CTP:phosphocholine cytidylyltransferase binds anionic phospholipid vesicles in a cross-bridging mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.     

Structural rationale for the affinity of pico- and femtomolar transition state analogues of Escherichia coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase

Immucillin and DADMe-Immucillin inhibitors are tight binding transition state mimics of purine nucleoside phosphorylases (PNP). 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) is proposed to form a similar transition state structure as PNP. The companion paper describes modifications of the Immucillin and DADMe-Immucillin inhibitors to better match transition state features of MTAN and have led to 5'-thio aromatic substitutions that extend the inhibition constants to the femtomolar range (Singh, V., Evans, G. B., Lenz, D. H., Mason, J., Clinch, K., Mee, S., Painter, G. F., Tyler, P. C., Furneaux, R. H., Lee, J. E., Howell, P. L., and Schramm, V. L. (2005) J. Biol. Chem. 280, 18265-18273). 5'-Methylthio-Immucillin A (MT-ImmA) and 5'-methylthio-DADMe-Immucillin A (MT-DADMe-ImmA) exhibit slow-onset inhibition with K(i)(*) of 77 and 2 pm, respectively, and were selected for structural analysis as the parent compounds of each class of transition state analogue. The crystal structures of Escherichia coli MTAN complexed with MT-ImmA and MT-DADMe-ImmA were determined to 2.2 A resolution and compared with the existing MTAN inhibitor complexes. These MTAN-transition state complexes are among the tightest binding enzyme-ligand complexes ever described and analysis of their mode of binding provides extraordinary insight into the structural basis for their affinity. The MTAN-MT-ImmA complex reveals the presence of a new ion pair between the 4'-iminoribitol atom and the nucleophilic water (WAT3) that captures key features of the transition state. Similarly, in the MTAN-MT-DADMe-ImmA complex a favorable hydrogen bond or ion pair interaction between the cationic 1'-pyrrolidine atom and WAT3 is crucial for tight affinity. Distance analysis of the nucleophile and leaving group show that MT-ImmA is a mimic of an early transition state, while MT-DADMe-ImmA is a better mimic of the highly dissociated transition state of E. coli MTAN.     

Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3' noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand synthesis during genome replication.     

Cytokinins induce photomorphogenic development in dark-grown gametophytes of Ceratopteris richardii

The cytokinins benzylaminopurine, kinetin and isopentenyladenine induce photomorphogenesis in dark-grown gametophytes of the fern Ceratopteris richardii. At sub-nanomolar concentrations each altered the rate and pattern of cell division, elongation and differentiation, mimicking aspects of the light-mediated transition from filamentous to prothallial growth. Untreated dark-grown gametophytes grow as narrow, elongate, asexual filaments with an apical meristem. Cytokinin treatments as low as 10(-12) M reduced the length-to-width ratio through decreased cell elongation, increased periclinal cell division and induced the formation of rhizoid initials in the cells immediately below the apical meristem. Higher concentrations (10(-9)-10(-8) M) induced conversion of the meristem from apical to notch morphology. Cytokinins induced both red- and blue-light-mediated photomorphogenic events, suggesting stimulation of both phytochrome and cryptochrome signaling; however, cytokinin treatment only partially substituted for light in that it did not induce hermaphroditic sexual development or spore germination in the dark. Additionally, cytokinins did not increase chlorophyll synthesis in dark-grown gametophytes, which unlike angiosperms are able to produce mature chloroplasts in the dark. Cytokinin treatment had only slight effects on light-grown gametophytes. These results suggest evolutionary conservation between angiosperms and pteridophytes in the role of cytokinins in regulating photomorphogenesis.     

Automated sample mounting and alignment system for biological crystallography at a synchrotron source

High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction. The sample-mounting subsystem has random access to 112 samples, stored under liquid nitrogen. Results of extensive tests regarding the performance and reliability of the system are presented. To further increase throughput, we have also developed a sample transport/storage system based on "puck-shaped" cassettes, which can hold sixteen samples each. Seven cassettes fit into a standard dry shipping Dewar. The capabilities of a robotic crystal mounting and alignment system with instrumentation control software and a relational database allows for automated screening and data collection to be developed.     

Organic nitrogen in precipitation: real problem or sampling artefact?

Published observations of organic nitrogen (N) compounds in precipitation go back almost a century. Several different methods have been used to measure both the total and ionic concentrations of N. There is therefore some uncertainty as to whether reported "organic N" is real, or simply the result of uncertainties in chemical analyses or inadequate sampling methods. We found that the materials from which the collector was made (polypropylene, steel, or glass) had no significant effect on the composition of dissolved organic N (DON). The use of a biocide was found to be very important during sampling and storage of samples before analysis. We set up a network of seven collectors across the U.K., from the Cairngorms to Dorset, all operating to the same protocol, and including a biocide. Samples were analysed centrally, using proven methods. Over 6 months, organic N contributed about 20% to the total N in U.K. precipitation, but with a large variation across the country. This means that current estimates of wet deposited N to the U.K., which are based only on the ammonium and nitrate concentrations, are too small. Organic N is not an artefact, but a real problem that needs to be addressed.     

Adoptive immunotherapy of cancer with pharmacologically activated lymph node lymphocytes: a pilot clinical trial

 Adoptive immunotherapy (AIT) of cancer with T lymphocytes may be limited by the need to activate tumor antigen-sensitized cells in vitro. In murine models, we have shown that AIT with tumor-sensitized T cells that have been pharmacologically activated with bryostatin 1 and ionomycin plus interleukin-2 can induce tumor regression. A Phase I clinical trial was carried out to assess the feasibility and toxicity associated with using tumor- or vaccine-draining lymph node cells, activated pharmacologically and expanded in culture with low-dose interleukin-2 and infused intravenously, followed by IL-2 infusion. Nine patients were entered into the trial, and six were treated as planned. Average expansion of cell numbers over 13 to 27 days in culture was 118-fold. No patient's cells reached the target cell number (2.5 × 10¹⁰). Infusion of these cells did not result in any unexpected toxicities. The toxicities observed were related to IL-2 infusion, and conformed to the expected range of side-effects. Based on these Phase I results, additional trials, with tumor antigen vaccine-sensitized DLN and technical modifications of the culture technique, are planned. Received: 18 January 2001 / Accepted: 26 April 2001     

Drug-resistant parasites and aggregated infection – early-season dynamics

We investigate the effect of spatial aggregation in the infection dynamics of nematode parasites in ruminants. We show that a high degree of spatial aggregation is likely to lead to a dramatically enhanced rate of invasion by drug-resistant strains. Received: 13 December 1999 / Revised version: 3 April 2000 / Published online: 4 October 2000