Localization to the proteasome is sufficient for degradation

The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.     

Control of the coagulation system by serpins. Getting by with a little help from glycosaminoglycans

Members of the serine protease inhibitor (serpin) superfamily play important roles in the inhibition of serine proteases involved in complex systems. This is evident in the regulation of coagulation serine proteases, especially the central enzyme in this system, thrombin. This review focuses on three serpins which are known to be key players in the regulation of thrombin: antithrombin and heparin cofactor II, which inhibit thrombin in its procoagulant role, and protein C inhibitor, which primarily inhibits the thrombin/thrombomodulin complex, where thrombin plays an anticoagulant role. Several structures have been published in the past few years which have given great insight into the mechanism of action of these serpins and have significantly added to a wealth of biochemical and biophysical studies carried out previously. A major feature of these serpins is that they are under the control of glycosaminoglycans, which play a key role in accelerating and localizing their action. While further work is clearly required to understand the mechanism of action of the glycosaminoglycans, the biological mechanisms whereby cognate glycosaminoglycans for each serpin come into contact with the inhibitors also requires much further work in this important field.     

Reduced contribution from Na+/H+ exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons

The effect of anoxia on Na+/H+ exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+-sensitive fluorophore, BCECF. Five-minute anoxia imposed under nominally HCO3-/CO2-free conditions induced a fall in pHi, the magnitude of which was smaller following prolonged exposure to medium in which N-methyl-D-glucamine (NMDG+) was employed as an extracellular Na+ (Na(+)(o)) substitute. Also consistent with the possibility that Na+/H+ exchange becomes inhibited soon after the induction of anoxia, rates of Na(+)(o)-dependent pHi recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na(+)(o)-dependent pHi recovery observed prior to anoxia. At the time at which rates of pHi recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na+/H+ exchange. In support, incubation of neurons with 2-deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na(+)(o)-dependent rates of pHi recovery from imposed acid loads; conversely, pre-treatment with 10 mm creatine attenuated the effects of anoxia to reduce both ATP levels and Na(+)(o)-dependent rates of pHi recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na+/H+ exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia-induced falls in intracellular ATP.     

Mathematical models of diffusion-constrained polymerase chain reactions: basis of high-throughput nucleic acid assays and simple self-organizing systems

DNA templates amplified by polymerase chain reaction in thin polyacrylamide gels form diffusion-constrained amplicons called "polonies" (polymerase colonies) that have been used to phase DNA haplotypes over long distances, to analyse RNA splice variants, and to assay other phenomena of biological interest. We present two sets of mathematical models, one for single polony growth (SPGM) and one for two polony interaction (TPIM), that will be used to optimize polony technology. The models provide detailed predictions of polony yield, concentration profiles, growth of isolated polonies, and the interaction of neighboring polonies. The TPIM explains an experimental observation that nearby polonies deform against each other rather than interpenetrate, an effect important for optimizing polony protocols. However, the TPIM also predicts that polonies may invade each other with a complex geometry when sufficiently close. Polonies are also of interest as simple abiotic systems that exhibit lifelike properties of self-organization, growth, and development, and the models may also apply to biological phenomena involving propagation through tethering and diffusion. Our polony modeling software is available at our web site.     

Efficient pyrimidine N-1-alkylation via activation of electron rich olefins with oxoammonium salts: synthesis of methoxy TEMPO substituted pyrimidine nucleoside analogs

Our work outlines the use of oxoammonium salts in a formal 1,2 addition process to olefins giving nucleoside analogs as products. Specifically, oxoammonium salts can be added to a solution of olefin and silylated heterocycle to give Methoxy TEMPO substituted nucleoside analogs after hydrolytic workup and chromatographic purification.     

Genetic regulation of endotoxin-induced airway disease

To identify novel genes regulating the biologic response to lipopolysaccharide (LPS), we used a combination of quantitative trait locus (QTL) analysis and microarray-based gene expression studies of C57BL/6J x DBA/2J(BXD) F2 and recombinant inbred (RI) mice. A QTL affecting pulmonary TNF-alpha production was identified on chromosome 2, and a region affecting both polymorphonuclear leukocyte recruitment and TNF-alpha levels was identified on chromosome 11. Microarray analyses of unchallenged and LPS-challenged BXD RI strains identified approximately 500 genes whose expression was significantly changed by inhalation of LPS. Of these genes, 28 reside within the chromosomal regions identified by the QTL analyses, implicating these genes as high priority candidates for functional studies. Additional high priority candidate genes were identified based on their differential expression in mice having high and low responses to LPS. Functional studies of these genes are expected to reveal important molecular mechanisms regulating the magnitude of biologic responses to LPS.     

A Change in Orientation: Recognition of Rotated Patterns by Bumble Bees

In three experiments, bumble bees were trained to discriminate between a reinforcing pattern (S+) and a nonreinforcing one (S–) which differed only in the configuration of four artificial petals. They were subsequently tested for recognition of the S+ rotated by 90° (S + 90). Experiment 1 used petals of four colors, and the other experiments used four symbols. The symbols either remained unchanged when the whole pattern was rotated (e.g., + in Experiment 2) or changed appearance (e.g., < in Experiment 3). The bees failed to recognize the S + 90 in the first two experiments, but in Experiment 3, the choice proportion for S + 90 in the presence of a New pattern was significantly higher than chance. Bumble bees can recognize a rotated pattern, possibly by using mental rotation, provided that a cue as to the extent of the pattern transformation is given.