The holin of bacteriophage lambda forms rings with large diameter

Holins control the length of the infection cycle of tailed phages (the Caudovirales) by oligomerizing to form lethal holes in the cytoplasmic membrane at a time dictated by their primary structure. Nothing is currently known about the physical basis of their oligomerization or the structure of the oligomers formed by any known holin. Here we use electron microscopy and single-particle analysis to characterize structures formed by the bacteriophage λ holin (S105) in vitro . In non-ionic or mild zwitterionic detergents, purified S105, but not the lysis-defective variant S105_A52V, forms rings of at least two size classes, the most common having inner and outer diameters of 8.5 and 23 nm respectively, and containing approximately 72 S105 monomers. The height of these rings, 4 nm, closely matches the thickness of the lipid bilayer. The central channel is of unprecedented size for channels formed by integral membrane proteins, consistent with the non-specific nature of holin-mediated membrane permeabilization. S105 present in detergent-solubilized rings and in inverted membrane vesicles showed similar sensitivities to proteolysis and cysteine-specific modification, suggesting that the rings are representative of the lethal holes formed by S105 to terminate the infection cycle and initiate lysis.     

Dynamic models of gene expression and classification

Powerful new methods, like expression profiles using cDNA arrays, have been used to monitor changes in gene expression levels as a result of a variety of metabolic, xenobiotic or pathogenic challenges. This potentially vast quantity of data enables, in principle, the dissection of the complex genetic networks that control the patterns and rhythms of gene expression in the cell. Here we present a general approach to developing dynamic models for analyzing time series of whole genome expression. In this approach, a self-consistent calculation is performed that involves both linear and non-linear response terms for interrelating gene expression levels. This calculation uses singular value decomposition (SVD) not as a statistical tool but as a means of inverting noisy and near-singular matrices. The linear transition matrix that is determined from this calculation can be used to calculate the underlying network reflected in the data. This suggests a direct method of classifying genes according to their place in the resulting network. In addition to providing a means to model such a large multivariate system this approach can be used to reduce the dimensionality of the problem in a rational and consistent way, and suppress the strong noise amplification effects often encountered with expression profile data. Non-linear and higher-order Markov behavior of the network are also determined in this self-consistent method. In data sets from yeast, we calculate the Markov matrix and the gene classes based on the linear-Markov network. These results compare favorably with previously used methods like cluster analysis. Our dynamic method appears to give a broad and general framework for data analysis and modeling of gene expression arrays. Electronic Publication     

Specific Localization of β-Arrestin2 in Myenteric Plexus of Mouse Gastrointestinal Tract

β-arrestin2 is a key molecule involved in signaling and internalization of activated G protein-coupled receptors including µ-opioid receptors (MOR). Previously we have shown that decreased expression of β-arrestin2 upon chronic morphine is associated with the development of opioid tolerance in the gastrointestinal tract. However, the localization of β-arrestin2 within the gastrointestinal wall is not known. In this study we found that β-arrestin2 is localized in the soma of a select group of neurons in the myenteric ganglia but not in smooth muscle. The density of β-arestin2 was significantly higher in the ileum than the colon. We identified four variants of β-arrestin2 in the ileum, with ARRB-005 and ARRB-013 being the most abundant. Further, the current study utilized multiple-labeling immunofluorescence to characterize the chemical coding of neurons expressing β-arrestin2 in the murine myenteric plexus and the co-localization of MOR₁ and β-arrestin2. β-arrestin2 co-localized with choline acetyltransferase and calretinin. In contrast, β-arrestin2 neither co-localized with substance P, nitric oxide synthase nor calbindin. Genetic deletion of β-arrestin2 did not affect cholinergic neuron activation by nicotine in the isolated ileum (-log M EC₅₀: wild type = 5.8 vs. β-arrestin2 knockout = 5.9). Our findings suggest specificity in the localization of β-arrestin2 in the myenteric plexus within MOR₁-expressing neurons and provide a relation for direct intracellular crosstalk between MOR₁ receptor activation and β-arrestin2 signaling in the myenteric neurons. β-arrestin2 deletion does not directly alter basal enteric cholinergic neuronal function.     

Rapid Stereoselective Hydrolysis of (+)-Cocaine in Baboon Plasma Prevents Its Uptake in the Brain: Implications for Behavioral Studies

The naturally occurring enantiomer of cocaine, (–)-cocaine, has been previously labeled with ¹¹C on the N-methyl group and used in conjunction with positron emission tomography to show that cocaine is rapidly taken up in the striata of human and baboon brain. In the present study, the behaviorally inactive (+)-cocaine was similarly labeled, with a view to its use for measuring the nonspecific binding of cocaine. No brain uptake was seen, although transport of cocaine into the brain is not expected to be stereoselective. The explanation for the lack of uptake was determined to be very rapid metabolism of (+)-cocaine in the blood. By 30s after administration of labeled (+)-cocaine, it was undetectable in plasma. In vitro studies demonstrated that (+)-cocaine is 50% debenzoylated to (+)-ecgonine methyl ester within 5 s of exposure to baboon plasma but not to washed erythrocytes. The hydrolysis of (–)-cocaine is at least 1,000 times slower. Serum butyrylcholinesterase (EC 3.1.1.8) appears to be responsible for this hydrolysis, as evidenced by its inhibition by physostigmine and catalysis by commercially available pseudocholinesterase from horse and human blood.     

Interactions between S and G1 cells : Effects on decay of synchrony

Chinese hamster ovary cells were synchronized by the mitotic selection technique and followed through 3 cell cycles. The maintenance of the observed high degree of synchrony, which would require a standard deviation in generation times of about 10%, could be partially attributed to the influence that S phase cells have on inducing G1 cells to initiate DNA synthesis. In fact, observations using two populations of synchronous cells revealed that conditioned medium collected from synchronous cells in the process of initiating DNA synthesis and/or the close proximity (within 15–100 μm) of S phase cells to G l cells accelerates (by 1.5 to 2.5 h) the entry of G1 cells into S. Thus, when synchronous cells are plated, the presence of cells entering S first could both shorten the average length of G 1 and sharpen synchrony by reducing the time required for the population of G 1 cells to cross the G 1/S boundary.     

ISOLATION AND CHARACTERIZATION OF PLASMA MEMBRANE FRACTIONS FROM GARFISH LEPISOSTEUS OSSEUS OLFACTORY NERVE

Garfish Lepisosteus osseus olfactory nerve, because of its large size and the unusually high concentration of axonal membrane, is an excellent source of axonal membrane. A procedure is described for the isolation of two types of plasma membranes from the nerve which are obtained in yields of about 20 mg (fraction I) and 1.5 mg (fraction II) per g of wet nerve. Both membrane fractions consist mostly of rounded membrane vesicles, with a unit membrane thickness of ~7.5 nm. The two membrane fractions are different in their lipid to protein ratios, Na-K ATPase activities, polypeptide patterns on sodium dodecyl sulfate (SDS) gel electrophoresis, and fatty acid compositions. They have similar phospholipid composition. On the basis of the relative concentration of axonal and Schwann cell plasma membranes in the nerve, the Na-K ATPase activities of the two membrane fractions and a comparison of the properties of the membrane fractions to those of squid and lobster nerve membrane preparations, fraction I seems to be the axonal membrane and fraction II the Schwann cell plasma membrane. Fraction I has a low protein to lipid ratio. Its polypeptide pattern on SDS gel appears to be much more complex as compared to that of fraction II membrane.