Element composition of inner ear lymphs in cats,lizards, and skates determined by electron probe microanalysis of liquid samples

Summary Siliconized, glass micropipets whose tips were filled with oil were used to obtain small (<100 nl) liquid samples from perilymphatic and endolymphatic regions of the inner ears of anesthetized animals: 3 cats, 19 alligator lizards (Gerrhonotus multicarinatus), and 8 skates (Raja erinacea). Samples of cerebrospinal fluid and seawater were also obtained for skates. Electron probe microanalysis was used to measure the concentrations of the following elements in each sample: K, Na, Cl, Ca, Mg, P, S. The Na and K concentrations in cat perilymph (Fig. 1 and Table 2) agree with previous estimates (Table 4) while endolymph samples show relatively low Na and high K concentrations. From a comparison of our results with previous work (Table 3), we infer that contamination of endolymph samples with perilymph is relatively low in our study, and that no large species difference in endolymph content is indicated by present data available for mammals. Our results show that Cl concentration is higher and Ca and Mg concentrations are lower in endolymph than in perilymph. The composition of perilymph in cats and alligator lizards is roughly the same (Figs. 1 and 2, Table 2). Uncontaminated endolymph samples in lizards were apparently difficult to obtain, although the compositions of a few samples suggest that endolymph K concentration is high and Na concentration is low. In skates the concentration of Na is nearly the same in the two inner ear lymphs (Fig. 3 and Table 2), in contrast to the roughly hundredfold ratio of perilymph to endolymph Na concentrations found in the higher vertebrates. The element composition of perilymph is correlated with the composition of seawater in which the skates were kept, whereas the endolymph composition shows no such correlation.Abbreviations CSF cerebrospinal fluid - EL samples, PL samples samples judged by visual criteria alone to be from the endolymphatic and perilymphatic spaces, respectively - SW sea water This work was supported by grants from the National Institutes of Health, National Aeronautics and Space Administration, and the Health Science Fund. We thank the following people for contributions to this work: D. Beil, K. Blouch, and E. Marr.     

Effect of aging and cellularity on lipolysis in isolated mouse fat cells

The effects of age and cellularity on lipolysis have been investigated in isolated epididymal fat cells from both Swiss albino mice and Sprague-Dawley rats. No significant lipolytic response to glucagon could be demonstrated with adipocytes from either young or old mice, while glycerol output was increased by this hormone with fat cells from young rats. Larger adipocytes from older mice showed significantly greater isoproterenol-stimulated lipolysis than those from younger animals if the glycerol output was expressed on a per cell basis. However, the lipolytic response per cell appeared to be equivalent in young and old rat adipocytes with either isoproterenol or ACTH-(1-24). In a complete aging study, relationships between body weight, epididymal fat pad weight and cellularity were examined covering the life span of the mouse. ACTH-(1-24)- and dibutyryl cyclic AMP-stimulated lipolysis increased with age and cell size but fell at senescence when adipocyte size diminished. Although an effect of aging per se cannot be ruled out with the experimental techniques used in the present study, a dominant influence of adipocyte size on the lipolytic process was demonstrated.     

Hypolimnetic anoxia hampers top–down food-web manipulation in a eutrophic lake

SUMMARY 1. A biomanipulation experiment was carried out in a small (10 ha), but relatively deep (17 m) and highly eutrophic lake in northern Poland. The lake had been stocked in 1996, 1997 and 1998 with a variety of piscivorous fish (pike, catfish, trout and pikeperch), in order to reduce numbers of cyprinid planktivores.
2. Piscivore stocking was associated with a threefold decrease in the offshore fish density (night echosounding). Despite this reduction, the large planktonic cladoceran, Daphnia hyalina , remained scarce, whereas the density of small-sized zooplankton increased greatly.
3. The lack of demographic response in D. hyalina was probably due to the anoxia in the hypolimnetic refuge of this vertically migrating species. The anoxic hypolimnion, below 3–4 m depth, was inhabited day and night by numerous Chaoborus flavicans larvae.
4. Changes in zooplankton were associated with shifts in the taxonomic composition (from single-cell green algae to filamentous cyanobacteria), size structure (from nano- to net phytoplankton) and seasonal dynamics of phytoplankton, but not in the average biomass of planktonic algae. A clear-water phase, which was absent in the prestocking years, developed in spring, with Secchi depth reaching 2.5 m, a value which had never been recorded in the 20 years preceding the biomanipulation. In general, the lake's status was switched from hypertrophic to eutrophic.
5. Deteriorating food conditions, resulting from qualitative changes in the phytoplankton community, combined with predation pressure by the remaining fish and Chaoborus larvae were associated with the ultimate elimination of D. hyalina from the lake.     

The Mechanisms of Fatty Acid-Induced Proton Permeability of the Inner Mitochondrial Membrane

Nonesterified long-chain fatty acids have long been known as uncouplers of oxidativephosphorylation. They are efficient protonophores in the inner mitochondrial membrane but not so inartificial phospholipid membranes. In the un-ionized form, they undergo a rapid spontaneoustransbilayer movement (flip-flop). However, the transbilayer passage of the dissociated(anionic) form is hindered by the negatively charged hydrophilic carboxylic group. In theinner mitochondrial membrane, the transfer of fatty acid anions is mediated by the adeninenucleotide translocase, the dicarboxylate carrier, and the glutamate/aspartate carrier. As a result,the passage of protons and electric charges is a concerted effect of the spontaneous flip-flopof the undissociated (protonated) form in one direction and carrier-facilitated transfer of theionized (deprotonated) form in the other direction. In addition, fatty acids also promote openingof the mitochondrial permeability transition pore, presumably due to their interaction with oneof its constituents, the adenine nucleotide translocase, thus forming an additional route fordissipation of the proton gradient. Structural prerequisites for these proton-conductingmechanisms are (1) a weakly ionized carboxylic group and (2) a hydrocarbon chain of appropriatelength without substituents limiting its mobility and hydrophobicity.     

Inhibition of Toll-like receptors TLR4 and 7 signaling pathways by SIGIRR: A computational approach

Toll-like receptors (TLRs) belong to the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily which is defined by a common cytoplasmic Toll/interleukin-1 receptor (TIR) domain. TLRs recognize pathogen-associated molecular patterns and initiate an intracellular kinase cascade to trigger an immediate defensive response. SIGIRR (single immunoglobulin interleukin-1 receptor-related molecule), another member of the TLR/IL-1R superfamily, acts as a negative regulator of MyD88-dependent TLR signaling. It attenuates the recruitment of MyD88 adaptors to the receptors with its intracellular TIR domain. Thus, SIGIRR is a highly important molecule for the therapy of autoimmune diseases caused by TLRs. So far, the structural mechanism of interactions between SIGIRR, TLRs and adaptor molecules is unclear. To develop a working hypothesis for this interaction, we constructed three-dimensional models for the TIR domains of TLR4, TLR7, MyD88 and SIGIRR based on computational modeling. Through protein–protein docking analysis, we developed models of essential complexes involved in the TLR4 and 7 signaling and the SIGIRR inhibiting processes. We suggest that SIGIRR may exert its inhibitory effect through blocking the molecular interface of TLR4, TLR7 and the MyD88 adaptor mainly via its BB-loop region.     

Mitochondria in cell life, death and disease

In animal cell, mitochondria are the main sites of the synthesis of ATP required for cell functioning and survival. On the other hand, mitochondria play a key role in initiating cell programmed death (apoptosis). In addition, defects in the mitochondrial genome and in the nuclear genome encoding mitochondrial proteins may result in malfunctioning of these organelles and, as result, in diseases of the whole organism. This article contains basic information on the functioning of oxidative phosphorylation and on mitochondrial production of reactive oxygen species. It also describes initiation of apoptosis at the mitochondrial level. Finally, it briefly presents some most common genetic defects responsible for "mitochondrial diseases".     

NMR structure of biosynthetic engineered human insulin monomer B31(Lys)-B32(Arg) in water/acetonitrile solution. Comparison with the solution structure of native human insulin monomer

A solution NMR-derived structure of a new long -acting, B31(Lys)-B32(Arg) (LysArg), engineered human insulin monomer, in H(2)O/CD(3)CN, 65/35 vol %, pH 3.6, is presented and compared with the available X-ray structure of a monomer that forms part of a hexamer (Smith, et al., Acta Crystallogr D 2003, 59, 474) and with NMR structure of human insulin in the same solvent (Bocian, et al., J Biomol NMR 2008, 40, 55-64). Detailed analysis using PFGSE NMR (Pulsed Field Gradient Spin Echo NMR) in dilution experiments and CSI analysis prove that the structure is monomeric in the concentration range 0.1-3 mM. The presence of long-range interstrand NOEs in a studied structure, relevant to the distances found in the crystal structure of the monomer, provides the evidence for conservation of the tertiary structure. Therefore the results suggest that this solvent system is a suitable medium for studying the native conformation of the protein, especially in situations (as found for insulins) in which extensive aggregation renders structure elucidations in water difficult or impossible. Starting from the structures calculated by the program CYANA, two different molecular dynamics (MD) simulated annealing refinement protocols were applied, either using the program AMBER in vacuum (AMBER_VC), or including a generalized Born solvent model (AMBER_GB). Here we present another independent evidence to the one presented recently by us (Bocian et al., J Biomol NMR 2008, 40, 55-64), that in water/acetonitrile solvent detailed structural and dynamic information can be obtained for important proteins that are naturally present as oligomers under native conditions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 820-830, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Metabolism of amino acids in germinating yellow lupin seeds III. Breakdown of arginine in sugar-starved organs cultivated in vitro

The pathways of arginine transformations in organs of yellow lupin (Lupinus luteus L.) cultivated in vitro in the presence and absence of sucrose were investigated. Isolated embryo axes, isolated cotyledons and seeds deprived of their coat were cultured for 96 h on Heller medium with 60 mM saccharose (the fed variant, +S), without sugar (the starved variant, −S) and for 72 h without sugar, followed by 24 h in its presence (the transferred variant, −S→+S). Activities of arginine decarboxylase [EC 4.1.1.19], arginase [EC 3.5.3.1], and urease [EC 3.5.1.5] were assessed in extracts from isolated embryo axes. They were the highest in the sugar-starved variant. Supplementation of the medium with saccharose resulted in decrease in enzyme activities. The level of urea was higher (of ca. 20 %) in starved embryos than in embryos grown in the saccharose-containing medium. Moreover, participation of transamination in arginine catabolism was evidenced.     

Metabolic responses of Lemna minor to lead ions I. Growth, chlorophyll level and activity of fermentative enzymes

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The growth of Lemna minor was inhibited by lead ions, but the dry to fresh weight ratio increased as the concentration of Pb²⁺ in the medium increased. With increased concentrations of Pb ions, the contents of chlorophyll a and chlorophyll b in roots and fronds were correspondingly lower in comparision with the control. The effect of lead upon activities of some glycolitic and fermentative enzymes in roots of duckweed was examined. The activity of pyruvate kinase decreased with increasing lead concentrations, but cytosolic malate dehydrogenase behaved in an opposite manner. The lowest concentration of Pb stimulated alcohol dehydrogenase; phosphoenolopyruvate carboxylase activity was maintained at relatively constant values at all tested lead concentrations.