Crystal Structure of the Extracellular Domain of a Bacterial Ligand-Gated Ion Channel

The crystal structure of the extracellular domain (ECD) of the pentameric ligand-gated ion-channel from Gloeobacter violaceus (GLIC) was solved at neutral pH at 2.3 Å resolution in two crystal forms, showing a surprising hexameric quaternary structure with a 6-fold axis replacing the expected 5-fold axis. While each subunit retains the usual β-sandwich immunoglobulin-like fold, small deviations from the whole GLIC structure indicate zones of differential flexibility. The changes in interface between two adjacent subunits in the pentamer and the hexamer can be described in a downward translation by one inter-strand distance and a global rotation of the second subunit, using the first one for superposition. While global characteristics of the interface, such as the buried accessible surface area, do not change very much, most of the atom-atom interactions are rearranged. It thus appears that the transmembrane domain is necessary for the proper oligomeric assembly of GLIC and that there is an intrinsic plasticity or polymorphism in possible subunit-subunit interfaces at the ECD level, the latter behaving as a monomer in solution. Possible functional implications of these novel structural data are discussed in the context of the allosteric transition of this family of proteins. In addition, we propose a novel way to quantify elastic energy stored in the interface between subunits, which indicates a tenser interface for the open form than for the closed form (rest state). The hexameric or pentameric forms of the ECD have a similar negative curvature in their subunit-subunit interface, while acetylcholine binding proteins have a smaller and positive curvature that increases from the apo to the holo form.     

Radioimmunoassay for γ-endorphin

A double antibody radioimmunoassay technique for γ-endorphin has been developed. The antisera have been raised in rabbits against synthetic γ-endorphin coupled to bovine serum albumin by carbodiimide. The best antibody has a working titer of $\text{1}\text{35,000}$ and can detect less than 9 pg of peptide. The usable range of the standard curve is between 9 to 2400 pg. This antiserum probably binds the Glu⁸-Leu¹⁷ region of γ-endorphin and shows only weak cross-reactivity with α-endorphin, β-endorphin and β-lipotropin. Parallelism is observed between the standard curve and the inhibition curves obtained with rat neurohypophysis-pars intermedia extracts or rat plasma.     

Immunolocalization of Fibronectin and Laminin in the Amphibian Intestine During Spontaneous and Triiodothyronine-Induced Metamorphosis

Fibronectin and laminin were detected by indirect immunofluorescence in the intestine of Alytes obstetricans (anuran amphibian) during triiodothyronine (T3)-induced metamorphosis and spontaneous post-embryonic development. Fibronectin was first detected between a small number of connective tissue cells. As T3-treatment and spontaneous development progressed, fibronectin became detectable as a fine network extending throughout the whole thickness of the connective tissue and particularly in the core of the developing epithelial folds. During the first week of T3-treatment and throughout the spontaneous larval period, laminin was present as a linear band within the basement membrane. Between day 6 and 12 of hormonal treatment, an increase in the laminin fluorescent staining was noted. After hormonal treatment for two weeks and at the end of spontaneous metamorphosis, laminin staining was localized within the basement membrane of the folded epithelium and around muscle fibers. These observations indicate that variations in the density and distribution of extracellular matrix molecules are closely related spatiotemporarily to the structural changes occurring in the connective and muscle tissues of the intestine during metamorphosis.     

Increased auxin efflux in the IAA-overproducing sur1 mutant of Arabidopsis thaliana: A mechanism of reducing auxin levels?

With the aim of investigating the mechanisms that maintain auxin homeostasis in plants, we have monitored the net uptake and metabolism of exogenously supplied indole-3-acetic acid (IAA) and naphthalene-1-acetic acid (NAA) in seedlings of wild type and the IAA-overproducing mutant sur1 of Arabidopsis thaliana . Tritiated IAA and NAA entered the seedling tissues within minutes and were mostly accumulated as metabolites, probably amino acid and sugar conjugates. The mutant seedlings were marked by a strong increase of [³H]IAA metabolism and a reduction of the accumulation levels of both free [³H]IAA and [³H]NAA. The same characteristics were observed in wild-type seedlings grown on 5 μ M picloram. We measured [³H]NAA uptake in the presence of high concentrations of unlabeled NAA or the auxin efflux carrier inhibitor naphthylphthalamic acid (NPA). This abolished the difference in free [³H]NAA accumulation between the mutant or picloram-treated seedlings and wild-type seedlings. These data indicated that active auxin efflux carriers were present in Arabidopsis seedling tissues. Picloram-treated seedlings and seedlings of the IAA-overproducing mutant sur1 displayed increased auxin efflux carrier activity as well as elevated conjugation of IAA. There is previous evidence to suggest that conjugation is a means to remove excess IAA in plant cells. Here, we discuss the possibility of efflux constituting an additional mechanism for regulating free IAA levels in the face of an excess auxin supply.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

A Re-Evaluation of the Place of Major Extirpation in the Management of Patients with Metastatic Mammary Carcinoma

Clearly defined indications for major extirpation procedures in metastatic mammary carcinoma are lacking. A reassessment of experience with 222 patients (133 after adrenalectomies and 89 after hypophysectomies) was therefore undertaken. This experience has changed our previous opinions that such procedures should be used only at the end of a sequential program of treatment.It is now felt that extirpation is only contraindicated in menopausal women with a duration of disease less than two years or when there is symptomatic disease of vital organs. Otherwise, early extirpation is recommended. When vital organ involvement is recognized in the asymptomatic stage or when screening of premenopausal women by castration indicates continuing hormonal dependency, then early extirpation is indicated.Both the qualitative and the quantitative responses achieved exceed those obtained by other measures when widely disseminated disease is present. The objective remission rate (34% after adrenalectomy and 19% after hypophysectomy) for longer than six months is worth while.     

Compressive Stress Inhibits Proliferation in Tumor Spheroids through a Volume Limitation

In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27^Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27^Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27^Kip1 is strongly influenced by this change.     

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos

Summary Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.