Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 M_r oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (α₂βγδ) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the δ chains.In agreement with previous work (Cartaud et al., 1980) the H-form appeared as “doublets” of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 ± 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublet are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the δ chain within the structure.Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified.Tentative assignment of structural details to individual subunits was deduced from an examination of α-bungarotoxin-labeled doublets. The α subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).     

A tripartite culture system for endomycorrhizal inoculation of micropropagated strawberry plantlets in vitro

The objective of the current investigation was to develop a reliable method to obtain vesicular arbuscular mycorrhizae (VAM) in micropropagated plantlets and to determine their influence on growth. An in vitro system for culturing the VA mycorrhizal fungus Glomus intraradices with Ri T-DNA-transformed carrot roots or nontransformed tomato roots was used in this study as a potential active source of inoculum for the colonization of micropropagated plantlets. After root induction, micropropagated plantlets grown on cellulose plugs (sorbarod) were placed in contact with the primary mycorrhizae in growth chambers enriched with 5000 ppm CO₂ and fed with a minimal medium. After 20 days of tripartite culture, all plantlets placed in contact with the primary symbiosis were colonized by the VAM fungus. As inoculum source, 30-day-old VA mycorrhizal transformed carrot roots had a substantially higher infection potential than 5-, 10-or 20-day-old VAM. Colonized plantlets had more extensive root systems and better shoot growth than control plants. The VAM symbiosis reduced the plantlet osmotic potential. This response may be a useful pre-adaptation for plantlets during transfer to the acclimatization stage.     

Formation of a Ternary Complex among NHERF1, β-Arrestin,and Parathyroid Hormone Receptor

β-Arrestins are crucial regulators of G-protein coupled receptor (GPCR) signaling, desensitization, and internalization. Despite the long-standing paradigm that agonist-promoted receptor phosphorylation is required for β-arrestin2 recruitment, emerging evidence suggests that phosphorylation-independent mechanisms play a role in β-arrestin2 recruitment by GPCRs. Several PDZ proteins are known to interact with GPCRs and serve as cytosolic adaptors to modulate receptor signaling and trafficking. Na⁺/H⁺ exchange regulatory factors (NHERFs) exert a major role in GPCR signaling. By combining imaging and biochemical and biophysical methods we investigated the interplay among NHERF1, β-arrestin2, and the parathyroid hormone receptor type 1 (PTHR). We show that NHERF1 and β-arrestin2 can independently bind to the PTHR and form a ternary complex in cultured human embryonic kidney cells and Chinese hamster ovary cells. Although NHERF1 interacts constitutively with the PTHR, β-arrestin2 binding is promoted by receptor activation. NHERF1 interacts directly with β-arrestin2 without using the PTHR as an interface. Fluorescence resonance energy transfer studies revealed that the kinetics of PTHR and β-arrestin2 interactions were modulated by NHERF1. These findings suggest a model in which NHERF1 may serve as an adaptor, bringing β-arrestin2 into close proximity to the PTHR, thereby facilitating β-arrestin2 recruitment after receptor activation.     

The evolution of groundwater Cladocera

Cladocera occur in various types of groundwater, but are most common in the underflow of rivers. Numerous surface water species occasionally venture into groundwater; some chydorids are more common in groundwaters than in surface waters; at least three groups within Alona, finally, have evolved exclusive groundwater species. The latter show few obvious adaptations to the subterranean habitat, except loss of an eye or total blindness. Some, however, have conserved an array of primitive characters (e.g. on the end-claw of the postabdomen, and the setation of the valve rims) which suggest that the physical protection and relative constancy of the hyporheic has permitted the survival of some ancient taxa.     

Effects of thyrotropin,prostaglandin E1 and iodide on cyclic 3′,5′-AMP concentration in dog thyroid slices

The kinetics and concentration effect on the relationship of thyrotropin (TSH) action on cyclic 3′,5′-AMP concentration has been studied in dog thyroid slices in vitro. TSH markedly increased cyclic 3′,5′-AMP level after 5 min, the effect reached a plateau after 10–60 min and slowly declined afterwards. TSH enhanced in parallel the cyclic 3′,5′-AMP level and the binding of iodide to proteins. For this latter effect of TSH, the four criteria of the validity of the Sutherland model for a hormonal action are therefore fulfilled. The effect of TSH on cyclic 3′,5′-AMP concentration in thyroid did not require the presence of a methylxanthine inhibitor of cyclic 3′,5′-AMP phosphodiesterase in the medium. Prostaglandin E1 increased cyclic 3′,5′-AMP levels in control and stimulated slices. The omission of Ca²⁺ in the incubation medium decreased the action of TSH but partial replacement of Na⁺ by K⁺ had little effect. Iodide, 1 μM to 100 μM, inhibited the action of TSH. This inhibitory effect was relieved by NaClO₄, methimazole and propylthiouracil (1 mM). The possible role of this inhibitory effect in an intracellular regulatory mechanism is discussed.     

The in vitro cellular synthesis of hemoglobin components in various media and its relevance to the concept of stenogenic and eurygenic states of differentiation.

The increase above normal in the rate of synthesis of human fetal hemoglobin in incubated cells from adults, as observed previously, is only relative. The addition of serum to the incubation medium tends to reestablish the normal ratio of rates. In the absence of serum the decrease in rates of synthesis affects most strongly the hemoglobin component, whichever it be, that is synthesized more intensively at the cell stage under consideration. This is brought out by the comparison of the effects of different media on fetal and adult cells. The data lead to the definition of the concept of stenogenic and eurygenic states of differentiation.     

Calorimetric study of water in muscle tissue

Differential scanning calorimetry has been used to investigate the state of water in intact single muscle fibers of the giant barnacle, Balanus nubilus. The shapes of the melting curves suggest the presence of three types of water: unfrozen (or bound), free (or bulk) and intermediate water. The amount of unfrozen water per g protein was constant within experimental error. An increase in water content changed almost exclusively the amounts of free water. The amount of intermediate water varies only slightly with the fiber water content.