Lost your orientation? Find your way with PtdIns(3,4,5)P3!

Faithful chromosome segregation requires correct positioning of the spindle during mitosis. In this issue of Developmental Cell, Toyoshima et al. describe a new mechanism for spindle orientation involving phosphatidylinositol-3,4,5-triphosphate [PtdIns(3,4,5)P3]. They found that in metaphase cells, dynactin was associated with the cortex through the actin cytoskeleton, and accumulated in the midsections in a PtdIns(3,4,5)P3-dependent manner. Thus, PtdIns(3,4,5)P3 regulates spindle orientation through dynein-dynactin motor complexes.     

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml⁻¹. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml⁻¹). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani. This revised version was published online in November 2006 with corrections to the Cover Date.     

Airway epithelial cell migration dynamics. MMP-9 role in cell-extracellular matrix remodeling.

Cell spreading and migration associated with the expression of the 92-kD gelatinase (matrix metalloproteinase 9 or MMP-9) are important mechanisms involved in the repair of the respiratory epithelium. We investigated the location of MMP-9 and its potential role in migrating human bronchial epithelial cells (HBEC). In vivo and in vitro, MMP-9 accumulated in migrating HBEC located at the leading edge of a wound and MMP-9 expression paralleled cell migration speed. MMP-9 accumulated through an actin-dependent pathway in the advancing lamellipodia of migrating cells and was subsequently found active in the extracellular matrix (ECM). Lamellipodia became anchored through primordial contacts established with type IV collagen. MMP-9 became amassed behind collagen IV where there were fewer cell-ECM contacts. Both collagen IV and MMP-9 were involved in cell migration because when cell-collagen IV interaction was blocked, cells spread slightly but did not migrate; and when MMP-9 activation was prevented, cells remained fixed on primordial contacts and did not advance at all. These observations suggest that MMP-9 controls the migration of repairing HBEC by remodeling the provisional ECM implicated in primordial contacts.     

Modern evolution of a single-copy gene: the immunoglobulin C kappa locus in wild mice

The immunoglobulin kappa light-chain constant region gene (C kappa) has been cloned and sequenced from five wild mouse species. Analysis of these data has permitted an assessment of single-copy gene evolution during a limited time period as defined by the genus Mus. Sequence conservation was found to be as high (or higher) in the 5' and enhancer regions as in the coding region. The pattern of substitutions throughout these genes suggests that parallel evolution has occurred frequently and that substitutions at replacement sites have not decreased significantly, owing to saturation during this period of approximately 10 Myr. Phylogenetic relationships have been determined among these wild species as well as among members of the genus Rattus.      

Adenovirus type 5 fiber knob binds to MHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells.

Adenovirus serotype 5 (Ad5) fiber receptor was investigated using reverse antibody biopanning of a phage-displayed hexapeptide library, and virus-neutralizing monoclonal antibodies (mAbs 1D6.3 and 7A2.7) raised against recombinant Ad5 fiber knob. Both mAbs inhibited attachment of Ad5 to HeLa cells. Mimotopes of 1D6.3 showed homology with the C-terminal segment of the alpha2 domain of the heavy chain of human MHC class I molecules (MHC-I alpha2), and mimotopes of 7A2.7 were consensus to human fibronectin type III (FNIII) modules. In vitro, GST-fused MHC-I alpha2- and FNIII-derived oligopeptides interacted with recombinant fibers in a subgroup-specific manner. In vivo, the MHC-I alpha2 synthetic icosapeptide RAIVGFRVQWLRRYFVNGSR showed a net neutralization effect on Ad5 in HeLa cells, whereas the FNIII icosapeptide RHILWTPANTPAMGYLARVS significantly increased Ad5 binding to HeLa cells. Daudi cells, which lack surface expression of HLA class I molecules, showed a weak capacity for Ad5 binding. In beta2-microglobulin-transfected Daudi cells, Ad5 attachment and permissivity were restored to HeLa cell levels, with 4000 receptors per cell and a binding constant of 1.4x10(10)/M. The results suggested that the conserved region of MHC-I alpha2-domain including Trp167 represents a high affinity receptor for Ad5 fiber knob, whereas ubiquitous FNIII modules would serve as auxiliary receptors.     

Modification of steroidogenesis in a mouse adrenal cell line (Y-1) transformed by simian adenovirus SA-7

Transformation of a steroidogenic mouse adrenal cell line (Y-1) by simian adenovirus SA7 produced a cell line with low apparent steroidogenic activity. The effect of ACTH and cholera toxin on cyclic AMP production was similar in both not transformed and virus-transformed cells and activity of cyclic AMP-dependent protein kinase was also similar in both cells. In transformed cells, cholesterol was metabolized to delta 5-3 beta-hydroxysteroids, mainly 20 alpha-dihydropregnenolone while in not transformed cells, the major metabolites were delta 4-3 ketosteroids (20 alpha-dihydro- and 11 beta-hydroxy-20 alpha-dihydroprogesterone). In both cell lines ACTH increased the metabolism of cholesterol. Further studies with labelled pregnenolone and progesterone revealed a loss of delta 5-3 beta-hydroxysteroid dehydrogenase/isomerase and 11 beta-hydroxylase activity in the transformed cells.