fbfB, a Gene Encoding a Putative Galactose Oxidase, Is Involved in Stigmatella aurantiaca Fruiting Body Formation

Stigmatella aurantiaca is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn5-derived transposon. The gene affected (fbfB) in one of the mutants (AP182) was studied further. Inactivation of fbfB results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in fbfB function are mixed with those of some independent mutants defective in fruiting before starvation. The fbfB gene is expressed about 14 h after induction of fruiting body formation as determined by measuring β-galactosidase activity in a merodiploid strain harboring the wild-type gene and an fbfB-Δtrp-lacZ fusion gene or by Northern (RNA) analysis with the Rhodobacter capsulatus pufBA fragment fused to fbfB as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus Dactylium dendroides. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.     

Transgenic Complementation of MARCKS Deficiency with a Nonmyristoylatable, Pseudo-Phosphorylated Form of MARCKS: Evidence for Simultaneous Positive and Dominant-Negative Effects on Central Nervous System Development

MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure–function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, “pseudo-phosphorylated” form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.     

Plasmodium vivax:A Monoclonal Antibody Recognizes a Circumsporozoite Protein Precursor on the Sporozoite Surface

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.     

Seasonal succession of Cladocerans in a ricefield in Italy

A biological ricefield in northern Italy, without periodic dry spells in its growing cycle and therefore more familiar to naturally humid zones, was studied for its heleoplankton community. The biocoenosis reached a greater level of complexity than reported in literature. In particular, the seasonal succession of Cladocerans, the dominant group throughout the study period, was analyzed. Wlassicsia pannonica (Daday, 1904; Anomopoda Macrothricidae), is new to Italy; its morphology is compared to that of other populations of the same species and its biological cycle is compared with that of other dominant Cladocerans.     

G Protein β Subunit–null Mutants Are Impaired in Phagocytosis and Chemotaxis Due to Inappropriate Regulation of the Actin Cytoskeleton

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein β subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type β subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In β subunit–null cells, and in a series of β subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein–actin transfected cells showed that β subunit– null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca²⁺ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.     

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria

The induction of a high-affinity state of the CO₂-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO₂ concentrations were resuspended in low-CO₂ buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO₂-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO₂ and HCO₃⁻ fluxes indicated that only the affinity of HCO₃⁻ transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO₂ uptake remained unchanged. Fast induction of high-affinity HCO₃⁻ transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO₃⁻ transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO₃⁻ transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.     

Spitsbergen glacial bays macrobenthos – a comparative study

Macrobenthos was studied in seven glacial bays situated along the Spitsbergen coast between 77 and 79°N. The fauna was dominated by deposit-feeding or carnivorous polychaetes and bivalves. Only 4 of 118 species identified in the collected material occurred in all the west Spitsbergen localities examined (the polychaetes Chaetozone/Tharyx sp., Cossura longocirrata, Lumbrinereis fragilis s.l. (sensu lato), and the bivalve Thyasira flexuosa). Clustering of samples showed a difference between the faunas of east and west Spitsbergen; the latter formed two subgroups, localities open to Atlantic waters and those from inner fjord basins. The fauna in open basins was dominated by cosmopolitan species, whereas arctic elements shares were higher in inner basins and predominated in the fauna in Bettybukta (east Spitsbergen). This indicates arctic, relict character of the inner fjords sites. The biomass ranged from 6 to 310 g/m² and Shannon diversities from 0.49 to 2.54. Received: 16 July 1997 / Accepted: 9 February 1998     

Design of a synthetic adhesion protein by grafting RGD tailed cyclic peptides on bovine serum albumin

A synthetic adhesion protein was designed by chemical grafting of the RGD tailed cyclic peptide cyclo[-d-Val-Arg-Gly-Asp-Glu(-Ahx-Tyr-Cys-NH₂)-] on the carrier protein bovine serum albumin (BSA). The cyclic conformation of the RGD motif grafted on the protein mimics the conformation of the motif displayed in native adhesion proteins such as fibronectin. The adhesion of the cells on polystyrene coated with the conjugate BSA–peptide was similar or even better than the one obtained when the proadhesive protein fibronectin was coated on the plates. Results also indicated that covalent coupling of the peptide on BSA is not absolutely required, since simple adsorption of the peptide on the protein coated on plates was efficient for enhancing cell adhesion. These results show that polystyrene support can be reconditioned with conformationally constrained RGD peptides to enhance cell adhesion on solid supports. The same methodology can be adapted for the development of new biomaterials based on the recognition of specific peptides.     

Regeneration in the spinal cord

Important advances have been made in our understanding of conditions that influence the intrinsic capacity of mature CNS neurons to initiate and maintain a regrowth response. The combination of exogenous neurotrophic support with strategies to alter the terrain at the injury site itself suggests that there are important interactions between them that lead to increased axonal regeneration. The ability of chronically injured neurons to initiate a regeneration response is unexpected. Our view of the role that inhibitors play in restricting axonal growth has also expanded. The findings indicate that the windows of opportunity for enhancing growth after spinal cord injury may be more numerous than previously thought.