Effect of DNAase I on muscle tropomyosin polymerization

DNAase I, an endonuclease which interacts with G-actin, also affects tropomyosin polymerization. With chicken pectoralis or bovine cardiac ventricle tropomyosin, DNAase I both prevents tropomyosin from polymerizing and disrupts already formed tropomysin filaments. DNAase I and filament tropomyosin can also form a precipitable complex. In the electron microscope, the complex is observed as irregularly margined stellate-shaped structures with a maximum size of 9 micron. Isolated DNAase I-tropomyosin stellate complex consists of a 2:1 molar ratio of DNAase I and tropomyosin, suggesting that each tropomyosin subunit can bind DNAase I.     

The determination of fresh organic carbon weight from formaldehyde preserved macrofaunal samples

Methodologies are presented whereby the fresh organic carbon weight of formaldehyde preserved macrofaunal samples may be estimated. Length-organic carbon weight regressions were determined for the four numerically dominant bivalves in Narragansett Bay, Rhode Island (Nucula annulata, Yoldia limatula, Mulinia lateralis, and Pandora gouldiana) and one commercially important, but less abundant species (Mercenaria mercenaria). Constants were determined to convert the dry weight of preserved softbodied organisms (polychaetes, oligochaetes, amphipods, etc.) to fresh (unpreserved) organic carbon weight. These results can be used by investigators studying the energetics of benthic communities similar to those in Narragansett Bay.     

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.     

Analyzing the substrate specificity of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase by co-expressing it with mammalian G protein alpha subunits in Escherichia coli

A dual plasmid system was used to examine the protein and acyl-CoA specificities of Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (NMT) by co-expressing it in Escherichia coli with each of four homologous alpha subunits of the signal-transducing, heterotrimeric G proteins. Exogenous [3H]myristate was incorporated into rat Gi alpha 1 and rat Go alpha but not into bovine Gs alpha or human Gz alpha. Oxygen for methylene group substitutions in myristate result in analogs with comparable chain length and stereochemistry but marked reductions in hydrophobicity. Metabolic labeling studies with 6-, 11-, or 13-[3H]oxatetradecanoic acid indicated that they were incorporated into rat Gi alpha 1 and Go alpha with an efficiency that could be correlated with their accumulation into E. coli and their interactions with purified NMT in vitro. Octapeptides derived from the NH2-terminal sequences of these four G alpha polypeptides were tested as substrates for purified S. cerevisiae NMT. None were bound by the enzyme. Acidic residues at positions 7 and 8 appear to contribute to this effect; deletion of these two amino acids or addition of the next 9 residues of rat Go alpha produced active substrates. These results imply that productive interactions between NMT and G alpha protein substrates in vivo require structural features that are not fully represented within their NH2-terminal 8 residues.     

Studies of the catalytic activities and substrate specificities of Saccharomyces cerevisiae myristoyl-coenzyme A: protein N-myristoyltransferase deletion mutants and human/yeast Nmt chimeras in Escherichia coli and S. cerevisiae.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential, 455-residue, monomeric enzyme. Amino- and carboxyl-terminal deletion mutants of Nmt1p were genetically engineered to determine the minimal domain necessary to maintain catalytic activity. Enzyme activity was assessed by (i) sequentially inducing Nmt1p or its mutant derivatives and one of two eukaryotic substrates for the wild type enzyme (S. cerevisiae Gpa1p and rat Go alpha) in Escherichia coli, a bacterium with no endogenous myristoyltransferase activity, and monitoring Nmt-dependent incorporation of exogenous [3H]myristate into the G protein alpha subunits or (ii) an in vitro enzyme assay using lysates prepared from bacteria producing wild type or mutant Nmts. The data indicate that the minimal catalytic domain of Nmt1p is located between Ile59-->Phe96 and Gly451-->Leu455. Analyses of the ability of mutant nmtps to rescue the lethal phenotype of an nmt1 null allele in a haploid strain of yeast grown on rich media, with or without blockade of cellular fatty acid synthetase, suggest that the amino-terminal 59 residues of Nmt1p may play an important noncatalytic role, functioning as a targeting signal so this cytosolic enzyme can access cellular myristoyl-CoA pools generated from activation of exogenous C14:0 by acyl-CoA synthetase(s). Moreover, there appear to be differences in the location or accessibility of myristoyl-CoA pools derived from fatty acid synthetase and acyl-CoA synthetases. The E. coli co-expression system was used to map structural elements that determine differences in the peptide substrate specificities of Nmt1p and the orthologous human Nmt. Rat Go alpha is a substrate for both enzymes, whereas human Gz alpha is a substrate only for human NMT. Studies of a series of chimeric enzymes composed of elements from the amino- or carboxyl-terminal portions of human and yeast Nmts indicate that (i) recognition/utilization of Gz alpha involves elements distributed from the amino-terminal half through the region defined by Leu352-->Lys410 of the 416 residue human enzyme and (ii) formation of a fully functional peptide binding site and a fully functional myristoyl-CoA binding site in either of these enzymes requires contributions from both their amino-terminal and carboxyl-terminal halves.     

Analysis of Serum Inflammatory Mediators Identifies Unique Dynamic Networks Associated with Death and Spontaneous Survival in Pediatric Acute Liver Failure

Background

Tools to predict death or spontaneous survival are necessary to inform liver transplantation (LTx) decisions in pediatric acute liver failure (PALF), but such tools are not available. Recent data suggest that immune/inflammatory dysregulation occurs in the setting of acute liver failure. We hypothesized that specific, dynamic, and measurable patterns of immune/inflammatory dysregulation will correlate with outcomes in PALF.

Methods

We assayed 26 inflammatory mediators on stored serum samples obtained from a convenience sample of 49 children in the PALF study group (PALFSG) collected within 7 days after enrollment. Outcomes were assessed within 21 days of enrollment consisting of spontaneous survivors, non-survivors, and LTx recipients. Data were subjected to statistical analysis, patient-specific Principal Component Analysis (PCA), and Dynamic Bayesian Network (DBN) inference.

Findings

Raw inflammatory mediator levels assessed over time did not distinguish among PALF outcomes. However, DBN analysis did reveal distinct interferon-gamma-related networks that distinguished spontaneous survivors from those who died. The network identified in LTx patients pre-transplant was more like that seen in spontaneous survivors than in those who died, a finding supported by PCA.

Interpretation

The application of DBN analysis of inflammatory mediators in this small patient sample appears to differentiate survivors from non-survivors in PALF. Patterns associated with LTx pre-transplant were more like those seen in spontaneous survivors than in those who died. DBN-based analyses might lead to a better prediction of outcome in PALF, and could also have more general utility in other complex diseases with an inflammatory etiology.     

Phosphorylation of threonine residue 276 is required for acute regulation of serotonin transporter by cyclic GMP

Cellular protein kinases, phosphatases, and other serotonin transporter (SERT) interacting proteins participate in several signaling mechanisms regulating SERT activity. The molecular mechanisms of protein kinase G (PKG)-mediated SERT regulation and the site of transporter phosphorylation were investigated. Treatment of rat midbrain synaptosomes with 8-bromo-cGMP increased SERT activity, and the increase was selectively blocked by PKG inhibitors. The V(max) value for serotonin (5-HT) transport increased following cGMP treatment. However, surface biotinylation studies showed no change in SERT surface abundance following PKG activation. (32)P metabolic labeling experiments showed increased SERT phosphorylation in the presence of cGMP that was abolished by selectively inhibiting PKG. Phosphoamino acid analysis revealed that cGMP-stimulated native SERT phosphorylation occurred only on threonine residues. When added to CHO-1 cells expressing SERT, 8-bromo-cGMP stimulated 5-HT transport and SERT phosphorylation. Mutation of SERT threonine 276 to alanine completely abolished cGMP-mediated stimulation of 5-HT transport and SERT phosphorylation. Although the T276A mutation had no significant effect on 5-HT transport or SERT protein expression, mutation to aspartate (T276D) increased the level of 5-HT uptake to that of cGMP-stimulated 5-HT uptake in wild-type SERT-expressing cells and was no longer sensitive to cGMP. These findings provide the first identification of a phosphorylation site in SERT and demonstrate that phosphorylation of Thr-276 is required for cGMP-mediated SERT regulation. They also constitute the first evidence that in the central nervous system PKG activation stimulates endogenous SERT activity by a trafficking-independent mechanism.     

A conformationally sensitive residue on the cytoplasmic surface of serotonin transporter

Serotonin transporter (SERT) contains a single reactive external cysteine residue at position 109 (Chen, J. G., Liu-Chen, S., and Rudnick, G. (1997) Biochemistry 36, 1479-1486) and seven predicted cytoplasmic cysteines. A mutant of rat SERT (X8C) in which those eight cysteine residues were replaced by other amino acids retained approximately 32% of wild type transport activity and approximately 56% of wild type binding activity. In contrast to wild-type SERT or the C109A mutant, X8C was resistant to inhibition of high affinity cocaine analog binding by the cysteine reagent 2-(aminoethyl)methanethiosulfonate hydrobromide (MTSEA) in membrane preparations from transfected cells. Each predicted cytoplasmic cysteine residue was reintroduced, one at a time, into the X8C template. Reintroduction of Cys-357, located in the third intracellular loop, restored MTSEA sensitivity similar to that of C109A. Replacement of only Cys-109 and Cys-357 was sufficient to prevent MTSEA sensitivity. Thus, Cys-357 was the sole cytoplasmic determinant of MTSEA sensitivity in SERT. Both serotonin and cocaine protected SERT from inactivation by MTSEA at Cys-357. This protection was apparently mediated through a conformational change following ligand binding. Although both ligands bind in the absence of Na(+) and at 4 degrees C, their ability to protect Cys-357 required Na(+) and was prevented at 4 degrees C. The accessibility of Cys-357 to MTSEA inactivation was increased by monovalent cations. The K(+) ion, which is believed to serve as a countertransport substrate for SERT, was the most effective ion for increasing Cys-357 reactivity.     

Serotonin transporter: gene, genetic disorders, and pharmacogenetics

The highly evolutionarily conserved serotonin transporter (SERT) regulates the entire serotoninergic system and its receptors via modulation of extracellular fluid serotonin concentrations. Differences in SERT expression and function produced by three SERT genes and their variants show associations with multiple human disorders. Screens of DNA from patients with autism, ADHD, bipolar disorder, and Tourette's syndrome have detected signals in the chromosome 17q region where SERT is located. Parallel investigations of SERT knockout mice have uncovered multiple phenotypes that identify SERT as a candidate gene for additional human disorders ranging from irritable bowel syndrome to obesity. Replicated studies have demonstrated that the SERT 5'-flanking region polymorphism SS genotype is associated with poorer therapeutic responses and more frequent serious side effects during treatment with antidepressant SERT antagonists, namely, the serotonin reuptake inhibitors (SRIs).