Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Cascade testing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,315 records · Page 73Linked to original sources
PubMed1985

Efficacy of discontinuous flow centrifugation compared with cascade filtration in Waldenstrom's macroglobulinemia: a pilot study.

In the treatment of Waldenstrom's macroglobulinemia (WM) and lipoprotein or immune complex mediated diseases, centrifugal and membrane plasma separation systems have been successfully employed over the past years. More recently, semiselective double filtration systems have been used in isolated cases. While it is hoped that cascade filtration (CF) will soon become a routine technique, a note of caution came from some investigators because of the lack of any evidence that CF does really deplete patients' plasma of known pathogenic macromolecules. The aim of this study was to test the efficacy of CF in the removal of IgM paraproteins in comparison with discontinuous flow centrifugation (DFC). Eight patients were treated by DFC and seven by CF; there were no significant differences between the two groups in terms of sex, age, clinical severity and IgM plasma concentration. After DFC or CF sessions, IgM concentration diminished by 50% and plasma viscosity by 60%, producing a comparable mean reduction in patients' clinical severity. Our in vivo results thus preliminarily answer the question whether CF is capable of removing known pathogenic macromolecules from patients' plasma, and the technique appears to warrant further investigations.

Centrifugation↗
PubMed2003

Treatment of metastatic melanoma with an orally available inhibitor of the Ras-Raf-MAPK cascade.

The Ras-Raf-MAPK pathway is constitutively activated in the majority of melanomas because of a mutation in the BRAF gene. It has been hypothesized that activation of this pathway is crucial for the genesis and maintenance of melanoma and therefore represents an attractive clinical target for metastatic disease. We synthesized a previously characterized MAP kinase kinase inhibitor to test the effect that blocking the Ras-Raf-MAPK pathway would have on the establishment and maintenance of melanoma metastases. Oral administration of CI 1040 inhibited formation of pulmonary metastases and caused rapid regression of established pulmonary metastases in the mouse. Our findings indicate that Ras-Raf-MAPK activation provides crucial signals for the survival of melanoma cells at ectopic sites and that the pharmacological inhibition of this pathway is a promising target for melanoma therapy.

Animals↗
PubMed2002

Role of deoxycytidine kinase (dCK) activity in gemcitabine's radioenhancement in mice and human cell lines in vitro.

BACKGROUND: Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is a deoxycytidine nucleoside analog which has a marked effect on several enzymes involved in DNA synthesis and repair. Gemcitabine has been tested as a radiosensitizer in various biological models, and radiation dose modification factors (DMF) have been reported in the range between 1.1 and 2.4. Gemcitabine is a prodrug that requires intracellular activation by phosphorylation into its active triphosphate dFdCTP form. Deoxycytidine kinase (dCK) is the enzyme involved in the first phosphorylation cascade, and several observations have suggested that dCK was a limiting factor for the cytotoxic activity of gemcitabine. OBJECTIVE: In the present article, we investigated the relationship between dCK activity and gemcitabine's radiosensitization in four mice and two human cell lines. MATERIALS AND METHODS: Four mice and two human tumor cell lines were investigated. Radiosensitization was assessed on confluent cell incubated with 5 microM gemcitabine for 3 h prior to a single radiation dose. Enzymatic activity was assessed using deoxycytidine as substrate with (specific activity) or without (total activity) inhibition of thymidine kinase 2 activity. dCK protein level was assessed by immunoblotting using a rabbit anti-human dCK antibody. mRNA expression was assessed with Northern blot using beta-actin as internal control. RESULTS: Gemcitabine's radiosensitization was heterogeneous with DMF ranging from 0.8 to 1.5. A good correlation was observed between the specific dCK activity and the protein level or the mRNA expression indicating that in our cell systems no post-transcriptional or post-translational activation occurred. An excellent correlation (r = 0.99) was observed between the specific enzymatic activity and gemcitabine's radiosensitization. Cell lines that expressed a high enzymatic activity were the more radiosensitized by gemcitabine. This correlation holds when radiosensitization was plotted against the dCK mRNA expression and protein level. CONCLUSIONS: The present study has suggested the role of dCK activity in gemcitabine's radioenhancement in human and mice cell lines. The study suggests that determination of the enzymatic activity prior to a concurrent gemcitabine and radiotherapy treatment might represent a good predictive assay for tumor response. Such concept should deserve further testing in pre-clinical and clinical settings.

Animals↗
PubMed2004

Metabotropic glutamate receptor type 4 is involved in autoinhibitory cascade for glucagon secretion by alpha-cells of islet of Langerhans.

In islets of Langerhans, L-glutamate is stored in glucagon-containing secretory granules of alpha-cells and cosecreted with glucagon under low-glucose conditions. The L-glutamate triggers secretion of gamma-aminobutyric acid (GABA) from beta-cells, which in turn inhibits glucagon secretion from alpha-cells through the GABAA receptor. In the present study, we tested the working hypothesis that L-glutamate functions as an autocrine/paracrine modulator and inhibits glucagon secretion through a glutamate receptor(s) on alpha-cells. The addition of L-glutamate at 1 mmol/l; (R,S)-phosphonophenylglycine (PPG) and (S)-3,4-dicarboxyphenylglycine (DCPG), specific agonists for class III metabotropic glutamate receptor (mGluR), at 100 micromol/l; and (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I) at 50 micromol/l inhibited the low-glucose-evoked glucagon secretion by 87, 81, 73, and 87%, respectively. This inhibition was dose dependent and was blocked by (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG), a specific antagonist of class III mGluR. Agonists of other glutamate receptors, including kainate and quisqualate, had little effectiveness. RT-PCR and immunological analyses indicated that mGluR4, a class III mGluR, was expressed and localized with alpha- and F cells, whereas no evidence for expression of other mGluRs, including mGluR8, was obtained. L-Glutamate, PPG, and ACPT-I decreased the cAMP content in isolated islets, which was blocked by CPPG. Dibutylyl-cAMP, a nonhydrolyzable cAMP analog, caused the recovery of secretion of glucagon. Pertussis toxin, which uncouples adenylate cyclase and inhibitory G-protein, caused the recovery of both the cAMP content and secretion of glucagon. These results indicate that alpha- and F cells express functional mGluR4, and its stimulation inhibits secretion of glucagon through an inhibitory cAMP cascade. Thus, L-glutamate may directly interact with alpha-cells and inhibit glucagon secretion.

Animals↗
PubMed2001

Role of stimulatory guanine nucleotide binding protein (GSalpha) in proliferation of PC-3M prostate cancer cells.

Previous studies have shown that calcitonin-like immunoreactive substances are secreted by primary prostate cells. Furthermore, exogenously added calcitonin stimulates proliferation of androgen-responsive LnCaP cells. To examine the possible effect of calcitonin on growth of invasive prostate cancer cells, we tested its effects on proliferation of PC-3M cells. Calcitonin stimulated DNA synthesis of PC-3M cells in a dose-dependent fashion, and also stimulated adenylyl cyclase and protein kinase C activities. To further delineate the role of these signaling cascades in proliferation of PC-3M prostate cancer cells, we selectively activated these pathways by transfecting cDNAs expressing constitutively active forms of either Gsalpha (Gsalpha-QL) or Gqalpha (Gqalpha-QL). cDNAs expressing wild-type forms of G-proteins (Gsalpha-WT and Gqalpha-WT) were used as vehicle controls. Gqalpha-QL transfectants exhibited growth inhibition and terminal differentiation. Those expressing Gsalpha-QL exhibited a dramatic increase in growth rate. Gsalpha-QL transfectants displayed an almost 3-fold increase in [3H]-thymidine incorporation and over a 4-fold increase in growth rate when compared with parental PC-3M cells or those expressing wild-type Gsalpha (Gsalpha-WT). The growth-promoting action of Gsalpha-QL could not be mimicked by either 8-bromo cAMP or forskolin. However, nifedipine, a calcium channel antagonist, potently and selectively inhibited DNA synthesis in Gsalpha-QL transfectants. These results suggest that the growth-promoting actions of Gsalpha on PC-3M cells may be mediated by nifedipine-sensitive proliferative events.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Characterization of complement-fixing antibodies to peripheral nerve myelin in Guillain-Barré syndrome.

Complement-fixing antibodies to peripheral nerve myelin (anti-PNM Ab) can be detected in the serum of patients with Guillain-Barré syndrome (GBS). Kinetics of these antibodies can be correlated with the changing clinical course; the appearance of activation products of the terminal complement cascade in cerebrospinal fluid, serum, and peripheral nerve of patients with GBS; and the ability of GBS serum to mediate complement-dependent demyelination of myelinating cultures of rodent dorsal root ganglion. Some of the anti-PNM Ab in all GBS serum tested thus far bind a neutral glycolipid of human PNM and cross react with Forssman antigen, a cross-species antigen found in many infectious agents. Studies suggest that an IgM antibody in GBS patients that could be triggered by multiple infectious agents binds a surface determinant of a Forssman-like lipid of human PNM and participates in demyelination of peripheral nerve through the activation of complement.

Autoantibodies↗
PubMed2005

Possible role of P-glycoprotein in cyclophosphamide resistance of transplanted mouse RLS lymphosarcoma.

The causes of different sensitivity of mouse LS lymphosarcoma and its resistant RLS variant to cyclophosphamide were studied. Division of LS and RLS cells stops in the G2/M phase 24 h after cyclophosphamide treatment, but this stop lasts for more than 48 h in LS cells and less than 24 h in RLS cells. DNA fragmentation, a marker of apoptosis, is observed only in LS cells starting from 24 h after cyclophosphamide treatment. LS and RLS strains do not differ by the expression of bcl-2, bcl-6, bax, bad, mdr1a, mdr1b genes and P-glycoprotein protein. The strains differ by transport activity of P-glycoprotein, tested by SYTO 16 substrate release from cells: activity of P-glycoprotein in RLS cells was 2-fold higher than in LS cells. Presumably, the resistance of RLS tumor to cyclophosphamide-induced apoptosis is a result of inhibition of the apoptotic cascade by P-glycoprotein which is functionally more active in these cells than in LS cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Arrestin binding determines the rate of inactivation of the G protein-coupled receptor rhodopsin in vivo.

G protein-coupled receptor inactivation is a crucial feature of cellular signaling systems; this process determines the catalytic lifetime of the activated receptor and is necessary for response termination. Although previous work has indicated a class of models in which several sequential steps are required for receptor inactivation, the rate-limiting event is still unclear. In this paper, we develop a theory that describes the kinetics of inactivation of the G protein-coupled receptor rhodopsin based on the rate of arrestin binding and test the theory using a combination of genetic and electrophysiological techniques in Drosophila photoreceptors. The theory quantitatively describes the inactivation kinetics of activated rhodopsin in vivo and can be independently tested with molecular and spectroscopic data. The results demonstrate that the rate of arrestin binding determines the kinetics of receptor inactivation in vivo and thus is the event that controls signal amplification at the first step of this G protein-coupled transduction cascade.

Animals↗
PubMed2004

Biomatrix/polymer composite material for heart valve tissue engineering.

BACKGROUND: Decellularized extracellular matrix has been suggested as a scaffold for heart valve tissue engineering or direct implantation. However, cell removal impairs the physical properties of the valve structure and exposes bare collagen fibers that are highly thrombogenic. Matrix/polymer hybrid valves with improved biological and mechanical characteristics may be advantageous. METHODS: Porcine aortic valves were decellularized enzymatically and impregnated with biodegradable poly(hydroxybutyrate) by a stepwise solvent exchange process. Biocompatibility was tested in vitro using cell proliferation and coagulation assays. Proinflammatory activity was assessed in vivo by implantation of matrix/polymer patches in the rabbit aorta. Biomechanic valve properties and fluid dynamics were tested in a pressure/flow-controlled pulse duplicating system. Matrix/polymer hybrid valves were implanted in pulmonary and aortic position in sheep. RESULTS: Biocompatibility assays indicated that human blood vessel cells survive and proliferate on matrix/polymer hybrid tissue. In vitro activation of cellular and plasmatic coagulation cascades was lower than with uncoated control tissue. After implantation in the rabbit aorta, matrix/polymer hybrid patches healed well, with complete endothelialization, mild leukocyte infiltration, and less calcification than control tissue. Matrix/polymer hybrid tissue had superior tensile strength and suture retention strength, and hybrid valves showed good fluid dynamic performance. The two valves in aortic position performed well, with complete endothelialization and limited inflammatory cell invasion after 12 weeks. Of the two valves in pulmonary position, one failed. CONCLUSIONS: Matrix/polymer hybrid tissue valves have good biological and biomechanic characteristics and may provide superior replacement valves.

Absorbable Implants↗
PubMed2005

Role of Ras/PKCzeta/MEK/ERK1/2 signaling pathway in angiotensin II-induced vascular smooth muscle cell proliferation.

The role of protein kinase C (PKC) and its cross talk with extracellular signal-regulated kinase (ERK) cascade in angiotensin II (AngII)-elicited vascular smooth muscle cell (VSMC) proliferation are still unclear. In this study, the PKC pathway of AngII to activate ERK1/2 and induce cell proliferation was investigated in rat aortic smooth muscle cells. The proliferation of VSMCs was tested by [3H]-thymidine incorporation assay. Phosphorylated and non-phosphorylated PKCzeta, ERK1/2, Elk-1, and mitogen-activated ERK-activating kinase (MEK) were estimated by Western blot analysis. The interactions of signal molecules were examined by immunoprecipitation. AngII-induced VSMC proliferation and activation of ERK1/2 and nuclear transcription factor Elk-1 were all down-regulated by PKC non-specific inhibitor (staurosporine) and PKCzeta pseudosubstrate inhibitor (PS-PKCzeta). Dominant negative Ras transfection into VSMCs decreased AngII-induced PKCzeta and ERK1/2 phosphorylation. AngII stimulated the association of PKCzeta with Ras. AngII-induced MEK phosphorylation was inhibited by PKCzeta pseudosubstrate inhibitor and the PKCzeta-MEK complex was detected by immunoprecipitation. These results suggest that PKCzeta isoform is involved in VSMC proliferation and Elk-1 activation. AngII can activate ERK1/2 by Ras/PKCzeta/MEK pathway, which may be one of the important signal transduction pathways in AngII-induced VSMC proliferation.

Angiotensin II↗
PubMed1990

Light adaptation in the primate retina: analysis of changes in gain and dynamics of monkey retinal ganglion cells.

The responses of monkey retinal ganglion cells to sinusoidal stimuli of various temporal frequencies were measured and analyzed at a number of mean light levels. Temporal modulation tuning functions (TMTFs) were measured at each mean level by varying the drift rate of a sine-wave grating of fixed spatial frequency and contrast. The changes seen in ganglion cell temporal responses with changes in adaptation state were similar to those observed in human subjects and in turtle horizontal cells and cones tested with sinusoidally flickering stimuli; "Weber's Law" behavior was seen at low temporal frequencies but not at higher temporal frequencies. Temporal responses were analyzed in two ways: (1) at each light level, the TMTFs were fit by a model consisting of a cascade of low- and high-pass filters; (2) the family of TMTFs collected over a range of light levels for a given cell was fit by a linear negative feedback model in which the gain of the feedback was proportional to the mean light level. Analysis (1) revealed that the temporal responses of one class of monkey ganglion cells (M cells) were more phasic at both photopic and mesopic light levels than the responses of P ganglion cells. In analysis (2), the linear negative feedback model accounted reasonably well for changes in gain and dynamics seen in three P cells and one M cell. From the feedback model, it was possible to estimate the light level at which the dark-adapted gain of the cone pathways in the primate retina fell by a factor of two. This value was two to three orders of magnitude lower than the value estimated from recordings of isolated monkey cones. Thus, while a model which includes a single stage of negative feedback can account for the changes in gain and dynamics associated with light adaptation in the photopic and mesopic ranges of vision, the underlying physical mechanisms are unknown and may involve elements in the primate retina other than the cone.

Adaptation, Ocular↗
PubMed2006

Subdiffraction photon guidance by quantum-dot cascades.

We report on a waveguide composed of a cascade of gain-enabled quantum dots with subwavelength dimensions. Fabrication is demonstrated through DNA-mediated self-assembly and a two-layer molecular self-assembly process that enables rapid prototyping. The device, which is identified with fluorescence microscopy and tested by optical near field detection, allows optical signal transfer at a well-defined wavelength in flexible routing geometry, such as straight paths and 90 degrees bends. The structure serves as a critical building block for nanophotonic systems with high integration density.

Adsorption↗
PubMed2003

CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants.

The significance of CD95/Fas ligand expression by melanoma cells has remained a controversial matter in recent years. On the other hand, CD95 activation may represent a powerful tool for eliminating tumor cells. Here, we demonstrate expression of CD95 in 15/17 human melanoma cell lines analysed, but complete lack of CD95 ligand (CD95L). Overexpression of CD95 in a tetracycline-inducible expression system enhanced melanoma cell sensitivity to CD95 ligation but was unable to trigger apoptosis by itself. In clear contrast, all melanoma cells tested responded with increased apoptosis to conditional expression of CD95L (2-10-fold), both after transient and after stable transfection. Activation of caspase-8, Bid cleavage, cytochrome c release and caspase-3 activation followed after CD95L induction indicating a functional CD95-signaling cascade. CD95L was also able to enhance the proapoptotic effect of chemotherapeutics applied in parallel. Nude mouse experiments revealed that tumorigenicity was lost when melanoma xenografts were triggered to express CD95L. In addition, further progression of pre-existing melanomas was inhibited and even regression was seen after induction of CD95L expression. Due to these data, transfection of CD95L proofs as a highly efficient tool against melanoma cells in vitro and in vivo, and targeted expression of CD95L may thus represent a suitable strategy for melanoma therapy.

Animals↗
PubMed2005

Two subclasses of lung squamous cell carcinoma with different gene expression profiles and prognosis identified by hierarchical clustering and non-negative matrix factorization.

Current clinical and histopathological criteria used to define lung squamous cell carcinomas (SCCs) are insufficient to predict clinical outcome. To make a clinically useful classification by gene expression profiling, we used a 40 386 element cDNA microarray to analyse 48 SCC, nine adenocarcinoma, and 30 normal lung samples. Initial analysis by hierarchical clustering (HC) allowed division of SCCs into two distinct subclasses. An additional independent round of HC induced a similar partition and consensus clustering with the non-negative matrix factorization approach indicated the robustness of this classification. Kaplan-Meier analysis with the log-rank test pointed to a nonsignificant difference in survival (P = 0.071), but the likelihood of survival to 6 years was significantly different between the two groups (40.5 vs 81.8%, P = 0.014, Z-test). Biological process categories characteristic for each subclass were identified statistically and upregulation of cell-proliferation-related genes was evident in the subclass with poor prognosis. In the subclass with better survival, genes involved in differentiated intracellular functions, such as the MAPKKK cascade, ceramide metabolism, or regulation of transcription, were upregulated. This work represents an important step toward the identification of clinically useful classification for lung SCC.

Biomarkers, Tumor↗
PubMed2005

Reactive blue prevented caffeine-induced neurotoxicity by an independent mechanism from intracellular calcium currents in cell culture from auditory cortex of rats.

Neurotoxicity induced by caffeine in auditory-neuron cultures was studied in rat pups. For possible protective effect, reactive blue (RB) alone and in combination with dantrolene were tested in subsequent doses. RB was found to have a U-shape neuroprotective effect in caffeine neurotoxicity. Dantrolene was also tested in combined application in caffeine neurotoxicity. Despite the existing neuroprotection, no additional protection was obtained with various doses of dantrolene. In conclusion, RB may exert neuroprotective effect by increasing intracellular ATP levels in caffeine toxicity. High ATP levels may postpone the toxic cascade. Dantrolene as an endoplasmic reticulum calcium release blocker had no additional protective effect, suggesting that the increased intracellular calcium levels may be involved in later states of the toxic cascade, occurring after the compensatory phase of the cell death.

Animals↗
PubMed2001

Human immunodeficiency virus type 1 entry inhibitors PRO 542 and T-20 are potently synergistic in blocking virus-cell and cell-cell fusion.

Human immunodeficiency virus type 1 (HIV-1) entry proceeds via a cascade of events that afford promising targets for therapy. PRO 542 neutralizes HIV-1 by blocking its attachment to CD4 cells, and T-20 blocks gp41-mediated fusion. Both drugs have shown promise in phase 1/2 clinical trials. Here, the drugs were tested individually and in combination in preclinical models of HIV-1 infection, and inhibition data were analyzed for cooperativity by using the combination index method. Synergistic inhibition of virus-cell and cell-cell fusion was observed for phenotypically diverse viruses for a broad range of drug concentrations, often resulting in > or = 10-fold dose reductions in vitro. Additional mechanism-of-action studies probed the molecular basis of the synergies. The markedly enhanced activity observed for the PRO 542:T-20 combination indicates that the multistep nature of HIV-1 entry leaves the virus particularly vulnerable to combinations of entry inhibitors. These findings provide a strong rationale for evaluating combinations of these promising agents for therapy in vivo.

Animals↗
PubMed2005

High-throughput screening using beta-lactamase reporter-gene technology for identification of low-molecular-weight antagonists of the human gonadotropin releasing hormone receptor.

G-protein coupled receptors (GPCRs) signal via G-proteins to intracellular second messengers. Assays that link transcription of a detectable reporter to promoters that are activated by such signaling cascades are highly sensitive and allow screening for compounds that either activate or inactivate a GPCR of interest. This study describes the development and performance of an antagonistic screen on the human gonadotropin releasing hormone receptor (GnRH-R). Compounds (245,000) were tested in a high-throughput screen using a Chinese hamster ovary cell line stably expressing the human GnRH-R and the Ca2+ sensitive reporter nuclear factor activated in T-cells/ activator protein-1-beta-lactamase. In total, 4,160 active compounds were identified. Colored and toxic compounds, as well as dust and compound aggregates, have been depicted as artifacts. To deselect non-target hits, several follow-up assays, including luminescent and fluorescent Ca2+ mobilization assays and radioligand binding, were developed for the GnRH-R. These assays were validated using peptide and low-molecular-weight GnRH-R reference compounds before hits from screening were also profiled in these assays. For several reference compounds the use of different assay technologies resulted in a poor correlation of potency values. In conclusion, beta-lactamase as a primary high-throughput screening assay is a powerful complementation to other screening technologies. The beta-lactamase technology has several advantages, including lack of cell lysis and ratiometric read-out, which augments assay robustness. Based on technology comparison, it is not adequate to assume that the same hits would be found regardless of which assay technology is used.

Animals↗
PubMed2003

Identification of protein tyrosine phosphatases with specificity for the ligand-activated growth hormone receptor.

Protein tyrosine phosphatases (PTPs) play key roles in switching off tyrosine phosphorylation cascades, such as initiated by cytokine receptors. We have used substrate-trapping mutants of a large set of PTPs to identify members of the PTP family that have substrate specificity for the phosphorylated human GH receptor (GHR) intracellular domain. Among 31 PTPs tested, T cell (TC)-PTP, PTP-beta, PTP1B, stomach cancer-associated PTP 1 (SAP-1), Pyst-2, Meg-2, and PTP-H1 showed specificity for phosphorylated GHR that had been produced by coexpression with a kinase in bacteria. We then used GH-induced, phosphorylated GH receptor, purified from overexpressing mammalian cells, in a Far Western-based approach to test whether these seven PTPs were also capable of recognizing ligand-induced, physiologically phosphorylated GHR. In this assay, only TC-PTP, PTP1B, PTP-H1, and SAP-1 interacted with the mature form of the phosphorylated GHR. In parallel, we show that these PTPs recognize very different subsets of the seven GHR tyrosines that are potentially phosphorylated. Finally, mRNA tissue distribution of these PTPs by RT-PCR analysis and coexpression of the wild-type PTPs to test their ability to dephosphorylate ligand-activated GHR suggest PTP-H1 and PTP1B as potential candidates involved in GHR signaling.

Amino Acid Sequence↗