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Results for “cell deconvolution”

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PubMed1995

Developmental increase in CA3-CA1 presynaptic function in the hippocampal slice.

1. We recorded extracellular and intracellular CA3-CA1 synaptic responses in hippocampal slices from neonatal rats [postnatal day (P) 15-21 and P29-35]. Presynaptic function was examined by measuring input-output relationships and paired-pulse facilitation and by quantal analysis of minimally evoked responses. 2. Extracellular recording revealed no difference in excitatory postsynaptic potential (EPSP) threshold or the fiber potential response for a given stimulus intensity between the two age groups. However, the slope of the field EPSP was consistently larger in older animals. The increase in EPSP slope was associated with a decrease in paired-pulse facilitation, suggesting an increase in presynaptic function with postnatal development. 3. Extracellular results were confirmed by intracellular recordings that revealed no difference in the minimal stimulation intensity needed to evoke a response, an increase in mean EPSP amplitude with development, and a decrease in paired-pulse facilitation. Quantal parameters were extracted by three separate methods including method of failures, coefficient of variance, and parameter optimization through noise deconvolution. All methods supported presynaptic mediation of facilitation. Comparison of quantal parameters during development indicated an increase in mean quantal content. 4. The results demonstrate that synaptic strength is altered over the course of development because of, at least in part, changes in presynaptic release mechanisms. Developmental differences in presynaptic function provide an explanation of differences in mechanisms for expression of long-term potentiation. The lower initial probability of transmitter release in neonates may permit increased presynaptic change.

Animals↗

PubMed1998

Pancreatic beta-cell responsiveness during meal tolerance test: model assessment in normal subjects and subjects with newly diagnosed noninsulin-dependent diabetes mellitus.

A model-based method was developed to quantify pancreatic beta-cell responsiveness during a meal tolerance test (MTT). C peptide secretion was related in a linear fashion to glucose concentration, whereas the standard population model was used to derive transfer rate constants of the two compartmental model of C peptide kinetics. Two indexes of pancreatic beta-cell responsiveness were defined: 1) postprandial sensitivity M(I) (ability of postprandial glucose to stimulate beta-cell), and 2) basal sensitivity M0 (ability of fasting glucose to stimulate beta-cell). The method was evaluated using plasma glucose and C peptide measured over 180 min with a 10- to 30-min sampling interval during a MTT (75 g carbohydrates; 500 Cal) performed in 16 normal subjects (7 men and 9 women; age, 50 +/- 10 yr; body mass index, 29.2 +/- 3.6 kg/m2; fasting plasma glucose, 5.1 +/- 0.5 mmol/L; mean +/- SD) and 16 body mass index-matched subjects with newly diagnosed noninsulin-dependent diabetes mellitus (NIDDM; 15 men and 1 woman; age, 50 +/- 9 yr; body mass index, 29.3 +/- 3.7 kg/m2; fasting plasma glucose, 12.6 +/- 3.2 mmol/L). M(I) and M0 indexes were estimated with very good precision (coefficient of variation, < 15%). Subjects with NIDDM demonstrated lower postprandial sensitivity M(I) (17.7 +/- 11.4 vs. 90.0 +/- 43.3 x 10(-9)/min; NIDDM vs. normal, P < 0.001) and basal sensitivity M0 (5.4 +/- 2.2 vs. 10.3 +/- 4.9 x 10(-9)/min; P < 0.005). Deconvolution analysis documented that the relationship between C peptide secretion and glucose concentration is approximately linear during MTT in both normal subjects (plasma glucose range, 5-8 mmol/L) and subjects with NIDDM (12-17 mmol/L). We conclude that pancreatic responsiveness during glucose stimulation (M(I)) and under basal conditions (M0) can be obtained from this novel method during MTT in healthy and disease states.

Adult↗

PubMed2002

High-resolution imaging of the human retina with a Fourier deconvolution technique.

A high-resolution retinal imaging camera is described that uses a Shack-Hartmann wave-front sensor and a Fourier deconvolution imaging technique. The operation of the camera is discussed in detail and high-resolution retinal images of the human cone mosaic are shown for a retinal patch approximately 10 arc min in diameter from two different retinal locations. The center-to-center cone spacing is shown to be approximately 2.5 microm for the retinal images recorded at 2 degrees temporal from the central fovea and approximately 4 microm for the retinal images recorded at 3 degrees temporal from the central fovea.

Diagnostic Imaging↗

PubMed2006

Acceleration of AMPA receptor kinetics underlies temperature-dependent changes in synaptic strength at the rat calyx of Held.

It is well established that synaptic transmission declines at temperatures below physiological, but many in vitro studies are conducted at lower temperatures. Recent evidence suggests that temperature-dependent changes in presynaptic mechanisms remain in overall equilibrium and have little effect on transmitter release at low transmission frequencies. Our objective was to examine the postsynaptic effects of temperature. Whole-cell patch-clamp recordings from principal neurons in the medial nucleus of the trapezoid body showed that a rise from 25 degrees C to 35 degrees C increased miniature EPSC (mEPSC) amplitude from -33 +/- 2.3 to -46 +/- 5.7 pA (n=6) and accelerated mEPSC kinetics. Evoked EPSC amplitude increased from -3.14 +/- 0.59 to -4.15 +/- 0.73 nA with the fast decay time constant accelerating from 0.75 +/- 0.09 ms at 25 degrees C to 0.56 +/- 0.08 ms at 35 degrees C. Direct application of glutamate produced currents which similarly increased in amplitude from -0.76 +/- 0.10 nA at 25 degrees C to -1.11 +/- 0.19 nA 35 degrees C. Kinetic modelling of fast AMPA receptors showed that a temperature-dependent scaling of all reaction rate constants by a single multiplicative factor (Q10=2.4) drives AMPA channels with multiple subconductances into the higher-conducting states at higher temperature. Furthermore, Monte Carlo simulation and deconvolution analysis of transmission at the calyx of Held showed that this acceleration of the receptor kinetics explained the temperature dependence of both the mEPSC and evoked EPSC. We propose that acceleration in postsynaptic AMPA receptor kinetics, rather than altered presynaptic release, is the primary mechanism by which temperature changes alter synaptic responses at low frequencies.

Animals↗

PubMed2004

Deconvolution of adaptive optics retinal images.

We quantitatively demonstrate the improvement to adaptively corrected retinal images by using deconvolution to remove the residual wave-front aberrations. Qualitatively, deconvolution improves the contrast of the adaptive optics images. In this work we demonstrate that quantitative information is also increased by investigation of the improvement to cone classification due to the reduction in confusion of adjacent cones because of the extended wings of the point-spread function. The results show that the error in classification between the L and M cones is reduced by a factor of 2, thereby reducing the number of images required by a factor of 4.

Algorithms↗

PubMed1998

Insulin secretion rates estimated by two mathematical methods in pancreas-kidney transplant recipients.

After pancreas-kidney transplantation, it is difficult to obtain an accurate estimate of the insulin secretion of the pancreas graft, since several pitfalls are involved using peripheral C-peptide and/or insulin measurements in this determination. In this study, the individual kinetic parameters of C-peptide and then the rates of insulin secretion were estimated by two mathematical methods, the deconvolution method and the "combined model" during slow (oral glucose) and fast (intravenous glucagon) changes in insulin secretion in six successful pancreas-kidney transplant recipients with systemic delivery of insulin (Px), six nondiabetic kidney-transplant recipients with portal insulin secretion (Kx), six nondiabetic controls (NS), and six C-peptide-negative insulin-dependent diabetes mellitus patients (IDDM). Decreased C-peptide clearance and basal and poststimulatory hyperinsulinemia were found in both Px and Kx compared with NS (P < 0.05). Similar glucose responses were observed after intravenous glucagon in all groups, whereas the responses after oral glucose were 30% higher in Px and Kx than in NS (P < 0.05). During oral glucose and after intravenous glucagon, both mathematical methods resulted in significantly lower maximal and incremental insulin secretion rates (ISR) in Px than in Kx (P < 0.05). In contrast, calculations of incremental ISR in NS and Px induced by the two beta-cell stimuli were about the same but significantly higher in Kx than in NS (P < 0.05). These results differed markedly from those obtained using peripheral measurements of insulin and C-peptide alone. In conclusion, when C-peptide clearance and insulin metabolism change, such as in pancreas-kidney transplant recipients, accurate evaluation of insulin secretion from the graft can be obtained only by using individual kinetics of the peptides before calculating the ISR. This study also clearly demonstrates that insulin secretion after pancreas transplantation is still defective.

Adult↗

PubMed1990

Interconversion of metarhodopsins I and II: a branched photointermediate decay model.

Flash photolysis experiments designed to monitor the establishment of the metarhodopsin I to metarhodopsin II equilibrium are interpreted according to a branched model in which two spectrally indistinguishable but kinetically distinguishable forms of metarhodopsin II are postulated to exist in equilibrium with a common pool of metarhodopsin I. This interpretation arises from the consistent requirement for at least three exponentials for a valid description of the observed growth of absorbance at 380 nm following bleaching of bovine rhodopsin in rod outer segment disk membranes. Analysis of the 380-nm transient absorbance data permitted direct determination of the five physically interpretable individual rate constants of the model. This analysis represents a more explicit interpretation of kinetic data than that employed in earlier experiments of this kind, which involved estimating only apparent rates and apparent amplitudes of discrete multiexponential functions. The 380-nm absorbance contributions of all relevant species contributing to the observed dynamic absorbance change were accounted for simultaneously during nonlinear least-squares estimation of the model rate parameters. Analysis of deconvoluted equilibrium spectra acquired from samples identical with those used in the kinetics experiments confirmed the metarhodopsin I-metarhodopsin II equilibrium constants, Keq, derived from the dynamic analyses. It is shown that Keq varies from 1.28 at 10 degrees C to 7.3 at 37 degrees C and that approximately 90% of the metarhodopsin II present is in the form of metarhodopsin IIslow over the temperature range 10-37 degrees C. A physical interpretation of this decay model is discussed in the context of a distribution of metarhodopsin II structural and energetic states.

Cell Membrane↗

PubMed2000

On the influence of pigment-protein interactions on energy transfer processes in photosynthetic membrane structures. 3. The FMO complex of Chlorobium tepidum at high pressure.

The low-temperature absorption spectra of the Chlorobium tepidum FMO bacteriochlorophyll-protein complex at various pressures have been calculated within the framework of mini-exciton theory. The dependences of the Qy transition energies of the monomeric pigments on pressure have been found by means of functional minimization. This functional includes the parameters of both theoretical and experimental absorption spectra at low temperatures and various pressures. The dependences obtained are compared with those derived for the exciton transition energies, which have been obtained by deconvoluting absorption spectra with seven Gaussian components at each pressure. The pressure increase has been shown to result in the increased coupling energy between both the pigment molecules themselves and pigments and amino acid residues. The pigment molecules capable of binding histidines and water molecules have been shown to have the greatest and smallest responses to increased pressure, respectively. The couplings of Bchl molecules with the surrounding amino acid residues have been shown to change both the exciton delocalization index and the exciton distribution between the pigment molecules within the protein subunit; the increased pressure does not change these parameters significantly.

Bacterial Proteins↗

PubMed1991

Nuclear calcium gradients in cultured rat hepatocytes.

Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.

Algorithms↗

PubMed2005

Development of a microplate-based scintillation proximity assay for MraY using a modified substrate.

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

Bacterial Proteins↗

PubMed2001

Numerical selection of optimal tumor imaging agents with application to engineered antibodies.

Three analytic indicators were used to compare five members of a monoclonal antibody (Mab) family. The cognates consisted of the genetically engineered intact chimeric IgGI (cT84.66) and related engineered fragments [scFv, diabody, minibody, F(ab')2] reactive against the same epitope of carcinoembryonic antigen (CEA). All analyses were based on radioiodinated Mabs targeting to colorectal xenografts of LS174T tumors in nude mice. Affinity constants were evaluated initially. A second indicator was the imaging figure of merit (IFOM) which determines how rapidly a statistically significant tumor image can be acquired. Finally, deconvolution was used to determine tumor temporal response to an arterial bolus. This last analysis gave the possible tumor accumulation in the absence of normal tissue sequestration. Affinities were all in excess of 10(8) M-1 and were highest for the divalent Mabs. Using the IFOM criterion, an 131I label was best suited as a radiolabel for the intact (IgG) T84.66, while an 123I label indicated optimal imaging with either minibody or F(ab')2. Deconvolution analyses showed that divalent members behaved similarly while the univalent member (scFv) had a tumor residence time smaller by an order of magnitude. The diabody had the largest impulse response function, but renal uptake may limit its present usefulness.

Animals↗

PubMed2002

The combined administration of GH-releasing peptide-2 (GHRP-2), TRH and GnRH to men with prolonged critical illness evokes superior endocrine and metabolic effects compared to treatment with GHRP-2 alone.

OBJECTIVE: Central hyposomatotrophism, hypothyroidism and hypogonadism are present concomitantly in men with prolonged critical illness. This study evaluated the impact of combined treatment with GH-releasing peptide-2 (GHRP-2), TRH and GnRH for 5 days compared with GHRP-2 + TRH and with GHRP-2 alone. PATIENTS AND DESIGN: Thirty-three men with prolonged critical illness participated at baseline compared to 50 age- and body mass index (BMI)-matched controls. Patients were randomly assigned to 5 days of placebo (n = 7), GHRP-2 (1 microg/kg/h; n = 9), GHRP-2 + TRH infusion (1 + 1 microg/kg/h; n = 9) or pulsatile GnRH (0.1 microg/kg every 90 min) together with GHRP-2 + TRH infusion (n = 8). MEASUREMENTS: GH, TSH and LH secretion were quantified by deconvolution analysis of serum concentration time series obtained by sampling every 20 min from 2100 to 0600 h at baseline and on nights 1 and 5 of treatment. Serum concentrations of IGF-I, IGFBPs, thyroid hormones, gonadal and adrenal steroids, proinflammatory cytokines and selected metabolic and inflammation markers were measured daily. RESULTS: Patients revealed suppressed pulsatile GH, TSH and LH secretion in the face of low serum concentrations of IGF-I, IGFBP-3 and the acid-labile subunit (ALS) (P < 0.0001 each), thyroid hormones (P < 0.0001) and total and estimated free testosterone (P < 0.0001) levels, whereas free oestradiol (E2) estimates were normal. Serum dehydroepiandrosterone sulphate (DHEAS) levels were also suppressed whereas morning cortisol was normal. Serum levels of type I procollagen (PICP) and bone alkaline phosphatase (sALP) were elevated whereas osteocalcin (OC) was low (P = 0.03). Ureagenesis (P < 0.0001) and breakdown of bone tissue (P < 0.0001) were increased. Baseline serum TNF-alpha, IL-6 and C-reactive protein level and white blood cell (WBC) count were elevated; serum lactate was normal. Only low T4 and high IGFBP-1 levels independently predicted mortality. GHRP-2 infusion reactivated GH secretion and normalized serum IGF-I, IGFBP-3 and ALS. GHRP-2 + TRH infusion reactivated both the GH axis and the thyroid axis, with normal levels of T4 and T3 reached within 1 day. Only GHRP-2 + TRH infusion combined with GnRH pulses reactivated the GH and TSH axis and at the same time increased pulsatile LH secretion compared to placebo. Only GnRH pulses together with GHRP-2 + TRH infusion increased testosterone significantly from day 2 (peak increase of + 312%) through day 5 and serum E2 with > 80% from day 1 through day 3 (all P = 0.05). Ureagenesis was reduced by GHRP-2 + TRH + GnRH (P = 0.01) and by GHRP-2 + TRH (P = 0.009) but not by GHRP-2 alone. Serum OC levels were increased only by GHRP-2 + TRH + GnRH (P = 0.03), with a trend for GHRP-2 + TRH (P = 0.09), but not by GHRP-2 alone. On day 5, serum lactate levels and WBC count were increased by GHRP-2 infused alone and in combination with TRH but not by GHRP-2 + TRH + GnRH. CONCLUSIONS: Coadministration of GHRP-2, TRH and GnRH reactivated the GH, TSH and LH axes in prolonged critically ill men and evoked beneficial metabolic effects which were absent with GHRP-2 infusion alone and only partially present with GHRP-2 + TRH. These data underline the importance of correcting the multiple hormonal deficits in patients with prolonged critical illness to counteract the hypercatabolic state.

Aged↗

PubMed2001

Gliadin nanoparticles as carriers for the oral administration of lipophilic drugs. Relationships between bioadhesion and pharmacokinetics.

PURPOSE: The aim of this work was to evaluate the bioadhesive properties of non-hardened gliadin nanoparticles (NPs) and cross-linked gliadin nanoparticles (CL-NP) in the carbazole pharmacokinetic parameters obtained after the oral administration of these carriers. METHODS: A deconvolution model was used to estimate the carbazole absorption when loaded in the different gliadin nanoparticles. In addition, the elimination rates of both adhered and non-adhered nanoparticulate fractions within the stomach were estimated. RESULTS: Nanoparticles dramatically increased the carbazole oral bioavailability up to 49% and provided sustained release properties related to a decrease of the carbazole plasma elimination rate. The carbazole release rates from nanoparticles (NP and CL-NP), calculated by deconvolution, were found to be of the same order as the elimination rates of the adhered fractions of nanoparticles in the stomach mucosa. In addition, good correlation was found between the carbazole plasmatic levels, during the period of time in which the absorption process prevails, and the amount of adhered carriers to the stomach mucosa. CONCLUSION: Gliadin nanoparticles significantly increased the carbazole bioavailability, providing sustained plasma concentrations of this lipophilic molecule. These pharmacokinetic modifications were directly related to the bioadhesive capacity of these carriers with the stomach mucosa.

Administration, Oral↗

PubMed1999

A Homogenous 384-Well High Throughput Screen for Novel Tumor Necrosis Factor Receptor: Ligand Interactions Using Time Resolved Energy Transfer.

The herpes virus entry mediator (HVEM) receptor and its ligand, HVEM-L, are involved in both herpes simplex virus type-1 (HSV-1) herpes simplex virus type-2 (HSV-2) infection, and in T-cell activation such that antagonists of this interaction are expected to have utility in viral and inflammatory diseases. In this report we describe the configuration of a homogeneous 384-well assay based on time-resolved energy transfer from a europium chelate on the HVEM receptor to an allophycocyanin (APC) acceptor on the ligand. Specific time resolved emission from the acceptor is observed on receptor:ligand complex formation. The results of various direct and indirect labeling strategies are described. Several assay optimization experiments were necessary to obtain an assay that was robust to automation and file compound interference while sensitive to the effect of potential inhibitors. The signal was stable for more than 24 h at room temperature using the Eu(3+) chelates, suggesting no dissociation of the lanthanide ion. The 384-well assay was readily automated and was able to identify more than 99.5% of known positive controls in the validation studies successfully. Screening identified both a series of known potent inhibitors and several structural classes of hits that readily deconvoluted to yield single compound inhibitors with the desired functional activity in secondary biological assays. The equivalence of the data in 384- and 1536-well formats indicates that routine implementation of 1536-well chelate-based energy transfer screening appears to be primarily limited by liquid handling rather than detection issues.

Journal Article↗

PubMed2004

The immune-enhancing enteral agents arginine and glutamine differentially modulate gut barrier function following mesenteric ischemia/reperfusion.

BACKGROUND: Immune-enhancing enteral diets have been shown to improve patient outcome. One contributing mechanism may be via maintenance of gut barrier function. While recent data has shown that glutamine is beneficial, arginine may be harmful. We therefore hypothesized that the immune-enhancing agents, glutamine and arginine, differentially modulate gut barrier function. METHODS: At laparotomy, rats had jejunal sacs filled with 10 mmol/L glutamine, arginine, fructose, or magnesium sulfate (osmotic control) followed by 60 minutes of superior mesenteric artery occlusion and 2 hours of reperfusion. Jejunum was harvested for histology, deconvolution microscopy, F:G actin, ATP, and permeability measurements. RESULTS: Glutamine and fructose minimized mucosal injury compared with controls and arginine. Deconvolution microscopy confirmed that glutamine and fructose preserved the actin cytoskeleton but there was disruption by arginine which correlated with F:G actin ratios and tissue ATP levels. Permeability was enhanced by arginine compared with the other groups. CONCLUSION: Arginine resulted in worsened mucosal injury, disruption of the actin cytoskeleton, decreased tissue ATP and enhanced permeability compared with glutamine which appeared protective. The immune-enhancing agent arginine results in breakdown of gut barrier function which may have important implications for critically injured patients.

Actins↗

PubMed2026

Immunopeptidomics-driven MHC class II peptide-binding motif discovery for 2 common canine DR alleles.

Despite the central role of major histocompatibility complex (MHC) class II in adaptive immunity, peptide-binding motifs have yet to be characterized for any canine MHC class II allele. Here, we report the first immunopeptidomics-derived binding motifs for DLA-DRB1*015:01 (DLA-DR15) and DLA-DRB1*012:01 (DLA-DR12), 2 alleles overrepresented in breeds predisposed to immune-mediated diseases. Because dogs co-express DLA-DR and DLA-DQ, the MHC class II Ab clone YKIX334.2 was validated to be DLA-DR-specific, enabling allele-selective immunoaffinity purification of DLA-DR molecules from homozygous DLA-DR15 and DLA-DR12 donor spleens. Mass spectrometry and GibbsCluster motif deconvolution of 838 DLA-DR15-associated and 644 DLA-DR12-associated peptides eluted from their respective peptide-binding grooves revealed distinct allele-specific binding motifs, with characterization of anchor residue preferences, peptide-length distributions, cross-species comparisons with human and murine MHC class II motifs, and source protein composition of the eluted self-peptidome. To evaluate the translational utility of these motifs, recombinant DLA-DR15 and DLA-DR12 molecules were used to screen rabies virus glycoprotein and nucleoprotein peptide libraries via fluorescence-based peptide competition assays, identifying high-affinity candidate binders for both alleles. Spearman rank correlation between immunopeptidomics-derived position-specific scoring matrix scores and peptide competition assay rankings demonstrated modest associations, consistent with these approaches capturing complementary dimensions of peptide-MHC class II interaction. Ultimately, these findings establish what we believe is the first allele-specific peptide-binding motif framework for canine MHC class II, providing a foundation for DLA-allele-informed CD4+ T-cell epitope discovery studies and Ag-specific immune response characterization in the dog.

Animals↗

PubMed2006

Hyperproinsulinaemia in normoglycaemic lipodystrophic HIV-infected patients.

BACKGROUND: We aimed to investigate whether the insulin precursors, intact (IP) and 32-33 split proinsulin (SP), which are elevated in states of insulin resistance and predict type 2 diabetes, would be elevated in human immunodeficiency virus (HIV)-infected patients with lipodystrophy (LIPO). MATERIALS AND METHODS: Forty-three normoglycaemic HIV-infected patients [18 LIPO and 18 without lipodystrophy (NONLIPO) receiving antiretroviral drugs, and seven patients naïve to antiretroviral drugs (NAIVE)] were examined. Insulin precursors were measured during fasting, during an intravenous glucose tolerance test and during a hyperinsulinaemic-euglycaemic clamp, respectively. Insulin secretion rates (ISR) were determined by deconvolution of C-peptide concentrations. Disposition index (DI) was calculated as insulin sensitivity (Si(RD)) multiplied by the first-phase insulin response to intravenous glucose. RESULTS: LIPO exhibited increased fasting IP and SP (P < 0.05), a higher proportion of elevated fasting IP (3.1 pmol L(-1), 66% vs. 33% and 28%, P < 0.05) and SP (7.2 pmol L(-1), 50%, 11% and 0%, P < 0.01), reduced Si(RD) (> 50%, P < 0.001) and increased ISR (P < 0.001) compared with NONLIPO and NAIVE. Fasting SP and IP correlated positively with ISR (P < 0.001) and inversely and hyperbolically with Si(RD) (P < 0.001). Fasting SP/insulin ratio correlated inversely with Si(RD) (P < 0.05). Incremental IP + SP/insulin ratio after an intravenous glucose bolus correlated inversely with DI (P < 0.01), but did not differ between study groups. CONCLUSIONS: Proinsulin appeared to be increased in HIV-lipodystrophy, but no more than caused by the increased ISR. Nevertheless, the inverse correlations between SP/insulin ratio versus Si(RD) and incremental total proinsulin/insulin ratio versus DI may argue for a subtle beta-cell dysfunction in those patients with insulin resistance and low DI.

Adult↗

PubMed2003

The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes.

Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.

Actin Cytoskeleton↗