Investigation of core machinery for biosynthesis of Vi antigen capsular polysaccharides in Gram-negative bacteria

Salmonella enterica serovar Typhi causes typhoid fever. It possesses a Vi antigen capsular polysaccharide coat that is important for virulence and is the basis of a current glycoconjugate vaccine. Vi antigen is also produced by environmental Bordetella isolates, while mammal-adapted Bordetella species (such as Bordetella bronchiseptica) produce a capsule of undetermined structure that cross-reacts with antibodies recognizing Vi antigen. The Vi antigen backbone is composed of poly-α-(1→4)-linked N-acetylgalactosaminuronic acid, modified with O-acetyl residues that are necessary for vaccine efficacy. Despite its biological and biotechnological importance, some central aspects of Vi antigen production are poorly understood. Here we demonstrate that TviE and TviD, two proteins encoded in the viaB (Vi antigen production) locus, interact and are the Vi antigen polymerase and O-acetyltransferase, respectively. Structural modeling and site-directed mutagenesis reveal that TviE is a GT4-family glycosyltransferase. While TviD has no identifiable homologs beyond Vi antigen systems in other bacteria, structural modeling suggests that it belongs to the large SGNH hydrolase family, which contains other O-acetyltransferases. Although TviD possesses an atypical catalytic triad, its O-acetyltransferase function was verified by antibody reactivity and ¹³C NMR data for tviD-mutant polysaccharide. The B. bronchiseptica genetic locus predicts a mode of synthesis distinct from classical S. enterica Vi antigen production, but which still involves TviD and TviE homologs that are both active in a reconstituted S. Typhi system. These findings provide new insight into Vi antigen production and foundational information for the glycoengineering of Vi antigen production in heterologous bacteria.     

Studies on the mechanism of cortisol inhibition of human natural killer cell activity: effects of calcium entry blockers and calmodulin antagonists

The role of Ca2+ in mediating the inhibition by glucocorticoids of human natural killer (NK) activity was investigated using Ca2+ entry blockers (verapamil and its desmethoxy-derivatives LU46973 and LU47093) and calmodulin antagonists (pimozide and two naphthalenesulfopamide derivatives, W-7 and W-13). Peripheral blood mononuclear (PBM) cell preparations were incubated for 20 h with 1 x 10(-6) M cortisol and these agents in various combinations (concentration range: 1 x 10(-7) - 1 x 10(-5) M) and then assayed in a direct 4-h cytolytic assay using 51Cr-labeled K 562 target cells. Exposure to cortisol led to a significant reduction of NK cell activity (about 50% with respect to the spontaneous activity). Ca2+ entry blockers displayed per se a dose-dependent depressive effect on cytotoxicity and gave significant enhancement of cortisol-dependent inhibition. Calmodulin antagonists were per se minimally effective but clearly amplified the cortisol-mediated inhibition. Raising extracellular Ca2+ by CaCl2 or intracellular Ca2+ by the ionophore A23187 yelded an appreciable reduction of these effects. Our data are compatible with the view that extracellular and intracellular Ca2+ play a role in the control of human NK cell activity. Moreover, it is conceivable that the mechanisms involved in glucocorticoid inhibition of NK cell activity involve Ca2+-dependent pathways.     

Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.     

Optimisation of the azinobis-3-ethyl-benzothiazoline-6-sulphonic acid radical scavenging assay for physiological studies of total antioxidant activity in woody plant germplasm.

A robust spectroscopic method for determining total antioxidant activity in aqueous extractions has been applied to tissues from diverse woody plant species, including seeds of Coffea arabica and in vitro shoots from Ribes nigrum, Picea sitchensis and Shorea leprosula. The assay involves scavenging of an ABTS [2,2'-azinobis-(3-ethyl-benzothiazoline-6-sulphonic acid)] radical generated by the reaction of potassium persulphate with ABTS to produce an ABTS*(+) chromophore (lambda=734 nm). Antioxidants reduce ABTS*(+) back to ABTS with a concomitant decrease in absorbance. Aqueous extractions from C. arabica and S. leprosula had considerably higher (110-205 micromol Trolox eq. g(-1) FW) total antioxidant activities than P. sitchensis and R. nigrum (6-11 micromol Trolox eq. g(-1) FW). Further studies in two of these species showed that the inclusion of water-insoluble polyvinylpyrrolidone during aqueous tissue extraction enabled the combined phenolic and alkaloid antioxidant activity to be determined. These fractions accounted for 85% and 60% of total antioxidant activity for C. arabica seeds and R. nigrum shoots, respectively. The ABTS radical scavenging assay is presented herein as a robust method for determining total antioxidant activity in germplasm from diverse woody plant tissues and species. Its applicability to study oxidative stress in tissue cultures and germplasm employed in plant biotechnology, breeding and stress physiology programmes is discussed.     

Characterization of non-8-17 sequences uncovers structurally diverse RNA-cleaving deoxyribozymes

RNA-cleaving deoxyribozymes (DNAzymes) can be isolated from random-sequence DNA pools via the process of in vitro selection. However, small and simple catalytic motifs, such as the 8-17 DNAzyme, are commonly observed in sequence space, presenting a challenge in discovering large and complex DNAzymes. In an effort to investigate underrepresented molecular species derived from in vitro selection, in this study we sought to characterize non-8-17 sequences obtained from a previous in vitro selection experiment wherein the 8-17 deoxyribozyme was the dominant motif. We examined 9 sequence families from 21 motifs by characterizing their structural and functional features. We discovered 9 novel deoxyribozyme classes with large catalytic domains (>40 nucleotides) utilizing three-way or four-way junction structural frameworks. Kinetic studies revealed that these deoxyribozymes exhibit moderate to excellent catalytic rates (k(obs) from 0.003 to 1 min(-1)), compared to other known RNA-cleaving DNAzymes. Although chemical probing experiments, site-directed mutational analyses, and metal cofactor dependency tests suggest unique catalytic cores for each deoxyribozyme, common dinucleotide junction selectivity was observed between DNAzymes with similar secondary structural features. Together, our findings indicate that larger, structurally more complex, and diverse catalytic motifs are able to survive the process of in vitro selection despite a sequence space dominated by smaller and structurally simpler catalysts.     

A Highlights from MBoC Selection: Synaptic neuropeptide release by dynamin-dependent partial release from circulating vesicles

Neurons release neuropeptides, enzymes, and neurotrophins by exocytosis of dense-core vesicles (DCVs). Peptide release from individual DCVs has been imaged in vitro with endocrine cells and at the neuron soma, growth cones, neurites, axons, and dendrites but not at nerve terminals, where peptidergic neurotransmission occurs. Single presynaptic DCVs have, however, been tracked in native terminals with simultaneous photobleaching and imaging (SPAIM) to show that DCVs undergo anterograde and retrograde capture as they circulate through en passant boutons. Here dynamin (encoded by the shibire gene) is shown to enhance activity-evoked peptide release at the Drosophila neuromuscular junction. SPAIM demonstrates that activity depletes only a portion of a single presynaptic DCV''s content. Activity initiates exocytosis within seconds, but subsequent release occurs slowly. Synaptic neuropeptide release is further sustained by DCVs undergoing multiple rounds of exocytosis. Synaptic neuropeptide release is surprisingly similar regardless of anterograde or retrograde DCV transport into boutons, bouton location, and time of arrival in the terminal. Thus vesicle circulation and bidirectional capture supply synapses with functionally competent DCVs. These results show that activity-evoked synaptic neuropeptide release is independent of a DCV''s past traffic and occurs by slow, dynamin-dependent partial emptying of DCVs, suggestive of kiss-and-run exocytosis.     

Identification of natural compounds tubercidin and lycorine HCl against small‐cell lung cancer and BCAT1 as a therapeutic target

Although small‐cell lung cancer (SCLC) accounts for a small fraction of lung cancer cases (~15%), the prognosis of patients with SCLC is poor with an average overall survival period of a few months without treatment. Current treatments include standard chemotherapy, which has minimal efficacy and a newly developed immunotherapy that thus far, benefits a limited number of patients. In the current study, we screened a natural product library and identified 5 natural compounds, in particular tubercidin and lycorine HCl, that display prominent anti‐SCLC activities in vitro and in vivo. Subsequent RNA‐sequencing and functional validation assays revealed the anti‐SCLC mechanisms of these new compounds, and further identified new cellular factors such as BCAT1 as a potential therapeutic target with clinical implication in SCLC patients. Taken together, our study provides promising new directions for fighting this aggressive lung cancer.