The ''Bayer bridges'' confronted with results from improved electron microscopy methods

In electron micrographs of conventionally prepared thin sections of Escherichia coli one observes (i) a wavy appearance of the two membranes showing frequent appositions (named adhesion sites) and (ii) intermembrane bridges after plasmolysis which, it is claimed, occur at the adhesion sites and are related to intermembrane protein transport (transmigration). When chemical fixation is replaced by cryofixation, the observations are very different. (a) The two membranes are equally spaced and no contacts, adhesions or other sorts of connections are visible. (b) After plasmolysis the protoplast is shrunken, but the typical bridges are no longer produced. (c) In addition, when peptidoglycan is stained on conventionally prepared sections, it is revealed as a 7-nm-thick sacculus which is not interrupted at the sites of apposition. In view of the new observations, the structural concepts derived from conventionally prepared material must be revised. It is proposed that the intermembrane space is entirely filled by a gel, the outer part of which is the 7 nm thick, very stable, chemically resistant peptidoglycan (or murein). The inner part is much less stable and is proposed to undergo rapid autolytic changes upon cell death. The large 'Bayer bridges' might then tentatively be explained as an artificial post-mortem enhancement of either a stream of proteins transmigrating across the periplasm or of a pre-existing, but not yet resolved, structure. This enhancement probably occurs during the 7-10 min between plasmolysis and fixation that are prescribed for the procedure necessary for revealing 'Bayer bridges'.     

Protoplasmatische Anatomie des Laubmooses Bryum capillare. I

Ohne ZusammenfassungDie Arbeit wurde am Pflanzenphysiologischen Institut der Universität Wien ausgeführt. Dem Vorstand des Institutes Herrn Professor Dr. Karl Höfler erlaube ich mir für die Anregung der Arbeit und die ständige Leitung der Untersuchungen meinen herzlichsten Dank auszusprechen. Für die Einführung in die Fluoreszenzmikroskopie bin ich Herrn Professor Dr. Christian Wimmer (Mödling) zu Dank verpflichtet. Herrn Dr. Lothar Hofmeister danke ich für seine ständige Hilfsbereitschaft herzlichst.     

Host specificity of DNA produced byEscherichia coli

Summary Phage , whose DNA was physically labelled with density markers²H and¹⁵N and biologically labelled with K-host specificity, was submitted to one growth cycle on a restriction deficient (r ^–) strain ofE. coli B exerting normally its modification function (m _B ⁺ ). All progeny phages, including those with nonreplicated, fully conserved parental DNA molecules, acquired the B-specificity in this passage, independently of DNA replication and of the presence of the K-specificity on the DNA. Phages with parental DNA had preserved the parental K-specificity. However, about half of the phages carrying a halfheavy DNA molecule (corresponding presumably to a semiconserved double helix) did only plate on B and onr ^– mutants, but not on K12. Experimental evidence is presented, that DNA degradation is the cause of this lack of growth in K12, while in infections initiated by the other half of the hybrids both strands (that with K and B specificity and that with only B specificity) are preserved and are recovered in the progeny with equal chance.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Modeling transitions in body composition: the approach to steady state for anthropometric measures and physiological functions in the Minnesota human starvation study

Background

This study evaluated whether the changes in several anthropometric and functional measures during caloric restriction combined with walking and treadmill exercise would fit a simple model of approach to steady state (a plateau) that can be solved using spreadsheet software (Microsoft Excel^®). We hypothesized that transitions in waist girth and several body compartments would fit a simple exponential model that approaches a stable steady-state.

Methods

The model (an equation) was applied to outcomes reported in the Minnesota starvation experiment using Microsoft Excel's Solver^® function to derive rate parameters (k) and projected steady state values. However, data for most end-points were available only at t = 0, 12 and 24 weeks of caloric restriction. Therefore, we derived 2 new equations that enable model solutions to be calculated from 3 equally spaced data points.

Results

For the group of male subjects in the Minnesota study, body mass declined with a first order rate constant of about 0.079 wk^-1. The fractional rate of loss of fat free mass, which includes components that remained almost constant during starvation, was 0.064 wk^-1, compared to a rate of loss of fat mass of 0.103 wk^-1. The rate of loss of abdominal fat, as exemplified by the change in the waist girth, was 0.213 wk^-1.On average, 0.77 kg was lost per cm of waist girth. Other girths showed rates of loss between 0.085 and 0.131 wk^-1. Resting energy expenditure (REE) declined at 0.131 wk^-1. Changes in heart volume, hand strength, work capacity and N excretion showed rates of loss in the same range. The group of 32 subjects was close to steady state or had already reached steady state for the variables under consideration at the end of semi-starvation.

Conclusion

When energy intake is changed to new, relatively constant levels, while physical activity is maintained, changes in several anthropometric and physiological measures can be modeled as an exponential approach to steady state using software that is widely available. The 3 point method for parameter estimation provides a criterion for testing whether change in a variable can be usefully modelled with exponential kinetics within the time range for which data are available.

     

Grouping horses according to gender—Effects on aggression, spacing and injuries

Many horse owners tend to group horses according to gender, in an attempt to reduce aggressive interactions and the risk of injuries. The aim of our experiment was to test the effects of such gender separation on injuries, social interactions and individual distance in domestic horses. A total of 66 horses were recruited from 4 different farms in Norway and Denmark and divided into six batches. Within each batch, horses were allotted into one mare group, one gelding group and one mixed gender group, with most groups consisting of three or four animals. After 4–6 weeks of acclimatisation, a trained observer recorded all social interactions using direct, continuous observation 1 h in the morning and 1 h in the afternoon for three consecutive days. Recordings of the nearest neighbour of each horse were performed using instantaneous sampling every 10 min. The horses were inspected for injuries before grouping, day 1 after grouping and after 4–6 weeks. No significant effect of gender composition was found on social interactions (P > 0.05), spacing (P > 0.07) or injuries (P > 0.23). Eighty percent of all aggressive interactions recorded were threats, not involving physical contact. Horses with the smallest space allowance showed the highest mean number of aggressive interactions (28.6 ± 6.1 interactions per 6 h) compared to the mean of all the other batches (8.3 ± 1.0 interactions per 6 h). Very few injuries were found and most were superficial. In conclusion, gender composition does not seem to have any effect on aggression level, spacing or injuries. However, the early social experience of horses, management of feeding and space allowance probably represents more important factors for successful group housing of domestic horses.     

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.     

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Isolation and reassembly of bacteriophage T4 core proteins

The products of genes 22, 67 and 68, and the internal proteins IPI, IPII and IPIII, as components of the scaffolding core of the bacteriophage T4 prohead, have been isolated and purified by hydroxylapatite column chromatography. Under conditions promoting reassembly in vitro, the proteins associated into elongated particles of practically constant width but variable length that we have called polycores. Preliminary optical diffraction experiments indicate that polycores may have an ordered structure, possibly helical, as has been suggested for the polyhead core. The coassembly of core proteins and the purified shell protein gp23 results in the formation of core-containing polyheads. Occasionally, prolate core-like particles have been observed but their reproducible formation has not been attained. Attempts to investigate the role of the minor prohead component gp20 in core assembly have been made through the cloning of the corresponding gene in an expression vector and subsequent purification of the protein.