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Adversarial attack of sequence-free enhancer prediction identifies chromatin architecture.

MOTIVATION: The wide range of cellular complexity created by multicellular organisms is due in large part to the intricate and synergistic interplay of regulatory complexes throughout the eukaryotic genome. These regulatory elements "enhance" specific gene programs and have been shown to operate in diverse networks that are distinct across cell states of the same organism. Attempts to characterize and predict enhancers have typically focused on leveraging information-dense DNA sequence in parallel with epigenomic assays. We examined the viability of enhancer prediction using only a minimal set of epigenomic datasets without direct DNA information. RESULTS: We demonstrate that chromatin datasets are sufficient to identify enhancers genome-wide with high accuracy. By training networks leveraging data from multiple cell types simultaneously, we generated a cell-type invariant enhancer prediction platform that utilized only the patterns of protein binding for inference. We also showed the utility of swarm-based adversarial attacks [adversarial particle swarm optimization (APSO)] to deconvolute trained genomic neural networks for the first time. Critically, unlike saliency mapping or other game-theory based approaches, APSO is completely network-architecture independent and can be applied to any prediction engine to derive the features that drive inference. AVAILABILITY AND IMPLEMENTATION: All software and code for data downloading, processing, enhancer inference, eXplainable AI (XAI), and complete figure generation are publicly available on GitHub at https://github.com/EpiGenomicsCode/ChromEnhancer and Zenodo at https://doi.org/10.5281/zenodo.15652797.

Enhancer Elements, Genetic

Differential responses of biologically active luteinizing hormone secretion in older versus young men to interruption of androgen negative feedback.

To investigate the responsiveness of the healthy aging male hypothalamo-pituitary-gonadal axis to short term interruption of androgen negative feedback, we administered a selective nonsteroidal competitive antagonist of the androgen receptor, flutamide hydrochloride (250 mg, orally, three times daily for 3.5 days), to five older (aged 63-72 yr) and eight young (aged 21-30 yr) men. Pulsatile bioactive LH release was assessed by the rat interstitial cell testosterone bioassay in plasma sampled at 10-min intervals for 8 h overnight at baseline and after flutamide administration. Pituitary responsiveness was evaluated after two successive i.v. injections of 10 micrograms GnRH. Deconvolution analysis was used to estimate the number, amplitude, duration, and mass of bioactive LH secretory bursts and the half-life of biologically active hormone. At baseline, older men exhibited a significantly lower spontaneous bioactive LH secretory burst frequency than young men, with a median of 5 events/8 h (older) vs. 7.5 bursts/8 h (younger, P < 0.05). In older men, mean 8-h plasma bioactive LH concentrations increased significantly in response to flutamide (P = 0.006), and the 8-h calculated secretion rate of bioactive LH rose concomitantly. These increases were similar to responses in young men. However, during antiandrogen administration, the frequency of bioactive LH secretory bursts failed to rise in older men to the baseline value seen in young men. Moreover, older (but not young) men showed a significant prolongation of the LH secretory burst duration in response to flutamide treatment. On the other hand, the estimated half-life of endogenous bioactive LH increased significantly after flutamide ingestion in young compared to older individuals. After GnRH injections, older and young men secreted similar amounts of LH before flutamide administration, but during flutamide treatment, older men released more biologically active LH after the first GnRH stimulus [older men, 104 +/- 11 IU/L (median, 110); younger men, 44 +/- 8.4 IU/L (median, 40); P < 0.05]. Serum free testosterone concentrations rose significantly during flutamide exposure in both young and older men, but estradiol concentrations increased significantly only in young men. In summary, healthy older men exhibit a reduced (overnight) spontaneous bioactive LH secretory burst frequency. Pharmacological attenuation of androgen-mediated negative feedback increases mean serum free testosterone concentrations and plasma bioactive LH concentrations to a similar degree in older and young individuals, but different mechanisms operate in the two age groups. In older men, flutamide treatment amplifies the mass of bioactive LH secreted per burst by prolonging the LH secretory burst duration, whereas in young men, flutamide administration increases the apparent half-life of biologically active LHG significantly relative to values in older men. We conclude that competitive nonsteroidal blockade of the androgen receptor unmasks qualitatively altered mechanisms of increased bioactive LH release in healthy older men.

Adult

Deconvoluting clonal and cellular architecture in IDH-mutant acute myeloid leukemia.

Isocitrate dehydrogenase 1/2 (IDH) mutations are early initiating events in acute myeloid leukemia (AML). The complex clonal architecture and cellular heterogeneity in IDH-mutant AML underlies the heterogeneous clinical presentation and outcomes. Integrating single-cell genotyping and transcriptomics, we demonstrate a stem-like and inflammatory phenotype of IDH-mutant AML and identify clone-specific programs associated with NPM1, NRAS, and SRSF2 co-mutations. Furthermore, these clones had distinct responses to treatment with combination IDH inhibitors and chemotherapy, including elimination, reconstitution of myeloid differentiation, or retention within progenitor populations. At relapse after IDH inhibitor monotherapy, we identify upregulated stemness, inflammation, mitochondrial metabolism, and anti-apoptotic factors, as well as downregulated major histocompatibility complex (MHC) class II antigen presentation. At the pre-leukemic stage, we observe upregulation of IDH2-associated pathways, including inflammation. We deliver a detailed phenotyping of IDH-mutant AML and a framework for dissecting contributions of recurrently mutated genes in AML at diagnosis and following therapy, with implications for precision medicine.

Leukemia, Myeloid, Acute

Estimation of in vivo reticuloendothelial system phagocytic activity in rats by direct blood clearance techniques and nuclear scintigraphy.

There has been considerable interest in the examination of reticuloendothelial system phagocytic blockade. In this study, the kinetics of phospholipid liposome-mediated and intraperitoneal silica-mediated phagocytic blockade were examined using five methods of analysis of in vivo 99mTc-labeled albumin clearance and reticuloendothelial cell uptake. Two direct blood sampling techniques revealed significant impairment in 99mTc-labeled albumin clearance after treatment with silica (P less than 0.05), while liposome treatment was not associated with such impairment. A method utilizing nuclear scintigraphy for the determination of blood clearance was incapable of detecting silica-mediated blockade but demonstrated significant impairment by liposomes at 2 hr (P less than 0.001), 6 hr (P less than 0.05), and 24 hr (P less than 0.001). Gamma camera imaging methods for determination of hepatic uptake demonstrated significant (P less than 0.05) and reversible impairment of 99mTc-labeled albumin uptake by liposomes. The most promising of these techniques utilizes deconvolutional analysis of liver region of interest time-activity curves to correct for continuously changing blood concentrations of tracer and for intracellular tracer processing and catabolism. Measurements of reticuloendothelial system phagocytic activity should include methods that take into account the observed discrepancies between blood clearance determinations and reticuloendothelial cell uptake.

Albumins

Electron probe microanalysis of biological soft tissues: principle and technique.

Electron probe microanalysis is a method based upon X-ray spectrometry used for analyzing the chemical element content of very small amounts of material. The concentration of electrolytes in the microenvironment of cells, in cells, and in intracellular organelles can be measured. The main difficulties in using this method in biological soft tissue lie in sample preparation and in proper interpretation of the data. Best tissue preparation seems to be to quench the sample and to analyze it either freeze dried in thin or ultrathin sections, or frozen hydrated in thin sections or bulk samples. In all cases analysis should be performed using a cold stage and an ultra clean vacuum in order to minimize mass loss due to beam damage and mass gain due to contamination trapping. Interpretation of the data relies upon the knowledge of both the localization of the volume excited by the electron beam and the origin of the continuum and characteristic X-ray signals received by the X-ray spectrometer. This knowledge can be complicated by two facts: 1) when the electron beam is used in an analytical mode, viewing of the analyzed microarea can be lost, and 2) the X-ray signals received by the spectrometer can originate not only from the volume directly excited by the electron beam but from areas that can be far apart, excited by the electron beam tail, scattered electrons or secondary fluorescence, particularly when using energy dispersive spectrometers. Theoretical quantitation of the results is well developed. Practical quantitation could be complicated by the possibility of mass gain, mass loss, standard inhomogeneity, non-uniformity of sample thickness, possibility of shrinkage during freeze drying and, when using energy dispersive spectrometry, by the low signal over background for low atomic number elements (Na), the possibility of overlap of characteristic X-ray lines, and the use of complex and empirical methods for background stripping and peak deconvolution. All these difficulties can be overcome, making electron probe microanalysis one of the most powerful tools available to the biologist.

Animals

Quantitative renal blood flow in urinary tract obstruction.

Technetium-labeled red blood cell scintigraphy using a bolus injection was used to obtain a noninvasive quantitative measurement of renal blood flow. The method used is an adaptation of the one used for calculating cerebral blood flow which involves deconvolution of the first pass of the isotope through the kidneys. The relative renal blood flow of 18 normal patients and 18 patients with unilateral urinary tract obstruction was performed. All patients with obstruction, but one, were reexamined after surgery. There was a significant difference between the normal and partially obstructed patients (p less than 0.001) and between those with partial and complete obstruction (p less than 0.01). After surgery a statistically observable improvement in the relative renal blood flow was obtained.

Adolescent

Artificial Intelligence-Driven Multi-Omics Analysis Reveals Hydroxytyrosol Targeting of the TXNIP-NLRP3 Inflammasome Axis in Traumatic Brain Injury.

Traumatic brain injury (TBI) induces secondary neuroinflammation driven by oxidative stress, inflammasome activation, and immune remodeling, yet specific mechanism-guided pharmacological interventions remain limited. This study established an artificial intelligence (AI)-integrated network pharmacology and multi-omics framework to evaluate whether hydroxytyrosol (HT), an olive-derived natural polyphenol, may regulate TBI-related neuroinflammatory targets centered on the TXNIP/NLRP3 inflammasome axis. Starting from the SMILES structure of HT, potential targets were predicted using PharmMapper, SwissTargetPrediction, and the Similarity Ensemble Approach and were standardized to UniProt identifiers. TBI-associated genes were integrated from GeneCards, DisGeNET, OMIM, and the Therapeutic Target Database. The overlapping target set was analyzed using STRING-based protein-protein interaction (PPI) networks, MCODE, CytoHubba, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Public GEO transcriptomic datasets (GSE123831 and GSE104687) were used for cross-platform expression validation, differential expression analysis, and exploratory CIBERSORT-based immune infiltration estimation. Random forest (RF), multilayer perceptron (MLP), graph convolutional network (GCN), graph attention network (GAT), SHAP/LIME explainability analysis, LASSO inflammatory-risk scoring, and two-sample Mendelian randomization (MR) were further applied for target prioritization, immune phenotype mapping, and genetic association analysis. Seventy-three overlapping HT-TBI targets were identified. PPI and topology analyses prioritized TXNIP, NLRP3, CASP1, MAPK1, and TP53 as key hubs enriched in inflammasome activation, oxidative stress, apoptosis, and NOD-like receptor signaling. TXNIP, NLRP3, and CASP1 were consistently upregulated in both TBI transcriptomic datasets. LM22-based immune deconvolution suggested increased pro-inflammatory immune signatures and a positive TXNIP-M1 macrophage association (r&#x202f;=&#x202f;0.63, p < 0.001), which should be interpreted as a transcriptome-derived hypothesis rather than validated murine immune-cell proportions. AI-based models consistently ranked TXNIP/NLRP3 as high-contribution features under internal validation, and removal of these targets reduced model performance. A five-gene inflammatory score achieved an internally evaluated AUC of 0.87, while two-sample MR supported positive genetic associations involving TXNIP expression, TBI risk, NLRP3 and IL-1&#x3b2; expression. Collectively, these findings prioritize the TXNIP/NLRP3/CASP1 module as a computationally supported candidate mechanism through which HT may influence oxidative stress-inflammasome-immune coupling in TBI. This study provides an interpretable drug-target-pathway-phenotype framework and identifies TXNIP, NLRP3, and CASP1 as priority nodes for future experimental validation.

Artificial Intelligence

Multiregion profiling of genomic and transcriptional heterogeneity in head and neck squamous-cell carcinoma.

BACKGROUND: Intratumoral heterogeneity (ITH) is thought to contribute to tumour evolution and treatment resistance but its biological and clinical significance in localised head and neck squamous-cell carcinoma (HNSCC) remains incompletely understood. PATIENTS AND METHODS: In the prospective SCANDARE study, we analysed 87 patients with resectable HNSCC treated with upfront surgery. Two to five spatially distinct tumour regions per patient underwent pathological evaluation, targeted DNA sequencing, and bulk RNA sequencing. Genomic ITH (gITH) was quantified using clonal deconvolution and Shannon diversity indices, whereas transcriptional heterogeneity (tITH) was assessed using the intratumour expression distance metric. Associations between ITH, molecular features, tumour microenvironment composition, and clinical outcomes were explored using multivariable statistical models. RESULTS: Pathology-based spatial heterogeneity showed limited prognostic value. gITH was common, with 37% of tumours displaying regionally heterogeneous pathogenic variants, including spatially actionable alterations in 10% of patients. In an initial multivariable Cox model, higher gITH was associated with shorter disease-free survival. However, after Ridge-penalised modelling and bootstrap internal validation, the effect size was attenuated [corrected hazard ratio 1.42, 95% confidence interval (CI) 0.91-2.75]. The overall model retained moderate discriminative performance (optimism-corrected C-index 0.69, 95% CI 0.59-0.79). gITH was associated with tumour cellularity, reduced estimated endothelial cell infiltration, and alterations in KMT2C and PIK3CA. tITH differed according to human papillomavirus (HPV) status, with lower tITH in HPV-positive tumours, and was associated with distinct biological pathways and genomic alterations. Genomic and tITH were not correlated. CONCLUSIONS: This prospective multiregion study provides a comprehensive characterisation of genomic and tITH in localised HNSCC. Our findings highlight substantial spatial molecular diversity within primary tumours and suggest potential associations between heterogeneity, tumour biology, and clinical outcome that warrant validation in independent cohorts.

head and neck squamous-cell carcinoma (HNSCC)

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study.

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

The beta cell glucose stimulus-response curve in normal humans assessed by insulin and C-peptide secretion rates.

Insulin and C-peptide secretion rates have been measured and compared in 12 nondiabetic subjects to characterize the glucose stimulus-response of B cell secretion in man. On three different days, glucose concentrations were clamped for 150 minutes at 7.5, 10, and 15 mmol/L, respectively. Plasma samples taken during the clamps were assayed for C-peptide and insulin. C-peptide secretion rates were estimated by the technique of deconvolution. Model-based estimation of insulin secretion rates from insulin concentrations yielded concordant results. In response to glucose, C-peptide concentrations rose less quickly than did insulin concentrations, but the estimated first- and second-phase secretion rates were similar when assessed from either the C-peptide or insulin concentrations. First-phase secretion peaks were larger than inspection of the plasma concentration data might suggest, with median values of 1.3, 2.0, and 2.9 nmol/min for C-peptide in response to 7.5, 10, and 15 mmol/L glucose clamp levels, respectively. The second-phase reached steady state by 90 to 120 minutes, with median C-peptide secretion rates of 0.31, 0.56, and 0.85 nmol/min after 120 minutes at 7.5, 10, and 15 mmol/L, respectively. The slopes of the curves of steady-state insulin and C-peptide secretion rates v the four glucose levels (basal plus the three clamp levels) were maximally steep between 7.5 and 10 mmol/L in the majority of subjects, consistent with in vitro sigmoidal responses. A characterization of the secretory response of the B cell of normal humans at different glucose concentrations has been obtained. With appropriate models, insulin secretion rates may be estimated from either plasma insulin or C-peptide concentration data.

Adult

Spatial proteomic mapping of the human and mouse retina using IBEX.

We generated a comparative spatial proteomic atlas of the human and mouse retina using a highly multiplexed immunohistochemistry technique called iterative bleaching extends multiplexity (IBEX). We refined the IBEX workflow by integrating an antibody dissociation option alongside chemical bleaching. This dual strategy enabled removal of the entire antibody complex, permitting the flexible use of antibodies from the same host species across iterative cycles. We coupled this workflow with super-resolution imaging via deconvolution and applied it to the retina of healthy humans and WT mice and the Crb1rd8 mouse model. We successfully imaged over 25 protein markers on human and mouse tissue sections, generating spatial atlases of the major retinal cell populations. Cross-species protein expression was compared to scRNA-seq datasets to identify protein and transcript disparities. Super-resolution IBEX delineated the ultrastructural features of the outer limiting membrane (OLM), identifying CD44 as a core structural component tightly colocalized with a highly organized F-actin belt within M&#xfc;ller glial endfeet. Using the Crb1rd8 mouse model, disruption of this complex was spatially associated with rosette formation and OLM structural failure. In summary, spatial proteomic atlases of the human and mouse retina were used to reveal insights into the arrangement of major retinal cell populations and OLM structure.

Animals

Three-dimensional intracellular calcium gradients in single human burst-forming units-erythroid-derived erythroblasts induced by erythropoietin.

We have previously shown that the intracellular free Ca2+ increase induced by erythropoietin is likely related to differentiation rather than proliferation in human BFU-E-derived erythroblasts (1989. Blood. 73:1188-1194). Since cell differentiation involves transcription of specific regions of the genome, and since nuclear endonucleases responsible for single strand DNA breaks observed in cells undergoing differentiation are Ca2+ dependent, we investigated whether the erythropoietin-induced calcium signal is transmitted from cytosol to nucleus in this study. To elucidate subcellular Ca2+ gradients, the technique of optical sectioning microscopy was used. After determining the empirical three-dimensional point spread function of the video imaging system, contaminating light signals from optical planes above and below the focal plane of interest were removed by deconvolution using the nearest neighboring approach. Processed images did not reveal any discernible subcellular Ca2+ gradients in unstimulated erythroblasts. By contrast, with erythropoietin stimulation, there was a two- to threefold higher Ca2+ concentration in the nucleus compared to the surrounding cytoplasm. We suggest that the rise in nuclear Ca2+ may activate Ca2(+)-dependent endonucleases and initiate differentiation. The approach described here offers the opportunity to follow subcellular Ca2+ changes in response to a wide range of stimuli, allowing new insights into the role of regional Ca2+ changes in regulation of cell function.

Calcium

A protocol for 3D image reconstruction from a single image of an oblique section.

Oblique section 3D reconstruction can produce a 3D image of a sectioned crystal from a single electron micrograph. We describe here in detail a reconstruction protocol applicable to an electron micrograph of an oblique section through a 3D crystal. The protocol is described in six steps: (1) selection criteria for images, (2) preprocessing steps to correct for image defects, (3) determination of unit cell coordinates, (4) interpolation of strip images with correction for image distortions and crystal disorder, (5) production of a crystallographic serial section reconstruction, (6) correction for skewed sampling to produce an oblique section reconstruction. In addition, we explore Wiener filter deconvolution of the section thickness. We describe a method for determining the section thickness by comparing data from projections of the oblique section reconstruction with corresponding data from a thick longitudinal section. Several schemes for Wiener filter deconvolution are described that differ in the way information on the signal-to-noise ratio is used in the filter.

Animals

A nuclear magnetic resonance imaging technique designed for studies of water in plant leaves.

A new imaging technique is described which uses nuclear magnetic resonance (NMR) to create a water profile of plant leaves. The water profile shows the average distribution of water as a function of depth into a leaf along a line perpendicular to the leaf surface; it can be used to measure the thickness of cell layers and the quantity of water in each layer. Two-dimensional NMR methods were used to avoid chemical shift distortion which degrades the resolution in leaf images made by conventional NMR techniques; image resolution was improved further by deconvolution analysis. To illustrate its application, the technique was used to follow changes in the internal structure of developing leaves.

Evaluation Studies as Topic

Alterations in the patterns of insulin secretion before and after diagnosis of IDDM.

OBJECTIVE: To study the natural history of beta-cell dysfunction in an individual who developed insulin-dependent diabetes mellitus (IDDM) over a 13-month period while under observation. RESEARCH DESIGN AND METHODS: Insulin secretion rates (ISR) in response to intravenous glucose and mixed meals were estimated by deconvolution of C-peptide levels. RESULTS: When fasting glucose and glycosylated hemoglobin concentrations were still within the normal range, insulin secretory responses to intravenous glucose infusion were reduced, but 80- to 100-min secretory oscillations could still be detected. Sequential glucose infusion studies over a 3-month period demonstrated a progressive reduction in insulin secretion. The tight temporal coupling between ultradian oscillations in ISR and glucose observed in nondiabetic subjects was lost. In response to mixed meals, the oscillatory pattern of secretion was preserved, but the magnitude of the secretory responses was reduced. CONCLUSIONS: Our results indicate that despite the lower absolute secretory rates, ultradian ISR oscillations persist in the period before and immediately after the onset of IDDM in this subject, but they are less tightly coupled to glucose than in nondiabetic subjects.

Adult

Insulin secretion and clearance during low-dose graded glucose infusion.

The present study was undertaken in normal volunteers to define the alterations in beta-cell responsiveness to glucose associated with different physiological states, including fasting and refeeding, and after prolonged intravenous glucose infusion. A low-dose graded glucose infusion protocol was used to explore the dose-response relationship between glucose and insulin secretion. Studies were performed in 10 normal volunteers, and insulin secretion rates (ISR) were calculated by deconvolution of peripheral C-peptide levels using a two-compartment model utilizing individual kinetic parameters. From 5 to 9 mmol/l glucose, the relationship between glucose and ISR was linear. After a 42-h glucose infusion at a rate of 4 mg.kg-1.min-1, the ISR increased by 53% over the same glucose concentration range (P < 0.002), resulting in a shift of the dose-response curve to the left. Insulin clearance rates decreased 27% after the 42-h glucose infusion (P < 0.001). After a 72-h fast, ISR decreased by 32% from baseline over the 5-8 mmol/l glucose range (P = 0.056), resulting in a shift of the dose-response curve to the right. This shift was reversed by a 42-h period of refeeding, after which ISR was increased by 77% compared with the fasting study (P < 0.02). Refeeding enhanced the beta-cell responsiveness, and ISR increased by 31% after refeeding compared with the baseline study (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult

Immobilized artificial membrane chromatography: supports composed of membrane lipids.

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.

Chromatography, High Pressure Liquid

Quantal parameters of "minimal" excitatory postsynaptic potentials in guinea pig hippocampal slices: binomial approach.

Binomial distributions of amplitudes of excitatory postsynaptic potentials (EPSPs) mixed with Gaussian noise were simulated. The objective of Monte Carlo simulations was, firstly, to study influences of sampling size (N) and noise standard deviation (Sn) on estimates of mean quantal content (m), quantal size (v) and binomial parameters (n and p) by four methods of quantal analysis (histogram, variance, failures and combined method) based on the binomial model and, secondly, to modify these methods on the basis of comparison of estimated with simulated parameters. Reliable estimates (within +/- 10% of the simulated values) were obtained for large sample sizes (N = 500-1000) with Sn less than or equal to v by the histogram (deconvolution) method and with Sn less than or equal to 2v by the other three methods. Similar results were obtained by averages from about 10 simulations if smaller samples were used (N = 50-200). In electrophysiological experiments on slices, "minimal" EPSPs were recorded from CA1 pyramidal cells after low-intensity stimuli to stratum radiatum or stratum oriens. Amplitudes of minimal EPSPs fluctuated in a manner predicted by the quantum hypothesis. Amplitude distributions of EPSPs in the non-facilitated state were adequately described either by binomial statistics with an average p equal to about 0.4 (a range of 0.3-0.7) and an average n of about 3 (range 2-6) or by Poisson statistics with m of about 1. The quantal analysis suggests that typical values of m and v for a single activated fibre in stratum radiatum might be about 0.5-1 and 300-400 microV, respectively, with low p (0.1-0.3) and n (2-4). However, the estimates of binomial parameters should be considered as coarse approximations in view of the simulation results and a possible nonuniformity of parameter p. The comparison of results of various methods based on the binomial model, in both simulation and physiological experiments, indicates the reliability of estimates of basic quantal parameters (m and v) under realistic conditions of physiological experiments. The methods are considered to be sufficiently sensitive to make use of them for studies on mechanisms of long-term synaptic plasticity.

Animals