Enzymatic Engineering of Polysialic Acid on Cells in Vitro and in Vivo Using a Purified Bacterial Polysialyltransferase

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST_Nm) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST_Nm synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST_Nm and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST_Nm requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST_Nm was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST_Nm-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST_Nm occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST_Nm-based approach may provide a valuable alternative to PST gene therapy.     

Insight into TPMT∗23 mutation mis-folding using molecular dynamics simulation and protein structure analysis

Thiopurine S-methyltransferase (TPMT) is an important enzyme that metabolizes thiopurine drugs. This enzyme exhibits a large number of interindividual polymorphism. TPMT^?23 polymorphism has been reported in a few cases in the world in co-dominance with TPMT^?3A. The phenotype has been reported to affect enzyme activity in vivo and in vitro. Its underlying structural basis is not clarified yet. In our study, the wild type (WT) protein structure was analyzed and the amino acids bordering water channels in thiopurine sites were identified. Molecular dynamics of both the WT and TPMT^?23 mutation was carried out. In addition, the effects of this mutation, especially on the thiopurine site which is closed with a pincer like mechanism, were investigated. We focused on explaining how a locally occurred A167G substitution propagated through hydrogen bonds alteration to induce structural modification which affects both thiopurine and S-adenosylmethionine receptors. Finally, a genetic prediction of mutation functional consequences has been conducted confirming altered activity.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:20     

Factors affecting reproduction of Woodpigeons Columba palumbus in North African forests: 1. Nest habitat selection

Capsule Woodpigeon Columba palumbus nest-tree selection is affected by human disturbance, grazing and tree characteristics.

Aims To determine the characteristics of trees used for nesting by Woodpigeons under two levels of grazing and disturbance in a Holm oak Middle Atlas forest, Morocco.

Methods Woodpigeon nest-tree selection was investigated in highly disturbed and less-disturbed sites over two years (2010 and 2011). The features of nest-tree characteristics were determined by comparing the characteristics of trees used for nesting to those of randomly selected trees.

Results Tree physical characteristics, grazing intensity and level of human disturbance were the main factors determining selection for nest-trees. Woodpigeons chose nesting trees that were greater in height, larger in DBH, lower in base of the live crown, in denser stands and at greater distance from tracks.

Conclusions Forest management for Woodpigeon nest habitat selection should focus on controlling the access of vehicles especially trucks, introducing a rotating system of forest utilization for grazing and maintaining (without extending) the cereal crops. These recommendations may assist land managers to plan for continued persistence of this species in Middle Atlas forests.     

Host plant defenses of black (Solanum nigrum L.) and red nightshade (Solanum villosum Mill.) against specialist Solanaceae herbivore Leptinotarsa decemlineata (Say)

Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in‐vitro glutathione S‐transferase (GST) inhibition activity, we used insecticide‐resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat‐body homogenate. In‐vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis‐hex‐3‐enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata.     

Spa32 regulates a switch in substrate specificity of the type III secreton of Shigella flexneri from needle components to Ipa proteins

Type III secretion systems (TTSS) are essential virulence determinants of many gram-negative bacteria and serve, upon physical contact with target cells, to translocate bacterial proteins directly across eukaryotic cell membranes. The Shigella TTSS is encoded by the mxi/spa loci located on its virulence plasmid. By electron microscopy secretons are visualized as tripartite with an external needle, a transmembrane domain, and a cytoplasmic bulb. In the present study, we generated a Shigella spa32 mutant and studied its phenotype. The spa32 gene shows low sequence homology to Salmonella TTSS1 invJ/spaN and to flagellar fliK. The spa32 mutant, like the wild-type strain, secreted the Ipas and IpgD, which are normally secreted via the TTSS, at low levels into the growth medium. However, unlike the wild-type strain, the spa32 mutant could neither be induced to secrete the Ipas and IpgD instantaneously upon addition of Congo red nor penetrate HeLa cells in vitro. Additionally, the Spa32 protein is secreted in large amounts by the TTSS during exponential growth but not upon Congo red induction. Interestingly, electron microscopy analysis of the spa32 mutant revealed that the needle of its secretons were up to 10 times longer than those of the wild type. In addition, in the absence of induction, the spa32 mutant secreted normal levels of MxiI but a large excess of MxiH. Taken together, our data indicate that the spa32 mutant presents a novel phenotype and that the primary defect of the mutant may be its inability to regulate or control secretion of MxiH.     

COMPARATIVE FUNCTIONAL ANALYSES OF ULTRABITHORAX REVEAL MULTIPLE STEPS AND PATHS TO DIVERSIFICATION OF LEGS IN THE ADAPTIVE RADIATION OF SEMI‐AQUATIC INSECTS

Invasion of new ecological habitats is often associated with lineage diversification, yet the genetic changes underlying invasions and radiations are poorly understood. Over 200 million years ago, the semi‐aquatic insects invaded water surface from a common terrestrial ancestor and diversified to exploit a wide array of niches. Here, we uncover the changes in regulation and function of the gene Ultrabithorax associated with both the invasion of water surface and the subsequent diversification of the group. In the common ancestor of the semi‐aquatic insects, a novel deployment of Ubx protein in the mid‐legs increased their length, thereby enhancing their role in water surface walking. In derived lineages that specialize in rowing on the open water, additional changes in the timing of Ubx expression further elongated the mid‐legs thereby facilitating their function as oars. In addition, Ubx protein function was selectively reversed to shorten specific rear‐leg segments, thereby enabling their function as rudders. These changes in Ubx have generated distinct niche‐specialized morphologies that account for the remarkable diversification of the semi‐aquatic insects. Therefore, changes in the regulation and function of a key developmental gene may facilitate both the morphological change necessary to transition to novel habitats and fuel subsequent morphological diversification.     

Association between plasmid carrying an expanded-spectrum cephalosporin resistance and biofilm formation in Escherichia coli

The formation of biofilm is a universal bacterial survival strategy. Biofilms occur on inert and living support in the natural environment and in industrial installations. This microenvironment leads to the horizontal transfer of genetic material between bacteria by physical contact. In order to evaluate the relationship between biofilm-forming capabilities, surface characteristics and plasmid content we purified from Salmonella a plasmid conferring resistance to cephalosporin and transferred it by electroporation to E.coli DH10B originally unable to form biofilm in inert surface. We demonstrated the association between a plasmid conferring resistance to expanded-spectrum cephalosporin and biofilm formation. We also noted that this plasmid influences the cell surface properties and cell motility.     

Une nouvelle observation d’une métastase testiculaire d’un adénocarcinome prostatique: Aspects diagnostiques et éthiopathogéniques

Testicular metastases from prostatic adenocarcinoma are rare. They are often asymptomatic and diagnosed incidentally or at autopsy after orchidectomy in more advanced stages of the disease. The authors report a new case and review the diagnosis and aetiopathogenesis of these metastases. A 67-year-old patient with prostatic adenocarcinoma presented with painless right testicular mass for two months with no inflammatory signs. Germ cell tumour serum markers were negative. Scrotal ultrasound showed a large testicular tumour measuring 7×4×3cm. Histological examination revealed a solid non-differentiated tumour, not suggestive of primary testicular tumour. The immunohistochemical panel confirmed the prostate as the primary site due to the positivity of cytokeratins, PSA and PSAP and the negativity of classical markers of germ cell tumours. Testicular metastases from prostatic adenocarcinoma are rare, but their incidence is currently on the increase. They are often asymptomatic and discovered after pulpectomy. This diagnosis must be considered in the case of a testicular mass in patients over the age of 50 with a history of prostatic adenocarcinoma. The diagnosis was established after orchidectomy by histological examination and immunohistochemical tests.