Basin-scale internal waves in the bottom boundary layer of ice-covered Lake Müggelsee, Germany

We performed high-resolution temperature measurements under ice cover in Lake Müggelsee, Germany, during the winter of 2005–2006. Intense seiche-like temperature oscillations developing after the ice-on have been encountered in a thin water layer above the sediments. The oscillations were initiated immediately after lake freezing by the release of the potential energy of the thermocline slope and existed for several weeks without appreciable external forcing. The oscillations were associated with a basin-scale internal waves existing in the lower stratified part of the water column. The weakness of the density stratification under ice ensured the long wave periods, exceeding the period of geostrophic inertial oscillations at the lake’s latitude. As a result, two frequency peaks were present in the oscillations corresponding to two rotational waves, one of Kelvin-wave type and another of Poincaré type wave. The rotational character provided long dissipation times of the waves and allowed the oscillations to persist in lake several weeks. Temperature measurements in the upper several centimeters of the sediment demonstrated that oscillations of the near-bottom temperature produced vertical density instability and pore-water convection in the upper sediments.     

Monkey Steering Responses Reveal Rapid Visual-Motor Feedback

The neural mechanisms underlying primate locomotion are largely unknown. While behavioral and theoretical work has provided a number of ideas of how navigation is controlled, progress will require direct physiolgical tests of the underlying mechanisms. In turn, this will require development of appropriate animal models. We trained three monkeys to track a moving visual target in a simple virtual environment, using a joystick to control their direction. The monkeys learned to quickly and accurately turn to the target, and their steering behavior was quite stereotyped and reliable. Monkeys typically responded to abrupt steps of target direction with a biphasic steering movement, exhibiting modest but transient overshoot. Response latencies averaged approximately 300 ms, and monkeys were typically back on target after about 1 s. We also exploited the variability of responses about the mean to explore the time-course of correlation between target direction and steering response. This analysis revealed a broad peak of correlation spanning approximately 400 ms in the recent past, during which steering errors provoke a compensatory response. This suggests a continuous, visual-motor loop controls steering behavior, even during the epoch surrounding transient inputs. Many results from the human literature also suggest that steering is controlled by such a closed loop. The similarity of our results to those in humans suggests the monkey is a very good animal model for human visually guided steering.     

Dynamin is required for recombinant adeno-associated virus type 2 infection

Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells.     

Second-strand genome conversion of adeno-associated virus type 2 (AAV-2) and AAV-5 is not rate limiting following apical infection of polarized human airway epithelia

Recombinant adeno-associated virus type 5 (rAAV-5) is known to efficiently transduce airway epithelia via apical infection. In contrast, rAAV-2 has been shown to be inherently ineffective at transducing airway epithelia from the apical surface. However, tripeptide proteasome inhibitors (such as LLnL) can dramatically enhance rAAV-2 transduction from the apical surface of human polarized airway epithelia by modulating the intracellular trafficking and processing of the virus. To further investigate potential differences between rAAV-2 and rAAV-5 that might explain their altered ability to transduce airway epithelia from the apical membrane, we examined the functional involvement of the ubiquitin/proteasome pathway and rate-limiting aspects of second-strand synthesis for these two rAAV serotypes. To this end, we conducted studies to compare the extent to which LLnL alters transduction efficiencies with both rAAV-2 and rAAV-2/5 by using luciferase and enhanced green fluorescent protein (EGFP) reporter vectors. Our results demonstrate that the coadministration of LLnL at the time of viral infection significantly enhanced transduction of both rAAV-2/5 and rAAV-2 from the apical surface of airway epithelia. Although rAAV-2/5 was slightly more effective at transducing epithelia from the apical membrane, rAAV-2 transduction was superior to that of rAAV-2/5 in the presence of proteasome inhibitors. Interestingly, the basolateral membrane entry pathways for both serotypes were not significantly affected by the addition of LLnL, which suggests that apical and basolateral infectious pathways possess distinctive intracellular processing pathways for both rAAV-2 and rAAV-5. Studies comparing the transduction of short self-complementary (scAAV) to full-length conventional AAV EGFP vectors suggested that second-strand synthesis of rAAV genomes was not rate limiting for either serotype or altered by proteasome inhibitors following apical infection of polarized airway epithelia. These findings suggest that both rAAV-2 and rAAV-5 share similar intracellular viral processing barriers that involve the ubiquitin/proteasome system, but do not appear to involve second-strand synthesis.     

The core of the tetrameric mycobacterial porin MspA is an extremely stable beta-sheet domain

MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel beta-strands organized in a coherent domain. Heating to 92 and 112 degrees C was required to dissociate the MspA tetramer and to unfold the beta-sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming beta-barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 m urea at 50 degrees C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the beta-sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are beta-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.     

Insulin-like growth factors and insulin-like growth factor binding proteins in adult patients with severe liver disease before and after orthotopic liver transplantation

INTRODUCTION: The liver is the main source of serum insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) and the concentration of these proteins might reflect liver function. METHODS: In a retrospective longitudinal study we examined serum levels of total and free IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3 and IGFBP-6 in 21 adult patients with end-stage liver disease before and after orthotopic liver transplantation (LTX) by sensitive and specific RIAs. In each patient, the mean value of at least three measurements before and after LTX was calculated. RESULTS: Before LTX, serum levels of total and free IGF-I, IGF-II, IGFBP-3 were low and showed a rapid and significant increase in almost all patients after successful LTX (total IGF-I: 30 +/- 7 vs. 256 +/- 30 ng/ml, p < 0.001; free IGF-I: 1.3 +/- 0.3 vs. 3.5 +/- 0.6 ng/ml, p < 0.01; IGF-II: 177 +/- 28 vs. 618 +/- 30 ng/ml, p < 0.001; IGFBP-3: 1,230 +/- 136 vs. 3,665 +/- 264 ng/ml, p < 0.001). In contrast, IGFBP-1 was found to be high immediately before LTX, and declined to normal levels after LTX (210 +/- 40 vs. 90 +/- 15 ng/ml, p < 0.01), while IGFBP-2 did not show any significant changes (1,154 +/- 296 vs. 1,303 +/- 192 ng/ ml). Positive correlations were found between IGF-I, IGF-II or IGFBP-3, and serum pseudocholinesterase (R = 0.50, 0.72 and 0.61 respectively, p < 0.001). Negative correlations were found between IGF-I, IGF-II or IGFBP-3, and prothrombin time (R = 0.50, 0.59 and 0.51 respectively, p < 0.001). CONCLUSION: Patients with severe liver disease show decreased levels of total and free IGF-I, IGF-II and IGFBP-3, and increased levels of IGFBP-1. These abnormalities are promptly normalized after successful LTX. Thus, serum levels of IGF-I, IGF-II and IGFBP-3 might be useful parameters for the assessment of liver function.     

Electrostatic properties of the anion selective porin Omp32 from Delftia acidovorans and of the arginine cluster of bacterial porins

The functional properties of the anion-selective porin Omp32 from the bacterium Delftia acidovorans, formerly Comamonas acidovorans, are determined by the particularly narrow channel constriction and the electrostatic field inside and outside the pore. A cluster of arginines (Arg 38, Arg 75, and Arg 133) determines the electrostatic field close to the constriction zone. Stacked amino acids carrying charges are prone to drastic pK(a) shifts. However, optimized calculations of the titration behavior of charged groups, based on the finite-difference Poisson-Boltzmann technique, suggest that all the arginines are charged at physiological pH. Protonation of the clustered arginines is stabilized by one buried glutamate residue (Glu 58), which is strongly interacting with Arg 75 and Arg 38. This functional arrangement of three charged amino acid residues is of general significance because it is found in the constriction zones of all known 16-stranded porins from the alpha-, beta-, and gamma-proteobacteria.     

Nonlinear and asymmetric open channel characteristics of an ion-selective porin in planar membranes.

The open channel characteristics of the bacterial porin Omp32 from Comamonas acidovorans were investigated by means of conductance measurements in planar lipid bilayers of the Montal-Mueller type. Particularly at low salt conditions (< or = 30 mM KCl) Omp32 exhibited some unusual asymmetric and nonlinear functional properties. Current-voltage relationship measurements showed that conductance depends on the orientation of porin molecules and is a nonlinear function of the applied membrane potential. Conductance also depends on the salt concentration in a manner not common to porins and the salt concentration modulates the nonlinearity of conductance-voltage relationships. Omp32 is strongly anion-selective. The nonlinear and asymmetric conductance of the open channel is a new observation in porins.