New data on the palaeobiology of the enigmatic yunnanozoans from the Chengjiang Biota,Lower Cambrian,China

Interpretation of the enigmatic soft‐bodied yunnanozoans from the Lower Cambrian (Stage 3, Series 2) Chengjiang Biota has remained controversial for decades because of their strange body plan and their variable taphonomic alteration. Proposed affinities have ranged from stem bilaterian to stem vertebrate. A study of over seven hundred slabs, many newly collected, from five sections at two localities demonstrates that yunnanozoans have sclerotized dorsal and axial segments, a body cavity, a unique feeding region and a coiled alimentary canal. Although two genera have been previously discriminated, all specimens have the same number of filamentous arches (seven pairs) and of ventral circular structures (four pairs); this indicates that Haikouella Chen, Huang and Li, 1999 is a junior synonym of Yunnanozoon Hou, Ramsköld and Bergström, 1991. Our analysis reveals new details of the putative pharyngeal pores of Yunnanozoon lividum, and although there are similarities with the gill slits of deuterostomes, the question of their homology remains moot. The filamentous arches and their supporting rods were probably sclerotized and represent a specialized feeding structure. Yunnanozoans exhibit characters that are open to a number of alternative interpretations of putative homology, encompassing comparisons with deuterostomes and protostomes (including members of the Platyzoa). Given their unique features, and the current lack of secure homologies, the affinities of yunnanozoans should continue to be considered in a wider bilaterian context.     

Expression and purification of recombinant human neuritin from <Emphasis Type="Italic">Pichia pastoris</Emphasis> and a partial analysis of its neurobiological activity in vitro

Neuritin (also known as candidate plasticity gene 15 (cpg15)) is a neurotrophic factor that was recently discovered in a screen aimed at identifying genes involved in activity-dependent synaptic plasticity. Neuritin plays multiple roles in both neural development (Chen et al. Zhonghua Yan Ke Za Zhi 46:978–983 2010; Corriveau et al. J Neurosci 19:7999-8008 1999; Lee and Nedivi J Neurosci 22:1807-1815 2002) and synaptic plasticity (Fujino et al. Gene Dev 25:2674-2685 2011; Leslie and Nedivi Prog 14 Neurobiol 94:223-237 2011; Loebrich and Nedivi Physiol Rev 89:1079 2009). In this study, to produce bioactive, soluble recombinant human neuritin protein, a portion of NRN1 was cloned into the Pichia pastoris expression vector pPIC9K. The recombinant vector was then transformed into the methylotrophic yeast strain P. pastoris GS115, and a shaking flask method and His-tag purification strategy were utilized to express and purify neuritin protein. The resulting protein had a molecular mass of approximately 11 kDa, and subsequent functional analyses indicated that the purified neuritin promoted neurite outgrowth from embryonic chicken dorsal root ganglions, while also prolonging the survival of these ganglions, and from PC12 cells. These findings suggest that neuritin was expressed effectively in vitro and that this protein may play a role in stimulating neurite outgrowth of both dorsal root ganglions and PC12 cells. This study provides a novel strategy for the large-scale production of bioactive neuritin, which will enable further study of the biological function of this protein.     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.