Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Circadian rhythms of sensitivity to insecticides in Musca domestica (Diptera, Muscidae)

Circadian rhythms of LD₅₀ values to DDT, dieldrin and malathion, topically applied, were determined for houseflies reared under LD 14:10 with dawn at 06.00 hr. There was a marked increase in susceptibility at 05.00 hr in each case. With dawn at 18.00 hr., DDT LD₅₀ values were lowest at 17.00 hr indicating independence of the flies' biological clocks from clock time of day. Flies reared under LD 18:6 and 10:14 also had circadian rhythms of sensitivity to DDT. Mean daily LD₅₀ values were inversely related to photophase length. The ratios of mean daily LD₅₀ to pre-dawn values were greatest for the longer photophases. Flies reared under LD 14:10 until the pupal stage, then DD until testing showed a normal circadian rhythm. Flies reared in total darkness (DD) showed no diel variations in susceptibility. W.H.O. standard strain flies were used for all the experiments. A fully susceptible (Cooper) and a DDT resistant (DEH-DOV) strain also showed significant circadian rhythms of sensitivity to DDT.

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Zusammenfassung Circadianrhythmen der LD 50-Werte gegenüber DDT, Dieldrin und Malathion-topical angewandt wurden bei Stubenfliegen ermittelt, die bei 14:10 h-Tag mit Tagesanbruch um 6.00 Uhr gezüchtet wurden. In allen Fällen war die Empfindlichkeit um 5.00 Uhr wesentlich erhöht. Bei Tagesanbruch um 18.00 Uhr waren die niedrigsten LD 50-Werte um 17.00 Uhr. Dies weist auf die Unabhängigkeit der biologischen Uhr der Fliegen von der Tageszeit hin. Fliegen, die bei 18:6 oder bei 10:14 LD gezüchtet wurden, zeigten ebenfalls einen Circadianrhythmus hinsichtlich der Empfindlichkeit gegenüber DDT. Die mittleren LD 50-Werte waren umgekehrt proportional zur Länge der Photophase. Das Verhältnis der mittleren täglichen LD 50-Werte zu den Vortagesanbruchwerten war am grössten bei längerer Photophase. Fliegen, die bei 14:10 LD bis zum Puppenstadium und anschliessend bei DD bis zur Testung gehalten wurden, zeigten einen normalen circadianen Rhythmus. Bei Züchtung in völliger Dunkelheit zeigten sie keine Tagesschwankungen in der Empfindlichkeit. Für alle Versuche wurde ein WHO-Standardstamm benutzt. Zwei andere Stämme, einer voll empfindlich (Cooper), der andere resistent (DEH-DOV) zeigten ebenfalls signifikante Circadianrhythmen in der DDT-Empfindlichkeir.

     

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility

The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: “Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?”. Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.     

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.