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Results for “cell deconvolution”

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PubMed2002

Allelic variation in exon 18 of the sulfonylurea receptor 1 (SUR1) gene, insulin secretion and insulin sensitivity in nondiabetic relatives of type 2 diabetic subjects.

BACKGROUND: We have previously observed associations of the T-allele of the exon 18 variant (ACC --> ACT; Thr759Thr) of the sulfonylurea receptor 1 (SUR1) gene with type 2 diabetes mellitus (T2DM). Here we assess beta-cell function and insulin sensitivity in carriers of different genotypes at this locus. METHODS: Pre-hepatic insulin secretion rates (ISR) derived by deconvolution of circulating C-peptide levels, and glucose clearance were assessed during graded infusions of intravenous glucose in CC-homozygous (n=6) and CT-heterozygous (n=6) nondiabetic relatives of CT-heterozygous type 2 diabetic subjects. RESULTS: Average ISR over the duration of the study, adjusted for sex, age, BMI and prevailing glucose levels, were lower in CT-heterozygous subjects as compared with CC-homozygous subjects (3.91 +/- 0.40 vs. 4.84 +/- 0.28 pmol/kg x min(-1); p=0.048). The correlation curves relating ISR to glucose levels were significantly different in the two groups (analyses of covariance p=0.029). Glucose clearance was similar in both groups. CONCLUSIONS: Insulin secretion rates, but not insulin sensitivity, assessed during graded infusion of glucose were mildly decreased in nondiabetic relatives of type 2 diabetic subjects, who carry the at risk T-allele of exon 18 variant of the SUR1 gene. These results suggest that the at-risk allele might have a small effect on pancreatic B-cell function and contribute to the development of T2DM in these families.

ATP-Binding Cassette Transporters↗

PubMed1998

Quantification of Ggamma- and Agamma-globins by electrospray ionisation mass spectrometry.

Elucidation of the molecular basis for persistent fetal haemoglobin (Hb F) production in adult life has important implications for the pathophysiology and treatment of human beta haemoglobinopathies. Electrospray ionisation mass spectrometry (ESMS) was applied to analyse the pattern of gamma-globin expression in patients with hereditary persistence of fetal haemoglobin (HPFH) and sickle cell anaemia (SCA). Ggamma and Agamma-globin chains were identified by their measured molecular masses and distinguished by mass difference (14 Da) following deconvolution of ESMS spectra using maximum entropy based software. Prediction of HPFH type by ESMS was confirmed by molecular analysis. Direct determination of Ggamma:Agamma globin chain ratio from whole blood by the novel application of ESMS provides a rapid and sensitive approach to characterisation of gamma-globins and facilitates correlation of gamma-globin level and polymorphism of cis-active elements at the beta-globin locus.

Adult↗

PubMed1994

Changes in the kinetics and biopotency of luteinizing hormone in hemodialyzed men during treatment with recombinant human erythropoietin.

To investigate the effect of recombinant human erythropoietin (rh-EPO) on the hypothalamo-pituitary-gonadal axis in end-stage renal failure, plasma luteinizing hormone (LH) concentration release was assessed by frequent blood sampling (every 10 min), both during an 8-h baseline period and after stimulation with an iv bolus of gonadotropin-releasing hormone (GnRH). Seven adult hemodialyzed men were studied before and after partial correction of anemia by rh-EPO treatment. LH was determined by an in vitro Leydig cell bioassay (bio-LH) and a highly sensitive immunoradiometric assay. Pulsatile bio-LH secretion and clearance characteristics were assessed by multiple-parameter deconvolution analysis. Although the rh-EPO treatment did not lead to a change in average concentrations of plasma bio-LH, the mass of hormone released per secretory burst more than doubled, and the estimated bio-LH production rate increased from 8.8 +/- 2.3 to 15.6 +/- 5.2 IU/L per hour (P = 0.05). The lack of change in mean plasma bio-LH is explained by a simultaneous decrease in plasma half-life from 106 +/- 27 to 67 +/- 19 min (P < 0.02). The decrease in the plasma half-life of bio-LH was closely associated with the rise in hematocrit, suggesting an effect of the increased red blood cell mass on LH distribution space and elimination kinetics. As a consequence of the changes in hormone kinetics, the incremental amplitudes of the plasma concentration pulses of bio-LH increased from 112 to 121% of nadir levels (P < 0.05), resulting in a more distinctly pulsatile pattern of hormone signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1999

A combinatorial approach to the identification of dipeptide aldehyde inhibitors of beta-amyloid production.

In an effort to rapidly identify potent inhibitors of Abeta production and to probe the amino acid sequence specificity of the protease(s) responsible for the production of this peptide, a large number of dipeptide aldehydes were combinatorially synthesized and manually evaluated for their inhibitory properties. The starting point for this study was the dipeptide aldehyde carbobenzoxyl-valinyl-phenylalanal previously shown to inhibit the production of Abeta in CHO cells stably transfected with the cDNA encoding betaAPP695. Pools of related dipeptide aldehydes were combinatorially synthesized, and the most active pool was deconvoluted, resulting in the identification of the most active inhibitor of this pool. Systematic optimization of this inhibitor resulted in a series of dipeptide aldehydes with enhanced potencies relative to carbobenzoxyl-valinyl-phenylalanal. The most active dipeptide aldehydes were those that possessed hydrophobic amino acids at both the P1 and P2 positions. The most potent compound identified in this study was 3, 5-dimethoxycinnamamide-isoleucinyl-leucinal with an IC(50) of 9.6 microM, approximately 10-fold more active than carbobenzoxyl-valinyl-phenylalanal. In immunoprecipitation experiments using antibodies directed toward either Abeta1-40 or Abeta1-42, 3,5-dimethoxycinnamamide-isoleucinyl-leucinal, like carbobenzoxyl-valinyl-phenylalanal, preferentially inhibited the shorter 1-40 form of Abeta, whereas the longer 1-42 form was not as strongly inhibited. These results suggest that dipeptide aldehydes related to carbobenzoxyl-valinyl-phenylalanal inhibit Abeta through similar mechanisms and demonstrate the utility of a combinatorial synthesis approach to rapidly identify potent inhibitors of Abeta production.

Aldehydes↗

PubMed2003

Contribution of abnormal insulin secretion and insulin resistance to the pathogenesis of type 2 diabetes in myotonic dystrophy.

OBJECTIVE: Myotonic dystrophy (MyD), the most common adult form of muscular dystrophy, is often complicated by diabetes. MyD is dominantly inherited and is due to heterozygosity for a trinucleotide repeat expansion mutation in a protein kinase gene able to induce derangement of RNA metabolism responsible of an aberrant insulin receptor expression. RESEARCH DESIGN AND METHODS: To assess insulin sensitivity and secretion before the onset of diabetes, we studied 10 MyD patients, 10 offspring of type 2 diabetes (OFF), and 10 healthy subjects with no family history of diabetes (control subjects) with dual X-ray energy absorption, euglycemic-hyperinsulinemic clamp (40 mU/[m(2). min]) combined with infusion of [6,6-D(2)]-glucose and oral glucose tolerance test (OGTT). RESULTS: MyD had reduced lean body mass, but peripheral insulin sensitivity was not different to that of control subjects in contrast to OFF, which showed insulin resistance. Insulin secretion, obtained by deconvolution of OGTT data, was also shown to be comparable with that of OFF and control subjects (index of beta-cell function = Phi; P = 0.91) even if increased parameters of insulin secretion were found during the first 30 min (Phi(30); P = 0.05) of the oral glucose challenge. Fasting plasma proinsulin concentrations (P = 0.01) and the ratio to insulin (P = 0.01) were increased in MyD patients. The proinsulin levels also failed to be suppressed during the clamp and showed exaggerated response after the OGTT. Increased proinsulin levels were shown to be peculiar of MyD patients when compared with OFF. CONCLUSIONS: In nondiabetic, young MyD patients, insulin sensitivity was preserved, and an increased early secretory response to oral glucose was detected. Abnormal plasma proinsulin levels in the fasting state, during the clamp, and during the OGTT were shown to be secretory dysfunctions peculiar of MyD patients and may be more important than insulin resistance in determining the high risk to develop diabetes in these patients.

Adult↗

PubMed1997

Cobalt and desferrioxamine reveal crucial members of the oxygen sensing pathway in HepG2 cells.

Cobalt and desferrioxamine, like hypoxia, stimulate the production of erythropoietin in HepG2 cells. It is believed that cobalt as well as desferrioxamine interact with the central iron atom of heme proteins by changing their redox state similar to hypoxia. A subsequent decrease of the intracellular H2O2 levels under hypoxia was presumed to be the key event for stimulating erythropoietin production. We therefore investigated whether cobalt and desferrioxamine control the intracellular H2O2 levels that regulate gene expression by interacting with hemeproteins. Deconvolution of light absorption spectra revealed respiratory heme proteins such as cytochrome c, b558 and cytochrome aa3, as well as cytochrome b558, which is a nonrespiratory heme protein found in HepG2 cells. Whereas respiratory heme proteins are located in mitochondria, cytochrome b558 similar to the one described for the neutrophil NADPH oxidase can be visualized in the cell membrane of HepG2 cells by immunohistochemistry. Incubation with cobalt (100 microM/24 hr) interacts predominantly with cytochrome b558 and cytochrome b558. The interaction of cobalt with the respiratory chain results in an increased oxygen consumption of HepG2 cells as revealed by PO2 microelectrode measurements. Desferrioxamine (130 microM/24 hr), however has no influence on the cytochromes. In response to an external application of NADH (1 mM), the membrane bound cytochrome b558 produces two times more O2- than to the external NADPH (1 mM) application. Neither desferrioxamine not cobalt has any influence on the NADH stimulated O2- generation. Incubation with cobalt or with desferrioxamine, however, leads to a decrease of the intracellular H2O2 level as revealed by the dihydrorhodamine 123 technique, perhaps causing the well-known enhanced erythropoietin production. The cobalt-induced H2O2 decrease seems to be caused by an increased activity of the glutathion peroxidase that is also induced under hypoxia. Desferrioxamine, however, leads to an apparent H2O2 decrease only because it seems to inhibit the iron catalyzed reaction of H2O2 with dihydrorhodamine 123, hinting at the occurrence of the Fenton reaction in HepG2 cells. Therefore, it must be determined whether or not degradation products of H2O2 by the Fenton reaction suppress erythropoietin production under normoxia.

Cell Line↗

PubMed2026

Pan-cancer multi-omics machine learning defines a lactylation-associated immune-excluded tumor state with proteomic and experimental corroboration.

BACKGROUND: Histone lactylation links lactate metabolism to chromatin regulation, but whether lactylation-program-associated transcriptional patterns delineate recurrent pan-cancer tumor states remains unclear. METHODS: We integrated mRNA, lncRNA, and miRNA profiles from 9712 TCGA tumors across 33 cancer types with GTEx references, six GEO cohorts, IMvigor210, and an institutional clear-cell renal cell carcinoma (ccRCC) cohort used for exploratory DIA-NN proteomic corroboration. Random-effects co-expression meta-analysis, multi-omics consensus clustering, regulon inference, immune deconvolution, TIDE, oncoPredict, and SHAP-based machine learning were applied. hsa-miR-431-5p was functionally evaluated as a proof-of-concept CS2-associated miRNA in bladder cancer models. RESULTS: LacCoEx-Atlas comprised 398,491 lactylation-related co-expression pairs across 24,667 RNA features under a random-effects framework (median I² = 88.6%). Consensus clustering identified two subtypes: CS2 showed glycolytic-mesenchymal-immune-excluded features, M2 macrophage enrichment, CD8⁺ T-cell depletion, elevated HDAC4/NSD3/KDM6B activity, and worse survival, whereas CS1 showed oxidative, sirtuin-active programs. CS2 had fewer predicted ICI responders (18.3% vs. 52.0%) and a lower observed ORR in IMvigor210 (15.3% vs. 24.0%). oncoPredict identified NU7441 as a hypothesis-generating CS2-associated sensitivity signal (Hedges' g = 1.17). DIA-NN proteomics in 50 ccRCC specimens provided exploratory support for CS2-associated hypoxia, ECM degradation, and metastasis programs. The 10-feature mRNA LARItools model achieved an apparent AUC of 0.9413, while a separate multi-omics model achieved 0.971; neither was independently validated. LARItools reproduced prognostic separation across six GEO cohorts. miR-431-5p promoted malignant phenotypes and EMT in bladder cancer cells, with concordant CMU4h expression findings. CONCLUSIONS: Lactylation-program-associated transcriptional patterns delineate a recurrent immune-excluded pan-cancer tumor state associated with adverse prognosis, reduced predicted immunotherapy responsiveness, exploratory single-cancer protein-level support, and testable DNA damage response-targeting hypotheses. LacCoEx-Atlas and LARItools provide open resources for lactylation-program-associated tumor-state stratification and future translational research.

Humans↗

PubMed2002

Cell surface targeting and clustering interactions between heterologously expressed PSD-95 and the Shal voltage-gated potassium channel, Kv4.2.

Kv4.2 is a voltage-gated potassium channel that is critical in controlling the excitability of myocytes and neurons. Processes that influence trafficking and surface distribution patterns of Kv4.2 will affect its ability to contribute to cellular functions. The scaffolding/clustering protein PSD-95 regulates trafficking and distribution of several receptors and Shaker family Kv channels. We therefore investigated whether the C-terminal valine-serine-alanine-leucine (VSAL) of Kv4.2 is a novel binding motif for PSD-95. By using co-immunoprecipitation assays, we determined that full-length Kv4.2 and PSD-95 interact when co-expressed in mammalian cell lines. Mutation analysis in this heterologous expression system showed that the VSAL motif of Kv4.2 is necessary for PSD-95 binding. PSD-95 increased the surface expression of Kv4.2 protein and caused it to cluster, as shown by deconvolution microscopy and biotinylation assays. Deleting the C-terminal VSAL motif of Kv4.2 eliminated these effects, as did substituting a palmitoylation-deficient PSD-95 mutant. In addition to these effects of PSD-95 on Kv4.2 distribution, the channel itself promoted redistribution of PSD-95 to the cell surface in the heterologous expression system. This work represents the first evidence that a member of the Shal subfamily of Kv channels can bind to PSD-95, with functional consequences.

Amino Acid Motifs↗

PubMed2003

Effects of IgM from rheumatic fever patients on intracellular calcium levels of neonatal rat cardiac myocytes.

Rheumatic fever (RF), a potential sequela of Streptococcus pyogenes pharyngitis, sometimes results in myocarditis and heart failure. Antibodies have been implicated in the pathogenesis of RF and anti-cardiac myosin antibody levels are elevated in RF patients. Since myocarditis is associated with altered cardiomyocyte calcium transients it was of interest to determine the direct effects of RF patient antibodies on calcium transients in cultured myocytes. RF patient polyclonal IgM treatment caused increased calcium retention by neonatal rat heart cells in vitro as determined with isotopically labeled calcium. Therefore, to further characterize this finding, calcium transients were evaluated by real time fluorescence spectroscopy and deconvolution imaging. RF patient polyclonal IgM produced increased calcium retention during the relaxation stage of the contraction cycle leading to a slowing of contraction rate, disorganized calcium transients, and eventual tetany. In contrast, calcium transient studies of cardiomyocytes following treatment with monoclonal anti-myosin antibodies revealed declining intracellular calcium levels, accompanied by disorganized transients and tetany. Treatment with both antibodies led to myocyte dysfunction and these novel findings suggest a role for antibodies in the pathogenesis of the myocarditis associated with rheumatic carditis.

Animals↗

PubMed2003

The GLP-1 derivative NN2211 restores beta-cell sensitivity to glucose in type 2 diabetic patients after a single dose.

Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion in a glucose-dependent manner, but its short half-life limits its therapeutic potential. We tested NN2211, a long-acting GLP-1 derivative, in 10 subjects with type 2 diabetes (means +/- SD: age 63 +/- 8 years, BMI 30.1 +/- 4.2 kg/m(2), HbA(1c) 6.5 +/- 0.8%) in a randomized, double-blind, placebo-controlled, crossover study. A single injection (7.5 micro g/kg) of NN2211 or placebo was administered 9 h before the study. beta-cell sensitivity was assessed by a graded glucose infusion protocol, with glucose levels matched over the 5-12 mmol/l range. Insulin secretion rates (ISRs) were estimated by deconvolution of C-peptide levels. Findings were compared with those in 10 nondiabetic volunteers during the same glucose infusion protocol. In type 2 diabetic subjects, NN2211, in comparison with placebo, increased insulin and C-peptide levels, the ISR area under the curve (AUC) (1,130 +/- 150 vs. 668 +/- 106 pmol/kg; P < 0.001), and the slope of ISR versus plasma glucose (1.26 +/- 0.36 vs. 0.54 +/- 0.18 pmol x l[min(-1) x mmol(-1) x kg(-1)]; P < 0.014), with values similar to those of nondiabetic control subjects (ISR AUC 1,206 +/- 99; slope of ISR versus plasma glucose, 1.44 +/- 0.18). The long-acting GLP-1 derivative, NN2211, restored beta-cell responsiveness to physiological hyperglycemia in type 2 diabetic subjects.

Aged↗

PubMed2003

Metabolic networks of Cucurbita maxima phloem.

Metabolomic analysis aims at a comprehensive characterization of biological samples. Yet, biologically meaningful interpretations are often limited by the poor spatial and temporal resolution of the acquired data sets. One way to remedy this is to limit the complexity of the cell types being studied. Cucurbita maxima Duch. vascular exudates provide an excellent material for metabolomics in this regard. Using automated mass spectral deconvolution, over 400 components have been detected in these exudates, but only 90 of them were tentatively identified. Many amino compounds were found in vascular exudates from leaf petioles at concentrations several orders of magnitude higher than in tissue disks from the same leaves, whereas hexoses and sucrose were found in far lower amounts. In order to find the expected impact of assimilation rates on sugar levels, total phloem composition of eight leaves from four plants was followed over 4.5 days. Surprisingly, no diurnal rhythm was found for any of the phloem metabolites that was statistically valid for all eight leaves. Instead, each leaf had its own distinct vascular exudate profile similar to leaves from the same plant, but clearly different from leaves harvested from plants at the same developmental stage. Thirty to forty per cent of all metabolite levels of individual leaves were different from the average of all metabolite profiles. Using metabolic co-regulation analysis, similarities and differences between the exudate profiles were more accurately characterized through network computation, specifically with respect to nitrogen metabolism.

Circadian Rhythm↗

PubMed2002

Peroxisome remnants in pex3delta cells and the requirement of Pex3p for interactions between the peroxisomal docking and translocation subcomplexes.

During peroxisomal matrix protein import, the peroxisomal targeting signal receptors recognize cargo in the cytosol and interact with docking and translocation subcomplexes on the peroxisomal membrane. Using immunoprecipitations of multiple protein components, we show that in Pichia pastoris the docking subcomplex consists of the unique peroxins Pex13p, Pex14p and Pex17p, whereas the putative translocation subcomplex has all three RING-finger peroxins, Pex2p, Pex10p and Pex12p, as unique constituents. We identify Pex3p as a shared component of both subcomplexes. In pex3delta cells, the unique constituents of the docking subcomplex interact as they do in wild-type cells, but the assembly of the translocation subcomplex is impaired and its components are present at reduced levels. Furthermore, several interactions detected in wild-type cells between translocation and docking subcomplex components are undetectable in pex3delta cells. Contrary to previous reports, pex3delta cells have peroxisome remnants that pellet during high-speed centrifugation, associate with membranes on floatation gradients and can be visualized by deconvolution microscopy using antibodies to several peroxins which were not available earlier. We discuss roles for Pex3p in the assembly of specific peroxisomal membrane protein subcomplexes whose formation is necessary for matrix protein import.

ATP-Binding Cassette Transporters↗

PubMed1980

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity.

The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO4-solution to which K4Fe(CN)6 had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M chi and L chi values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L chi values were reliable, whereas the M chi values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L chi values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.

Acid Phosphatase↗

PubMed1999

Monte Carlo simulation of the heterotypic aggregation kinetics of platelets and neutrophils.

The heterotypic aggregation of cell mixtures or colloidal particles such as proteins occurs in a variety of settings such as thrombosis, immunology, cell separations, and diagnostics. Using the set of population balance equations (PBEs) to predict dynamic aggregate size and composition distributions is not feasible. The stochastic algorithm of Gillespie for chemical reactions (. J. Comput. Phys. 22:403-434) was reformulated to simulate the kinetic behavior of aggregating systems. The resulting Monte Carlo (MC) algorithm permits exact calculation of the decay rates of monomers and the temporally evolving distribution of sizes and compositions of the aggregates. Moreover, it permits calculation of all moments of these distributions. Using this method, we explored the heterotypic aggregation of fully activated platelets and neutrophils in a linear shear flow of shear rate G = 335 s(-1). At plasma concentrations, the half-lives of homotypically aggregating platelet and neutrophil singlets were 8.5 and 2.4 s, respectively. However, for heterotypic aggregation, the half-lives for platelets and neutrophils decreased to 2.0 and 0.11 s, respectively, demonstrating that flowing neutrophils accelerate capture of platelets and growth of aggregates. The required number of calculations per time step of the MC algorithm was typically a small fraction of Omega(1/2), where Omega is the initial number of particles in the system, making this the fastest MC method available. The speed of the algorithm makes feasible the deconvolution of kernels for general biological heterotypic aggregation processes.

Blood Platelets↗

PubMed2026

Association of MPO Expression with the Immune Microenvironment in Breast Cancer: Insights from Bioinformatics and Single-Cell Analyses.

Breast cancer remains a major cause of cancer-related mortality, and exploratory computational workflows can help prioritize immune-associated markers for further investigation. Here, we used the cancer genome atlas breast invasive carcinoma (TCGA-BRCA) bulk transcriptomic data and the public single-cell dataset GSE161529 to examine associations between myeloperoxidase (MPO) expression, clinical outcomes, immune infiltration, methylation, upstream-regulator annotations, single-cell expression patterns, virtual knockdown sensitivity outputs, drug-gene interaction retrieval, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) annotation. MPO expression was lower in breast cancer tissues than in adjacent non-tumor tissues. Higher MPO expression was associated with a longer progression-free interval, whereas its associations with overall survival and disease-specific survival were not statistically significant. Receiver operating characteristic (ROC) analysis suggested tumor-normal separation within the analyzed public dataset, but this should not be interpreted as clinical diagnostic validation. Immune deconvolution and enrichment analyses indicated that MPO expression mainly tracked with immune- and myeloid-related transcriptional features, rather than establishing tumor-intrinsic regulation of the immune microenvironment. At single-cell resolution, the MPO signal was sparse, with only 85 MPO-positive cells detected before k-nearest neighbor (KNN)-based neighborhood expansion. Detectable MPO signal and MPO-associated scores were interpreted cautiously because they may be influenced by sparse expression, cell-type annotation uncertainty, dropout, doublets, or ambient RNA. In silico virtual knockdown suggested candidate immune- and inflammatory-related transcriptional changes, but these results were considered exploratory and require validation. Drug-gene interaction database (DGIdb)-based drug-gene retrieval and ADMET annotation were used only as preliminary chemical annotations and were not interpreted as therapeutic evidence. Overall, this study provides a reproducible in silico workflow for generating hypotheses about MPO-associated immune/myeloid features in breast cancer, which require external cohort validation and experimental confirmation.

Humans↗

PubMed2001

Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen.

Tumor microvessels differ in structure and metabolic function from normal vasculature, and neoangiogenesis is associated with quantitative and qualitative changes in expression of endothelial proteins. Such molecules could serve as molecular addresses differentiating the tumor vasculature from those of the normal brain. We have applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against transformed endothelial cells as a complex target to select single-stranded DNA-ligands (aptamers) that function as histological markers to detect microvessels of rat experimental glioma, a fatal brain tumor that is highly vascularized. Both the SELEX selection procedure as well as subsequent deconvolution-SELEX were analyzed by fluorescence based methods (flow cytometry and fluorescence microscopy). Of 25 aptamers analyzed, one aptamer was selected that selectively bound microvessels of rat brain glioblastoma but not the vasculature of the normal rat brain including peritumoral areas. The molecular target protein of aptamer III.1 was isolated from endothelial cells by ligand-mediated magnetic DNA affinity purification. This protein was identified by mass spectrometry as rat homologue of mouse pigpen, a not widely known endothelial protein the expression of which parallels the transition from quiescent to angiogenic phenotypes in vitro. Because neoangiogenesis, the formation of new blood vessels, is a key feature of tumor development, the presented aptamer can be used as a probe to analyze pathological angiogenesis of glioblastoma. The presented data show that pigpen is highly expressed in tumor microvessels of experimental rat brain glioblastoma and may play an important role in warranting blood supply, thus growth of brain tumors.

Animals↗

PubMed1981

Material from the internal surface of squid axon exhibits excess noise. Implications in modeling membrane noise.

A fluid material from a squid (Loligo pealei) axon was isolated by mechanical application of two types of microcapillary (1-3-mum Diam) to the internal surface of intact and cut-axon preparations. Current noise in the isolated material exceeded thermal levels and power spectra were 1/f in form in the frequency range 1.25-500 Hz with voltage-dependent intensities that were unrelated to specific ion channels. Whether conduction in this material is a significant source of excess noise during axon conduction remains to be determined. Nevertheless, a source of excess noise external to or within an ion channel may not be properly represented solely as an additive term to the spectrum of ion channel noise; a deconvolution of these spectral components may be required for modeling purposes.

Animals↗

PubMed2001

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal and adult cardiomyocytes.

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.

Age Factors↗