Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.     

Identifying the membrane proteome of HIV-1 latently infected cells

Profiling integral plasma membrane proteins is of particular importance for the identification of new biomarkers for diagnosis and for drug development. We report in this study the identification of surface markers by performing comparative proteomics of established human immunodeficiency virus-1 (HIV-1) latent cell models and parental cell lines. To this end we isolated integral membrane proteins using a biotin-directed affinity purification method. Isolated proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) after in gel digestion. Seventeen different proteins were found to vary on the surface of T-cells due to HIV-1 infection. Of these proteins, 47% were integral membrane proteins, and 18% were membrane-associated. Through the use of complementary techniques such as Western blotting and fluorescent staining, we confirmed the differential expression of some of the proteins identified by MALDI-TOF including Bruton's tyrosine kinase and X-linked inhibitor of apoptosis. Finally, using phosphatidylinositol 3-kinase inhibitors and flavopiridol to inhibit Bruton's tyrosine kinase localization at the membrane and X-linked inhibitor of apoptosis protein expression, respectively, we showed that HIV-1 latently infected cells are more sensitive to these drugs than uninfected cells. This suggests that HIV-1 latently infected cells may be targeted with drugs that alter several pathways that are essential for the establishment and maintenance of latency.     

Optimism and pessimism in optimised replay

The replay of task-relevant trajectories is known to contribute to memory consolidation and improved task performance. A wide variety of experimental data show that the content of replayed sequences is highly specific and can be modulated by reward as well as other prominent task variables. However, the rules governing the choice of sequences to be replayed still remain poorly understood. One recent theoretical suggestion is that the prioritization of replay experiences in decision-making problems is based on their effect on the choice of action. We show that this implies that subjects should replay sub-optimal actions that they dysfunctionally choose rather than optimal ones, when, by being forgetful, they experience large amounts of uncertainty in their internal models of the world. We use this to account for recent experimental data demonstrating exactly pessimal replay, fitting model parameters to the individual subjects’ choices.     

Bidirectional communication system in katydids: the effect on chorus structure

Unlike most acoustic systems evolved for pair formation whereonly males signal, the katydidPhaneroptera nana has a bidirectional communication system where both males and females sing. Despiteextensive study on male chorusing behavior in different communicationsystems, this behavior has rarely been explored in duettingspecies. I examined how this bidirectional communication systemaffects the collective pattern of male signaling.P. nana malesalternate their songs, and in response to synthetic stimulidelay their calls, according to the phase of stimulation. Pairsof synthetic calls (simulating alternating males) presentedto females elicited equal female response, as long as the intercallinterval was 200 ms. Thus, male alternation is imposed by thefemale's responsiveness and may be interpreted as a "jammingavoidance reaction." Further evidence suggests that chorusstructure is not merely constrained by the female sensory temporalresolution, but rather is adaptively related to female choicein this species.     

Functional restitution of cardiac control in heart transplant patients

Cardiovascular control is fundamentally altered after heart transplantation (HT) because of surgical denervation of the heart. The main goal of this work was the noninvasive characterization of cardiac rate control mechanisms after HT and the understanding of their nature. We obtained 25 recordings from 13 male HT patients [age = 28-68 yr, time after transplant (TAT) = 0.5-62.5 mo]. The control group included 14 healthy men (age = 28-59 yr). Electrocardiogram, continuous blood pressure (BP), and respiration were recorded for 45 min in the supine position and then during active change of posture (CP) to standing. The signals were analyzed in the time domain [mean and variance of heart rate (HR) and rise time of HR in response to CP] and the frequency domain [low and high frequency (LF and HF)]. Our principal finding was the consistent pattern of evolution of the HR response to standing: from no response, via a slow response (>40 s, TAT > 6 wk), to a fast increase (<20 s, TAT > 24 mo). HR response correlated with TAT (P < 0.001). LF correlated with HR response to CP (P < 0.0001); HF and HR did not. An important finding was the presence of very-high-frequency peaks in the power spectrum of HR and BP fluctuations. Extensive arrhythmias tended to appear at the TAT that corresponds to the transition from slow to fast HR response to CP. Our results indicate a biphasic evolution in cardiac control mechanisms from lack of control to a first-order control loop followed by partial sympathetic reinnervation and, finally, the direct effect of the old sinoatrial node on the pacemaker cell of the new sinoatrial node. There was no indication of vagal reinnervation.     

Enzymatic synthesis and purification of aromatic coenzyme a esters

Two recombinant His-tagged proteins, a plant 4-coumarate:coenzyme A ligase (EC 6.2.1.12) and a bacterial benzoate:coenzyme A ligase (EC 6.2.1.25), were expressed in Escherichia coli and purified in a single step using Ni-chelating chromatography. Purified enzymes were used to synthesize cinnamoyl-coenzyme A (CoA), p-coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, and benzoyl-CoA. Conversions up to 95% were achieved. Using a rapid solid-phase extraction procedure, the target CoA esters were isolated with yields of up to 80%. Structures were confirmed by analytical comparison with chemically synthesized reference compounds and electrospray ionization-mass spectrometry. The recombinant enzymes were stable for several months at -80 degrees C, thus providing a reliable and facile method to produce these delicate biological intermediates.     

Axoplasmic importins enable retrograde injury signaling in lesioned nerve

Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.