Functional Characterization and Cyclization Mechanism of a Diterpene Synthase Catalyzing the Skeleton Formation of Cephalotane-Type Diterpenoids.

CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane-type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for the unique carbocation-driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane-type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.

Progranulin deficiency decreases gross neural connectivity but enhances transmission at individual synapses.

Frontotemporal dementia (FTD) has been linked to mutations in the progranulin gene (GRN) that lead to progranulin (PGRN) haploinsufficiency. Thus far, our understanding of the effects of PGRN depletion in the brain has been derived from investigation of gross pathology, and more detailed analyses of cellular function have been lacking. We report that knocking down PGRN levels in rat primary hippocampal cultures reduces neural connectivity by decreasing neuronal arborization and length as well as synapse density. Despite this, the number of synaptic vesicles per synapse and the frequency of mEPSCs are increased in PGRN knockdown cells, suggesting an increase in the probability of release at remaining synapses. Interestingly, we demonstrate that the number of vesicles per synapse is also increased in postmortem brain sections from FTD patients with PGRN haploinsufficiency, relative to controls. Our observations show that PGRN knockdown severely alters neuronal connectivity in vitro and that the synaptic vesicle phenotype observed in culture is consistent with that observed in the hippocampus of FTD patients.

Progranulin deficiency leads to enhanced cell vulnerability and TDP-43 translocation in primary neuronal cultures.

Null mutations in the progranulin gene (PGRN) have been identified as a major cause of frontotemporal dementia with ubiquitinated inclusions. In this disorder, ubiquitinated, aggregated protein inclusions of a normally nuclear-located RNA processing protein called TAR DNA binding protein (TDP-43) accumulate in the neuronal cytoplasm (FTLD-TDP). To determine whether aspects of this clinical pathology can be established in primary cultures of mouse cortical neurons, PGRN levels were knocked down in neuronal cultures using lentiviral vectors to introduce mouse PGRN-siRNA constructs and subsequently rescued by overexpressing PGRN using a human PGRN-expressing lentiviral vector. The depletion of PGRN enhanced caspase-3 activation, and the PGRN-deficient neurons demonstrated enhanced vulnerability to normally sublethal doses of N-methyl-D-aspartic acid (NMDA) and hydrogen peroxide (H(2)O(2)). TDP-43 protein levels were markedly increased in the cytoplasm of PGRN-deficient neurons relative to nuclear levels, which is similar to observations in the brains of FTLD-TDP patients. Our results establish a neuronal culture model of the PGRN deficiency, which displays some of the important phenotypic characteristics of the early stages of the disease. The results further suggest that the seeds of this form of frontotemporal dementia may be sown early in life.

New stemona alkaloids from the roots of Stemona sessilifolia.

From the roots of Stemona sessilifolia, three new stemona-type alkaloids, namely stemosessifoine (1), isooxymaistemonine (2), and isomaistemonine (3), along with eight known alkaloids (bisdehydrostemoninine, isobisdehydrostemoninine, tuberostemonine, bisdehydrotuberostemonine, bisdehydrostemoninine, isobisdehydrostemoninine, stemoninine, and protostemonine), were isolated. Their structures were determined on the basis of extensive 2D-NMR spectroscopic-data analysis and by comparison with reported values in the literature. Compound 1 is a structurally unprecedented alkaloid, and it is depicted to be bioconverted from tuberostemonine as the precursor. Isooxymaistemonine (2) showed a positive effect on the human high-density lipoprotein (HDL) receptor gene CD36 and LIMP II analogous-1 (CLA-1) at the dosage of 10 microg/ml.

Mechanism for the TtDnaA-Tt-oriC cooperative interaction at high temperature and duplex opening at an unusual AT-rich region in Thermoanaerobacter tengcongensis.

Thermoanaerobacter tengcongensis is an anaerobic low-GC thermophilic bacterium. To further elucidate the replication initiation of chromosomal DNA at high temperature, the interaction between the replication initiator (TtDnaA) and the putative origin (Tt-oriC) in this thermophile was investigated. We found that efficient binding of TtDnaA to Tt-oriC at high temperature requires (i) at least two neighboring DnaA boxes, (ii) the specific feature of the TtDnaA Domain IV and (iii) the self-oligomerization of TtDnaA. Replacement of the TtDnaA Domain IV by the counterpart of Escherichia coli DnaA or disruption of its oligomerization by amino acid mutations (W9A/L20S) abolished the oriC-binding activity of TtDnaA at 60 degrees C, but not at 37 degrees C. Moreover, ATP-TtDnaA, but not ADP-TtDnaA or the oligomerization-deficient mutants was able to unwind the Tt-oriC duplex. The minimal oriC required for this duplex opening in vitro was demonstrated to consist of DnaA boxes 1-8 and an unusual AT-rich region. Interestingly, although no typical ATP-DnaA box was found in this AT-rich region, it was exclusively bound by ATP-TtDnaA and acted as the duplex-opening and replication-initiation site. Taken together, we propose that oligomerization of ATP-DnaA and simultaneously binding of several DnaA boxes and/or AT-rich region may be generally required in replication initiation at high temperature.