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PubMed1999

Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors.

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

Adaptor Protein Complex alpha Subunits↗

PubMed2002

Automated postprocessing of electrospray LC/MS data for profiling protein expression in bacteria.

We describe an integrated approach for automating protein analysis of bacterial cell extracts. The method uses electrospray LC/MS to generate chromatographic profiles of proteins present in an extract, along with a software program that automates the data analysis. The software program, Retana, automates the sequential summing, centroiding, and deconvolution of multiply charged proteins present in consecutive scans of the LC/MS analysis. This procedure generates a concise, single spectrum of proteins present in the extract, along with their retention time and relative abundance. A comparison of the method with "whole cell" MALDI analysis demonstrates improved mass resolution and mass accuracy, along with the appearance of a greater number of proteins. Additionally, it is possible to compare protein expression among strains of bacteria by normalizing the relative abundance of similar proteins in each analysis.

Automation↗

PubMed1996

Quantitative analysis of three-dimensional distribution of AgNOR proteins during interphase in leukemic cells.

Acidic proteins of the nucleolar organizer regions, selectively stained by silver (AgNOR-proteins), were investigated during interphase in leukemia cells with a confocal scanning laser microscope (CSLM). Simultaneous confocal fluorescence (for specific labeling of DNA, using propidium iodide) and transmitted light microscopy combined with digital deconvolution (for the location of the AgNOR proteins in nonconfocal mode) were used. The distribution of the AgNOR proteins measured by 3D microscopy was described by their number, the volume occupation of the nucleus by the AgNOR aggregates, the distance between each AgNOR, the distance of each AgNOR to the nucleolar border, and their anisotropy. The results of the 3D analysis were compared to those obtained by conventional 2D analysis, cytogenetical analysis of metaphase nucleolar organiser regions (NORs), and cell duplication rate. The descriptive power of these 3D parameters were assessed for nine leukemic cell lines. The measurements of the 3D spatial distribution of AgNORs was a better discriminant parameter than the morphological parameters (i.e., number and volume). The 3D expression of AgNORs is also a reliable parameter for assessing proliferative activity of leukemic cells and seems to be in relation with the differentiation stage of these leukemic cells.

Cell Division↗

PubMed1994

Quantitative three-dimensional confocal microscopy of synaptic structures in living brain tissue.

In order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi-like staining of small numbers of cells and processes that can be resolved clearly using confocal microscopy; viability of stained cells is established by exclusion of the fluorescent DNA-binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high-resolution images are combined in an automated procedure to generate three-dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations.

Animals↗

PubMed1991

Estrogen regulates the gonadotropin-releasing hormone-stimulated secretion of biologically active luteinizing hormone.

Estrogen produces time-dependent bidirectional effects on the GnRH-stimulated release of immunoactive LH in various species. To examine estrogen's regulation of biologically active LH secretion in response to pulsatile stimulation by GnRH, we studied estrogen-deficient postmenopausal women basally and during treatment with diethlystilbesterol (DES; 1 mg, orally, daily). Basal and GnRH-stimulated plasma concentrations of bioactive LH were assayed by the in vitro rat interstitial cell testosterone bioassay. GnRH-promoted LH secretory bursts in response to two consecutive stimuli were quantitated by multiple parameter deconvolution analysis. Basal half-lives of LH averaged 171 +/- 17 min (immunoactive) and 223 +/- 10 min (bioactive). Analysis of variance revealed a significant decrease in mean basal plasma bioactive LH concentrations on days 10 and 30 of DES treatment. Mean serum immunoactive LH concentrations fell similarly. DES significantly increased the half-life of immunoactive LH (days 5 and 10), but did not change that of bioactive LH. GnRH self-priming of bioactive LH secretion (increased LH secretory peak 2 compared to peak 1) was demonstrated, with a maximal value on day 10 of DES treatment. In addition, the ratio of the mass of bioactive to immunoactive LH secreted in response to the first GnRH pulse was significantly enhanced by estrogen on day 5, whereas that after the second pulse of GnRH was significantly suppressed on day 30 of DES. The self-priming action of GnRH on bioactive LH release evident in the presence of oral DES was corroborated in a separate group of six women, who were treated for 30 days with 17 beta-estradiol via an intravaginally placed Silastic ring. In conclusion, we infer that estrogen exerts a highly selective effect on the gonadotroph secretory process, such that successive GnRH stimuli result in an increase in the maximal rate and mass of secretion of biologically active LH.

Administration, Intravaginal↗

PubMed2026

A Practical Workflow for Spatial Transcriptomics Data Analysis: From Data Acquisition to Advanced Analyses.

Spatial transcriptomics (ST) profiles genome-wide gene expression while preserving the two-dimensional spatial context of mRNA molecules within tissue sections, enabling studies of tissue architecture and microenvironment-associated biology. However, ST analysis remains challenging because data import, quality control, integration, deconvolution, spatial statistics, and visualization often require multiple software environments and reproducible parameter choices. This protocol presents a practical computational workflow for public ST datasets in R, beginning with data acquisition and software setup and proceeding through Seurat-based data loading, quality control, normalization, multi-sample integration, clustering, and spatially variable gene analysis. The workflow then applies complementary deconvolution strategies, including reference-guided SPOTlight analysis and unsupervised STdeconvolve topic modeling, followed by Giotto-based spatial cell-cell communication analysis and interactive region-of-interest (ROI) selection using a custom Python Dash application. By emphasizing script-based execution, explicit parameter rationales, expected outputs, and troubleshooting checkpoints, the protocol provides an adaptable framework for standard array-based ST datasets and related platforms after dataset- and platform-specific parameter evaluation.

Spatial Transcriptomics↗

PubMed1999

A comparison between direct determination of in vivo dissolution and the deconvolution technique in humans.

AIM: The primary objective of this study was to investigate the in vivo dissolution of carbamazepine in humans and to compare it with the dissolution estimated by deconvolution of plasma concentrations as well as the in vitro dissolution. METHODS: The in vivo study included six healthy volunteers, and consisted of two sequential parts. In part 1 the dissolution was measured by perfusing a semi-open segment in the proximal jejunum in humans. In part 2 the volunteers were given a solution of carbamazepine orally. In both parts of the study, plasma samples were collected up to 48 h after administration of the dose. The in vitro dissolution was measured in a flow-through cell using dissolution medium with and without the addition of bile acids (3 mM). RESULTS: The direct measured in vivo dissolution profile of carbamazepine and the deconvoluted profile were found to be similar. The two dissolution profiles of carbamazepine obtained in vitro were statistically lower than the two in vivo dissolution profiles. The higher in vivo dissolution rate is probably due to efficient sink conditions as a consequence of the high permeability of carbamazepine and more pronounced intestinal motility. CONCLUSION: The jejunal perfusion system was successfully used for in vivo dissolution measurements of carbamazepine and agreed with the deconvoluted plasma profile regarding rate and extent of dissolution. Single-pass perfusion is therefore a meaningful tool for further studies of in vivo dissolution.

Algorithms↗

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↗

PubMed2004

Robust conversion of marrow cells to skeletal muscle with formation of marrow-derived muscle cell colonies: a multifactorial process.

OBJECTIVE: Murine marrow cells are capable of repopulating skeletal muscle fibers. A point of concern has been the "robustness" of such conversions. We have investigated the impact of type of cell delivery, muscle injury, nature of delivered cell, and stem cell mobilizations on marrow-to-muscle conversion. METHODS: We transplanted green fluorescence protein (GFP)-transgenic marrow into irradiated C57BL/6 mice and then injured anterior tibialis muscle by cardiotoxin. One month after injury, sections were analyzed by standard and deconvolutional microscopy for expression of muscle and hematopoietic markers. RESULTS: Irradiation was essential to conversion, although whether by injury or induction of chimerism is not clear. Cardiotoxin- and, to a lesser extent, PBS-injected muscles showed significant number of GFP(+) muscle fibers, while uninjected muscles showed only rare GFP(+) cells. Marrow conversion to muscle was increased by two cycles of G-CSF mobilization and to a lesser extent by G-CSF and steel or GM-CSF. Transplantation of female GFP to male C57BL/6 and GFP to ROSA26 mice showed fusion of donor cells to recipient muscle. High numbers of donor-derived muscle colonies and up to 12% GFP(+) muscle cells were seen after mobilization or direct injection. These levels of donor muscle chimerism approach levels that could be clinically significant in developing strategies for the treatment of muscular dystrophies. CONCLUSION: In summary, the conversion of marrow to skeletal muscle cells is based on cell fusion and is critically dependent on injury. This conversion is also numerically significant and increases with mobilization.

Animals↗