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Repair of damaged DNA is a dynamic process that requires careful orchestration of a multitude of enzymes, adaptor proteins, and chromatin constituents. In this issue of Cell, Lisby et al. (2004) provide a visual glimpse into how the diverse signaling and repair machines are organized in space and time around the deadliest genetic lesions--the DNA double-strand breaks.
Two fundamental properties of an ion channel are its single channel current (i) and its open probability (Po), which are most directly defined by single channel recordings. However, under some circumstances either practical limitations (e.g., extreme voltages) or unique channel properties may preclude the use of single channel recordings for definition of such properties. As an alternative, the variance in macroscopic currents (non-stationary noise analysis or variance-mean analysis) has been exploited to define fundamental elementary properties of the underlying channels. Although some limitations of the variance-mean approach have been considered in previous work by others, here simulation methods were used to define the conditions under which variance analysis can be suitable for providing estimates of i and Po. Of particular interest is the extent to which reasonably reliable estimates of Po can be obtained, even under conditions of Po less than 0.5. Empirically, the analysis indicates that, with sufficient numbers of sweeps and with constraints on initial estimates of i, reasonably reliable estimates of Po can be made down to 0.2. The impact of a number of other factors on the utility of variance-mean analysis are also considered, including effects of inactivation, filtering, and the consequences of particular gating schemes.
Magnetic resonance methodology has made a significant impact in helping us understand the physics of porous media. Among an important class of experiments is that set of techniques designed to measure fluid dispersion. This paper provides some background on some of the underlying physics of dispersion, and outlines some of the NMR approaches that have proven successful. The local and nonlocal dispersion tensors are described and the prospects for future NMR advances considered.
The present study investigates the performance of population codes when the fluctuations in neural activity have mutual correlation with strength being proportional to the neuronal firing rate (multiplicative noise). The neural field is used to calculate the Fisher information, which is decomposed in two parts, one due to the tuning function and spatial correlation, and the other due to the multiplicative structure. Their different characteristics are studied. The paper also investigates three types of maximum likelihood method, namely, decoding by using faithful and unfaithful models and the Center of Mass strategy, and compares their performances in terms of decoding accuracy and computational complexity.
The motivation to start or terminate a meal involves the continual updating of information on current body status by central gustatory and reward systems. Previous electrophysiological and neuroimaging investigations revealed region-specific decreases in activity as the subject's state transitions from hunger to satiety. By implanting bundles of microelectrodes in the lateral hypothalamus, orbitofrontal cortex, insular cortex, and amygdala of hungry rats that voluntarily eat to satiety, we have measured the behavior of neuronal populations through the different phases of a complete feeding cycle (hunger-satiety-hunger). Our data show that while most satiety-sensitive units preferentially responded to a unique hunger phase within a cycle, neuronal populations integrated single-unit information in order to reflect the animal's motivational state across the entire cycle, with higher activity levels during the hunger phases. This distributed population code might constitute a neural mechanism underlying meal initiation under different metabolic states.
7-12 Hz Oscillations, characterized by spindle-like high-voltage rhythmic spike components, appear in quiet immobile states of rats. However, it remains unclear what their relationships with preceding behavioral activities are and how prefrontal neuronal dynamics during these oscillations is. In the present study, we first determined the relationship of 7-12 Hz oscillations with the wake-sleep cycle and preceding behavioral activities in several normal rat strains by recording electroencephalograms from the multiple cortical regions. Prolonged awake period transiently enhanced the following appearance of 7-12 Hz oscillations, which were frequently followed by slow-wave sleep. The degree of transient enhancement under the task condition was similar to that by prolonged wakefulness under the no-task condition. In addition, by recording local-field potential and multi-unit activities in the medial prefrontal cortex, we determined the temporal dynamics of prefrontal neuronal activities in relation to 7-12 Hz oscillations. Collective neuronal activities in medial prefrontal cortex were gradually organized into phase-locked patterns and showed highly synchronization during these oscillations. These dynamics were in temporal proximity to those of slow-wave activities (<4 Hz). Since slow-wave activities are thought to synchronize large spatial domains, these results suggest that 7-12 Hz oscillations are involved in the transition from the awake to sleep states by oscillatory entrainment of global cortical networks including the prefrontal neurons.
A novel way is proposed to describe the evolution of nuclear magnetic polarization and the induced NMR spectrum. In this method, the effect of a high-intensity external static magnetic field and the effects of proper Hamiltonian left over interaction components, which commute with the first, are taken into account simultaneously and equivalently. The method suits any concrete NMR problem. This brings forth the really existing details in the registered spectra, evoked by Hamiltonian secular terms, which may be otherwise smoothed due to approximate treatment of the effects of the secular terms. Complete analytical expressions are obtained describing the NMR spectra including the rotational sideband sets of single quadrupolar nuclei in rotating solids.
The predominant equilibrium in proteins is not between native and unfolded states, it is between the native and multiple partially unfolded forms. Some of these partially unfolded forms can be energetically close to the native state and, therefore, have the potential to become appreciably populated. This could have an important role in protein function or misfolding diseases. The recent identification and characterization of the partially unfolded forms of apoflavodoxin furthers our understanding of their formation.
BACKGROUND: Lung adenocarcinoma (LUAD) shows prognostic heterogeneity, and tumor-node-metastasis (TNM) staging is limited for individualized management. Chaperone-mediated autophagy (CMA) maintains proteostasis, but its role during adenocarcinoma in situ (AIS)-minimally invasive adenocarcinoma (MIA)-invasive adenocarcinoma (IAC) progression remains unclear. METHODS: Single-cell RNA sequencing (scRNA-seq) data from GSE189357 and bulk transcriptomes from The Cancer Genome Atlas (TCGA)-LUAD and Gene Expression Omnibus (GEO) cohorts were integrated. CMA activity, cell-cell communication, weighted gene co-expression network analysis (WGCNA), tumor-normal differential expression, machine-learning survival modeling, tumor microenvironment (TME) features, drug sensitivity, and EPC1 function were analyzed. RESULTS: CMA-high tumor epithelial cells increased from AIS (58.1%) to MIA (65.7%) but declined in IAC (44.4%; p < 0.001). CMA-low cells preferentially received fibroblast-derived extracellular matrix cues. A CMA-negatively correlated module identified 69 core genes. Random survival forest (RSF) performed best among 117 machine-learning combinations (mean concordance index > 0.873). High-risk patients had worse survival across cohorts, and the risk score was independently associated with overall survival (hazard ratio = 16.013, 95% confidence interval: 9.579-26.768, p < 0.001). High-risk tumors showed proliferative activation and M0 macrophage enrichment, whereas low-risk tumors showed stronger immune-related signaling. EPC1 overexpression suppressed malignant phenotypes in A549 cells. CONCLUSION: CMA dynamics are associated with stromal and immune remodeling during LUAD progression. A CMA-based model provides robust prognostic stratification and may offer a basis for future TME-guided studies.
Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins.
We have studied the spectral properties of the voltage-sensitive dye, 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphtyl]vinyl] pyridinium betaine (di-8-ANEPPS), and the Ca(2+)-sensitive dye, fura-2, in azolectin liposomes and in isolated taste buds from mouse. We find that the fluorescence excitation spectra of di-8-ANEPPS and fura-2 are largely nonoverlapping, allowing alternate ratio measurements of membrane potential and intracellular calcium ([Ca2+]i). There is a small spillover of di-8-ANEPPS fluorescence at the excitation wavelengths used for fura-2 (340 and 360 nm). However, voltage-induced changes in the fluorescence of di-8-ANEPPS, excited at the fura-2 wavelengths, are small. In addition, di-8-ANEPPS fluorescence is localized to the membrane, whereas fura-2 fluorescence is distributed throughout the cytoplasm. Because of this, the effect of spillover of di-8-ANEPPS fluorescence in the [Ca2+]i estimate is < 1%, under the appropriate conditions. We have applied this method to study of the responses of multiple taste cells within isolated taste buds. We show that membrane potential and [Ca2+]i can be measured alternately in isolated taste buds from mouse. Stimulation with glutamate and glutamate analogs indicates that taste cells express both metabotropic and ionotropic receptors. The data suggest that the receptors responding to 2-amino-4-phosphonobutyrate (L-AP4), presumably metabotropic L-glutamate receptors, do not mediate excitatory glutamate taste responses.
The correlation between sequence diversity and enzymatic function was studied in a library of Theta class glutathione transferases (GSTs) obtained by stochastic recombination of fragments of cDNA encoding human GST T1-1 and rat GST T2-2. In all, 94 randomly picked clones were characterized with respect to sequence, expression level, and catalytic activity in the conjugation reactions between glutathione and six alternative electrophilic substrates. Out of these six different compounds, dichloromethane is a selective substrate for human GST T1-1, whereas 1-menaphthyl sulfate and 1-chloro-2,4-dinitrobenzene are substrates for rat GST T2-2. The other three substances serve as substrates for both enzymes. Through this broad characterization, we have identified enzyme variants that have acquired novel activity profiles that differ substantially from those of the original GSTs. In addition, the expression levels of many clones were improved in comparison to the parental enzyme. A library of mutants can thus display a distribution of properties from which highly divergent evolutionary pathways may emerge, resembling natural evolutionary processes. From the GST library, a clone was identified that, by the point mutation N49D in the rat GST T2-2 sequence, has a 1700% increased activity with 1-menaphthyl sulfate and a 60% decreased activity with 4-nitrophenethyl bromide. Through the N49D mutation, the ratio of these activities has thus been altered 40-fold. An extensive characterization of a population of stochastically mutated enzymes can accordingly be used to find variants with novel substrate-activity profiles and altered catalytic properties. Recursive recombination of selected sequences displaying optimized properties is a strategy for the engineering of proteins for medical and biochemical applications. Such sequential design is combinatorial protein chemistry based on remodeling of existing structural scaffolds and has similarities to evolutionary processes in nature.
We develop in two ways an existing spin-glass model of prebiotic polymer evolution. First, by choosing the environment J in a prescribed manner, similar to neural network presciptions, we may create an environment which favors a linearly independent set of evolutionary niches (Ea). That is, we may control which polymer "species" will evolve in our system. Computer simulations confirm this result. We obtain a quantitative value for the sharpness of a niche. Second, we extend the model by allowing a surviving polymer to act upon--to "remold"--its environment; the nature of the environmental action is governed by the "molding" matrix M. When the mold M is the identity matrix, the feedback algorithm reduces to a Hebb learning algorithm form, and a surviving polymer acts to enhance its own survival prospects. Molds having a structure analogous to (temporal) associative memories in neural networks can generate autocatalytic species or can exhibit symbiotic interspecies relationships.
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The persistent sodium current density (I(NaP)) at the soma measured with the 'whole-cell' patch-clamp recording method is linearized about the resting state and used as a current source along the dendritic cable (depicting the spatial distribution of voltage-dependent persistent sodium ionic channels). This procedure allows time-dependent analytical solutions to be obtained for the membrane depolarization. Computer simulated response to a dendritic current injection in the form of synaptically-induced voltage change located at a distance from the recording site in a cable with unequally distributed persistent sodium ion channel densities per unit length of cable (the so-called 'hot-spots') is used to obtain conclusions on the density and distribution of persistent sodium ion channels. It is shown that the excitatory postsynaptic potentials (EPSPs) are amplified if hot-spots of persistent sodium ion channels are spatially distributed along the dendritic cable, with the local density of I(NaP) with respect to the recording site shown to specifically increase the peak amplitude of the EPSP for a proximally placed synaptic input, while the spatial distribution of I(NaP) serves to broaden the time course of the amplified EPSP. However, in the case of a distally positioned synaptic input, both local and nonlocal densities yield an approximately identical enhancement of EPSPs in contradiction to the computer simulations performed by Lipowsky et al. [J. Neurophysiol. 76 (1996) 2181]. The results indicate that persistent sodium channels produce EPSP amplification even when their distribution is relatively sparse (i.e. , approximately 1-2% of the transient sodium channels are found in dendrites of CA1 hippocampal pyramidal neurons). This gives a strong impetus for the use of the theory as a novel approach in the investigation of synaptic integration of signals in active dendrites represented as ionic cables.
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Explore the source record for details and available documents.