The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.     

Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the Mg-dependent enzyme

The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17°C, in media containing 125 mM Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgCl₂, and 1 mM Pb(NO₃)₂. After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoate, and lack of inhibition by ouabain.     

Steroids and related products. LIV. The synthesis of 11-oxa steroids. VI. The synthesis of 11-oxatestosterone

The synthesis of 11-oxatestosterone from 11-oxa-5 alpha-androstane-3,17-dione, which is available from hecogenin, is described. The product shows, in comparison with the natural hormone, diminished androgenic and anabolic activities.     

Cartilage proteoglycans. Assembly with hyaluronate and link protein as studied by electron microscopy.

Aggregates formed by the interaction of cartilage proteoglycan monomers and fragments thereof with hyaluronate were studied by electron microscopy by use of rotary shadowing [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333]. The differences in shape and packing of the proteins bound along the hyaluronate strand in aggregates formed in the presence and in the absence of link protein were examined in detail. The high resolution of the method allowed examination of the involvement in hyaluronate binding of the globular core-protein domains G1, G2 and G3 [Wiedemann, Paulsson, Timpl, Engel & Heinegård (1984) Biochem. J. 224, 331-333; Paulsson, Mörgelin, Wiedemann, Beardmore-Gray, Dunham, Hardingham, Heinegård, Timpl & Engel (1987) Biochem. J. 245, 763-772]. Fragments comprising the globular hyaluronate-binding region G1 form complexes with hyaluronate with an appearance of necklace-like structures, statistically interspaced by free hyaluronate strands. The closest centre-to-centre distance found between adjacent G1 domains was 12 nm. Another fragment comprising the binding region G1 and the adjacent second globular domain G2 attaches to hyaluronate only by one globule. Also, the core protein obtained by chondroitinase digestion of proteoglycan monomer binds only by domain G1, with domain G3 furthest removed from the hyaluronate. Globule G1 shows a statistical distribution along the hyaluronate strands. In contrast, when link protein is added, binding is no longer random, but instead uninterrupted densely packed aggregates are formed.     

In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β- -glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

Pressure-diameter relationships of the upper airway in awake supine subjects

In awake supine normal subjects, dimensional changes of the oropharyngeal airway were measured during exposure to negative intraluminal pressures. The pressure was generated 1) "actively" by subjects inspiring against an externally occluded airway or 2) "passively" by external suction at the mouth during voluntary glottic closure with no inspiratory effort. Airway dimensions were imaged with X-ray fluoroscopy and anteroposterior diameters measured at levels corresponding to cervical vertebra 3 and 4 (C3 and C4). Cephalad axial displacement of the hyoid bone (CDHY) was also measured. During the "active" maneuver, airway diameters and position were maintained at resting levels despite airway pressure up to -15 cmH2O. In contrast, during the passive maneuver at -15 cmH2O, C3 was only 15 +/- 9% and C4 only 47 +/- 8% of control; CDHY was 5.6 +/- 1.8 mm. In three subjects airway wall apposition occurred and persisted until an active inspiratory effort. We conclude that, in the absence of inspiratory effort, negative oropharyngeal airway pressures result in marked narrowing and cephalad displacement of the upper airway, even during wakefulness. Therefore, our data suggest that the complex interaction of upper airway and thoracic muscle activity is critical in determining the effective compliance and patency of the upper airway, which is readily collapsible even in normal subjects.     

Haploid male germ cells show no susceptibility to transformation by simian virus 40 large tumour antigen in transgenic mice.

Cell-type specific tumorigenesis can be induced in transgenic mice by the directed expression of simian virus 40 (SV 40) large tumour antigen (TAg). In an attempt to determine the susceptibility of haploid male germ cells to neoplastic transformation by this oncogene, transgenic mice were generated that harboured a chimeric gene composed of the SV40 T antigen genes fused to the 2.3-kb 5' flanking sequences of the rat proacrosin gene. It was previously shown that this regulatory sequence is able specifically to direct the expression of CAT reporter gene in male germ cells with the onset of translation in early haploid male germ cells. The transgene showed regulated expression in male germ cells. Although T antigen immunostaining was detected specifically in spermatids, no testicular pathology was observed. This indicates that spermatids show no susceptibility to transformation by oncogene TAg. However, in about 10% of animals of two independent transgenic lines, we could find non-testicular tumours in abdomen with a sarcoma-like structure in advanced age which showed SV40 TAg expression.     

The ATP binding cassette transporter A1 contributes to the secretion of interleukin 1beta from macrophages but not from monocytes

Deficiency of ABCA1 causes high density lipoprotein deficiency and macrophage foam cell formation in Tangier disease. ABCA1 was also postulated to mediate the secretion of IL-1beta from monocytes and macrophages. We investigated the contribution of ABCA1 to IL-1beta secretion from human monocytes and macrophages of normal donors and Tangier disease patients. Neither an anti-ABCA1 antisense oligonucleotide nor ABCA1 deficiency interfered with LPS-induced secretion of IL-1beta from full blood or freshly isolated monocytes. By contrast, anti-ABCA1 antisense oligonucleotides decreased the LPS-induced secretion of IL-beta from macrophages by 30-50%. The secretion of the precursor pro-IL-1beta and TNFalpha was not inhibited. Compared to normal macrophages, LPS-stimulated Tangier disease macrophages secreted less IL-1beta relative to TNFalpha. Also the spontaneous secretion of IL-1beta by Tangier macrophages was lower than by control cells. We conclude that IL-1beta is secreted from monocytes by an ABCA1-independent pathway and from macrophages by ABCA1-dependent and -independent pathways.