Purification of coated vesicles by agarose gel electrophoresis

We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. Coated vesicles from three different cell types have different mobilities. In each case, however, all of the major polypeptides previously attributed to coated vesicles comigrate with the now homogeneous particles, even though a powerful ATPase activity is completely removed.     

Human Brain γ-Aminobutyric Acid Levels and Seizure Control Following Initiation of Vigabatrin Therapy

Abstract: Vigabatrin is a novel antiepileptic drug designed to control seizures by raising brain γ-aminobutyric acid (GABA) concentrations. Seizure control is not improved significantly when the daily dose is increased beyond 50 mg/kg. Serial, in vivo measurements of GABA levels in human occipital lobe were made using ¹H NMR spectroscopy before and after the start of vigabatrin treatment. We used a 2.1-T magnetic resonance imagerspectrometer and an 8-cm surface coil to examine serially a 14-cm³ volume in the occipital lobe of 26 patients with complex partial seizures. Brain GABA content increased following the start of vigabatrin treatment up to a daily dose of 60 mg/kg. Additional increases in dose failed to increase brain GABA content further. GABA synthesis may decrease with sustained elevations of human brain GABA levels. Starting vigabatrin treatment reduced seizure frequency by >50%, from six to seven per month to three. Improved seizure control was not associated with further increases of vigabatrin dose. Increased brain GABA concentration was associated with improved seizure control. Starting vigabatrin treatment improved seizure control twofold when GABA levels increased above 1.8 mmol/kg. Further increases in brain GABA content above 2.5 mmol/kg provided less protection. Measuring occipital lobe GABA concentrations may predict improved seizure control when using antiepileptic drugs designed to increase brain GABA levels.     

A consensus view of the proteome of the last universal common ancestor

The availability of genomic and proteomic data from across the tree of life has made it possible to infer features of the genome and proteome of the last universal common ancestor (LUCA). A number of studies have done so, all using a unique set of methods and bioinformatics databases. Here, we compare predictions across eight such studies and measure both their agreement with one another and with the consensus predictions among them. We find that some LUCA genome studies show a strong agreement with the consensus predictions of the others, but that no individual study shares a high or even moderate degree of similarity with any other individual study. From these observations, we conclude that the consensus among studies provides a more accurate depiction of the core proteome of the LUCA and its functional repertoire. The set of consensus LUCA protein family predictions between all of these studies portrays a LUCA genome that, at minimum, encoded functions related to protein synthesis, amino acid metabolism, nucleotide metabolism, and the use of common, nucleotide‐derived organic cofactors.     

Uncoating ATPase is a member of the 70 kilodalton family of stress proteins

The synthetic peptide, VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp70, S. cerevisiae YG100, and E. coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts. One of these proteins, 71 kd, has previously been identified as uncoating ATPase, an enzyme that releases clathrin from coated vesicles. This immunological data complemented the result that uncoating ATPase was indistinguishable from the constitutive mammalian 71 kd stress protein by either partial proteolytic mapping or two-dimensional gel analysis. In addition, affinity-purified uncoating ATPase antibodies recognize proteins in yeast identified as the gene products of the heat shock or heat shock cognate genes YG100 and YG102. The results show that uncoating ATPase is a member of the 70 kd heat shock protein family.     

Interaction of leucine enkephalin with (3H)naloxone binding in rat brain: Evidence for an opioid receptor complex

We recently presented evidence that distinct morphine and enkephalin receptors coexist in an opioid receptor complex (Mol. Pharmacol. 21:548-557, 1982). In this paper, we present data which demonstrate that in the presence of sodium leucine enkephalin noncompetitively inhibits the binding of [3H]naloxone to a crude particulate fraction of rat brain. Since the binding site labeled by [3H]naloxone in the presence of sodium may be an alternate conformation of the morphine receptor, these data provide further evidence that morphine and enkephalin receptors are allosterically coupled.     

Correction to: Reevaluation of Astrocyte‑Neuron Energy Metabolism with Astrocyte Volume Fraction Correction: Impact on Cellular Glucose Oxidation Rates,Glutamate–Glutamine Cycle Energetics,Glycogen Levels and Utilization Rates vs. Exercising Muscle,and Na+/K+ Pumping Rates

Neurochemical Research -     

Probing the opioid receptor complex with (+)-trans-SUPERFIT. II. Evidence that mu ligands are noncompetitive inhibitors of the delta cx opioid peptide binding site.

Previous studies delineated two classes of δ binding sites; a δ binding site not associated with the opioid receptor complex, termed the δ_ncx site, and a δ site associated with the opioid receptor complex, termed the δ_cx site. The δ_ncx site has high affinity for [ -Pen², -Pen⁵]enkephalin, and is synonymous with what is now identified as the δ₁ binding site. Pretreatment of membranes with the δ-selective acylating agents FIT, or (+)-trans-SUPERFIT, deplete membranes of the δ_ncx binding site, which permits the selective labeling of the δ_cx binding site with [³H][ -Ala²,Leu⁵]enkephalin. The present study compared the properties of the δ_cx binding site present in brain membranes pretreated with (+)-trans-SUPERFIT with the properties of the δ_cx site present in untreated membranes. The major findings are: 1) pretreatment of membranes with (+)-trans-SUPERFIT decreased the IC₅₀ values of δ-preferring drugs, and increased the IC₅₀ values of μ-preferring drugs, for the δ_cx binding site; 2) the degree of δ selectivity was highly correlated with the magnitude of the (+)-trans-SUPERFIT-induced shift in the IC₅₀ values; 3) the ligand-selectivity patterns of the μ and δ_cx sites present in (+)-trans-SUPERFIT-pretreated membranes were poorly correlated; 4) whereas μ-preferring drugs were noncompetitive inhibitors of [³H][ -Ala²,Leu⁵]enkephalin binding to the δ_cx site, δ-preferring drugs were competitive inhibitors. Viewed collectively, these data support the hypothesis that the μ and δ_cx binding sites are distinct, provide additional evidence for δ receptor heterogeneity, and suggest that ( (+)-trans-SUPERFIT-pretreated membranes will provide a useful preparation for studying the δ_cx binding site.     

Time domain analysis of diaphragmatic electromyogram during fatigue in men

Diaphragmatic fatigue has been correlated with a change in the electromyogram recorded from the diaphragm (EMGdi), which suggests that the electromyogram is a potential clinical tool to detect respiratory muscle fatigue. Changes in the EMGdi have previously been quantified by using the power spectral parameters high-low ratio or mean frequency. In this study, we developed an autoregressive model of the EMG in an attempt to improve the analysis of the EMGdi. This model was tested on recordings of the EMGdi that were obtained from an esophageal electrode in five normal subjects breathing to fatigue through an inspiratory resistor. The data obtained from the autoregressive model were directly compared with data from the high-low ratio and mean frequency techniques. The autoregressive model showed an excellent correlation with mean frequency. Both techniques were superior to the high-low ratio measurement. Because the autoregressive model requires much less computation than mean frequency and can be easily implemented in real time on a minicomputer, we propose this as a preferable approach.