Linkage analysis of the myosin heavy chain gene in Dictyostelium discoideum using a mutation generated by homologous recombination

Summary A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL, couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.     

Protein kinase Akt2/PKBβ is involved in blastomere proliferation of preimplantation mouse embryos

Activation of Akt/Protein Kinase B (PKB) by phosphatidylinositol-3-kinase (PI3K) controls several cellular functions largely studied in mammalian cells, including preimplantation embryos. We previously showed that early mouse embryos inherit active Akt from oocytes and that the intracellular localization of this enzyme at the two-cell stage depends on the T-cell leukemia/lymphoma 1 oncogenic protein, Tcl1. We have now investigated whether Akt isoforms, namely Akt1, Akt2 and Akt3, exert a specific role in blastomere proliferation during preimplantation embryo development. We show that, in contrast to other Akt family members, Akt2 enters male and female pronuclei of mouse preimplantation embryos at the late one-cell stage and thereafter maintains a nuclear localization during later embryo cleavage stages. Depleting one-cell embryos of single Akt family members by microinjecting Akt isoform-specific antibodies into wild-type zygotes, we observed that: (a) Akt2 is necessary for normal embryo progression through cleavage stages; and (b) the specific nuclear targeting of Akt2 in two-cell embryos depends on Tcl1. Our results indicate that preimplantation mouse embryos have a peculiar regulation of blastomere proliferation based on the activity of the Akt/PKB family member Akt2, which is mediated by the oncogenic protein Tcl1. Both Akt2 and Tcl1 are essential for early blastomere proliferation and embryo development.     

Application of Hazard Analysis and Critical Control Points (HACCP) to the Cultivation Line of Mushroom and Other Cultivated Edible Fungi

The Hazard analysis and critical control points (HACCP) is a preventive system which seeks to ensure food safety and security. It allows product protection and correction of errors, improves the costs derived from quality defects and reduces the final overcontrol. In this paper, the system is applied to the line of cultivation of mushrooms and other edible cultivated fungi. From all stages of the process, only the reception of covering materials (stage 1) and compost (stage 3), the pre-fruiting and induction (step 6) and the harvest (stage 7) have been considered as critical control point (CCP). The main hazards found were the presence of unauthorized phytosanitary products or above the permitted dose (stages 6 and 7), and the presence of pathogenic bacteria (stages 1 and 3) and/or heavy metals (stage 3). The implementation of this knowledge will allow the self-control of their productions based on the system HACCP to any plant dedicated to mushroom or other edible fungi cultivation.     

Isolation of Sporothrix schenckii GDA1 and functional characterization of the encoded guanosine diphosphatase activity

Sporothrix schenckii is a fungal pathogen of humans and the etiological agent of sporotrichosis. In fungi, proper protein glycosylation is usually required for normal composition of cell wall and virulence. Upon addition of precursor oligosaccharides to nascent proteins in the endoplasmic reticulum, glycans are further modified by Golgi-glycosyl transferases. In order to add sugar residues to precursor glycans, nucleotide diphosphate sugars are imported from the cytosol to the Golgi lumen, the sugar is transferred to glycans, and the resulting nucleoside diphosphate is dephosphorylated by the nucleoside diphosphatase Gda1 before returning to cytosol. Here, we isolated the open reading frame SsGDA1 from a S. schenckii genomic DNA library. In order to confirm the function of SsGda1, we performed complementation assays in a Saccharomyces cerevisiae gda1? null mutant. Our results indicated that SsGDA1 restored the nucleotide diphosphatase activity to wild-type levels and therefore is a functional ortholog of S. cerevisiae GDA1.     

Evidence for branching in cryptococcal capsular polysaccharides and consequences on its biological activity

The encapsulated fungus Cryptococcus neoformans is a common cause of life-threatening disease in immunocompromised individuals. Its major virulence determinant is the polysaccharide (PS) capsule. An unsolved problem in cryptococcal biology is whether the PSs composing the capsule are linear or complex branched polymers, as well as the implications of this structural composition in pathogenesis. In this study we approached the problem by combining static and dynamic light scattering, viscosity analysis, and high-resolution microscopy and correlated the findings with biological properties. Analysis of the dependence of capsular PS molecular mass and the radius of gyration provided strong evidence against a simple linear PS configuration. Shape factors calculated from light scattering measurements in solution revealed values consistent with polymer branching. Furthermore, viscosity measurements provided complementary evidence for structural branching. Electron microscopy showed PS spherical-like structures similar to other branched PS. Finally, we show that the capacity of capsular PS to interfere in complement-mediated phagocytosis, inhibit nitric oxide production by macrophage-like cells, protect against reactive oxygen species, antibody reactivity and half-life in serum were influenced by the degree of branching, providing evidence for the notion that PS branching is an important parameter in determining the biological activity of C. neoformans PS.     

Natural rock roughness as a key parameter for optimizing seedling adhesion and restoration of the seagrass Posidonia oceanica in its native environment

Seagrasses provide various ecosystem functions in coastal areas of the world. In the Mediterranean Sea, Posidonia oceanica is an endemic species threatened by several activities despite being protected by national and international laws. Currently, several transplanting initiatives have been carried out using different methods, among which those including seeds and seedlings are considered the most ecological and low-cost ones. Beach-cast fruits and seeds can be found in spring and their appearance can easily be reported, through a citizen science approach, by the community. One of the obstacles in using these methods is identifying the best substrate in which to place P. oceanica seeds to facilitate root adhesion of the seedlings prior to their transplantation into the sea. In the present study, we analyzed, using a 3D surface optical microscope, the roughness of natural rocks to identify the availability of specific roughness ranges suitable for adhesion and root anchoring of P. oceanica seedlings. Conventional roughness parameters and roughness power spectral density were calculated for the inner and outer surfaces of 9 different rock samples. Among the rock samples examined, the calcarenitic ones and in particular marsala calcarenite, due to the presence of the “ideal roughness for seedlings” can be considered one of the best consolidated substrates to be used for the construction of ad hoc devices on which plantlet of P. oceanica can grow for the purpose of restoration.     

One-step synthesis of Gly-Gly-PheNH2 from N-unprotected amino acid derivatives by papain in one-phase liquid media

Summary Papain was able to catalyze the one-step synthesis of Gly-Gly-PheNH₂, from N-unprotected amino acid derivatives. Maximum synthetic activity was obtained for a pH value of 6.5 and for a ]PheNH₂]/[Gly-GlyOEt] ratio of 6. The presence of an organic cosolvent, such as ethylene glycol, influenced the synthetic activity. Synthetic yield was higher than 65% for a 12.5 M cosolvent concentration.     

Chitin-Like Molecules Associate with Cryptococcus neoformans Glucuronoxylomannan To Form a Glycan Complex with Previously Unknown Properties

In prior studies, we demonstrated that glucuronoxylomannan (GXM), the major capsular polysaccharide of the fungal pathogen Cryptococcus neoformans, interacts with chitin oligomers at the cell wall-capsule interface. The structural determinants regulating these carbohydrate-carbohydrate interactions, as well as the functions of these structures, have remained unknown. In this study, we demonstrate that glycan complexes composed of chitooligomers and GXM are formed during fungal growth and macrophage infection by C. neoformans. To investigate the required determinants for the assembly of chitin-GXM complexes, we developed a quantitative scanning electron microscopy-based method using different polysaccharide samples as inhibitors of the interaction of chitin with GXM. This assay revealed that chitin-GXM association involves noncovalent bonds and large GXM fibers and depends on the N-acetyl amino group of chitin. Carboxyl and O-acetyl groups of GXM are not required for polysaccharide-polysaccharide interactions. Glycan complex structures composed of cryptococcal GXM and chitin-derived oligomers were tested for their ability to induce pulmonary cytokines in mice. They were significantly more efficient than either GXM or chitin oligomers alone in inducing the production of lung interleukin 10 (IL-10), IL-17, and tumor necrosis factor alpha (TNF-α). These results indicate that association of chitin-derived structures with GXM through their N-acetyl amino groups generates glycan complexes with previously unknown properties.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

The origin of a novel gene through overprinting in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Overlapped genes originate by a) loss of a stop codon among contiguous genes coded in different frames; b) shift to an upstream initiation codon of one of the contiguous genes; or c) by overprinting, whereby a novel open reading frame originates through point mutation inside an existing gene. Although overlapped genes are common in viruses, it is not clear whether overprinting has led to new genes in prokaryotes.