy had been performed. Following surgery, although mild enophthalmos remained in three of the five cases, esthetically satisfactory results were obtained for all cases. Costal cartilage chip grafts were shown to be an effective method for the treatment of late posttraumatic enophthalmos.

Adult↗
PubMed2001

Approach to a multiparametric sensor-chip-based tumor chemosensitivity assay.

Although not widely practiced by oncologists, in vitro tumor chemosensitivity assays (TCA) have proved to increase the lifetime of tumor patients in prospective clinical trials. By individualizing cancer therapy, they can support the clinician's decision which is usually based on empirically retrieved data and thereby prevent inadequate chemotherapy. We present the first results of a new sensor-chip-based technology which might be useful for a multiparametric TCA. In particular, the aspect of dynamic on-line data generation on intact cellular specimens is a major difference to alternative assays. A series of experiments has been performed on cell lines and human tumor explants. Cell cultures and tumor tissue explants were placed on miniaturized silicon and glass sensor chips. The sensor data currently analyze metabolic profiles (rates of extracellular acidification and cellular oxygen consumption) and changes in cell morphology (monitoring of electric impedance). With the cell lines, drug-associated cellular signals have been detected with all three parameters, while primary explants so far caused metabolic responses only. In particular, cellular respiration or mitochondrial activity seems to be a most sensitive indicator of acute cytotoxic effects. The experimental results were achieved using different test versions. Besides giving a status report, the theoretical potential and current problems of sensor chip technology in TCA is discussed.

Acetaldehyde↗
PubMed1997

Pulmonary responses after wood chip mulch exposure.

Organic Dust Toxic Syndrome (ODTS) is a flu-like syndrome that can occur after inhalation of cotton, grain, wood chip dusts, or other organic dusts or aerosols. We investigated whether inflammatory pulmonary responses occur, even after relatively brief, low-level wood chip mulch exposure. Six volunteers were exposed to wood chip mulch dust. Total dust and/or endotoxin levels were measured in five subjects. Pulmonary function and peripheral blood counts were measured before and after exposure in each subject. Bronchoalveolar lavage (BAL) was performed in each subject after exposure, and cell, cytokine, and protein concentrations were measured. Control BAL without previous exposure was also performed on three of the subjects. Three of six subjects had symptoms consistent with ODTS. No clinically relevant or statistically significant changes in pulmonary function tests after exposure were found. Three subjects manifested a marked elevation in neutrophil percentage in their BAL (range, 10 to 57%). When these three subjects underwent control BAL, the postexposure comparison demonstrated an increase in neutrophil levels of 154 +/- 89 x 10(3)/mL (mean +/- standard error; P = 0.22). The mean increase in BAL interleukin-8 levels after exposure, compared with paired control values, was 11.2 +/- SE 2.5 pg/mL (P = 0.047). There was also an increase in BAL interleukin-6 levels that reached borderline significance (6.4 +/- SE 2.0 pg/mL; P = 0.08). Tumor necrosis factor levels were increased in all three subjects' BAL as well (0.4 +/- SE 0.2 pg/mL), but this change was not statistically significant (P = 0.2). Our findings of increased BAL proinflammatory cytokine and neutrophil levels are consistent with the theory that cytokine networking in the lung may mediate ODTS.

Adult↗
PubMed2002

Impact of the coronary health improvement project (CHIP) on several employee populations.

The greatest potential to improve public health lies in the ability of individuals to adopt healthful behaviors. The purpose of this study was to determine whether participation in a facilitator-based video version of the Coronary Health Improvement Project (CHIP) would improve health behaviors and significantly reduce employee health risks. Employees (n = 442) from six worksites in metropolitan Rockford, Illinois, were used in a pretest/posttest design. Employees self-selected to participate in a facilitator-based, CHIP video program. Participants received instruction twice a week, for 8 weeks, via 15 videos shown at each participating worksite. Demographic and biometric data (body weight, body mass index, blood lipids, blood pressure, and fasting blood glucose) were evaluated at baseline and at 8 weeks. All sites individually and collectively demonstrated significant and meaningful reductions in body weight, body mass index, total cholesterol, low-density lipoprotein cholesterol, triglycerides, and fasting blood glucose. Men demonstrated greater improvement than women, and individuals with higher baseline health risks experienced the greatest reductions in risk. The CHIP video program appears to be an effective method of lowering employee health risks. Future research is needed to determine how long these reductions may persist.

Blood Pressure↗
PubMed2003

Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa.

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. We show here that this communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes and we show that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. Our study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication.

Animals↗