Influence of template strandedness on in vitro replication of mutagen-damaged DNA

We analyzed the ability of DNA polymerases to bypass damage on single- and double-stranded templates. In vitro DNA synthesis was studied on UV-irradiated and polyaromatic hydrocarbon reacted (benzo[a]pyrenediol epoxide and oxiranylpyrene) double-stranded templates by a protocol involving initiation on a uniquely nicked circular double-stranded template. The template was prepared by treating single-stranded (+)M13mp2 circular strands with mutagen and then hybridizing with restricted M13 RFmp2, followed by isolation of the nicked RFII forms. The protocol permits either (+), (-), or both strands to carry lesions. We found that the rules for termination and bypass of lesions previously observed with single-stranded DNA templates also hold for double-stranded templates. Termination of synthesis occurs primarily one nucleotide 3' to the lesion in the template strand. Bypass of UV-induced lesions can be followed in a series of three partial reactions in the presence of Mn2+ and dGMP, which relax the specificity of nucleotide insertion and 3'----5' exonuclease activity, respectively. There is no evidence for greater permissivity of bypass in double-as opposed to single-stranded templates. As with single-stranded templates, purines and preferentially deoxyadenosine (dA) are inserted opposite lesions. Lesions in the nontemplate strand elicit neither termination nor pausing. The addition of Rec A protein resulted in a measurable increase of bypass in this system.     

Structural Basis for Type VI Secretion Effector Recognition by a Cognate Immunity Protein

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three effector proteins, type VI secretion exported 1–3 (Tse1–3), to recipient Gram-negative cells. The Tse2 protein is a cytoplasmic effector that acts as a potent inhibitor of target cell proliferation, thus providing a pronounced fitness advantage for P. aeruginosa donor cells. P. aeruginosa utilizes a dedicated immunity protein, type VI secretion immunity 2 (Tsi2), to protect against endogenous and intercellularly-transferred Tse2. Here we show that Tse2 delivered by the T6SS efficiently induces quiescence, not death, within recipient cells. We demonstrate that despite direct interaction of Tsi2 and Tse2 in the cytoplasm, Tsi2 is dispensable for targeting the toxin to the secretory apparatus. To gain insights into the molecular basis of Tse2 immunity, we solved the 1.00 Å X-ray crystal structure of Tsi2. The structure shows that Tsi2 assembles as a dimer that does not resemble previously characterized immunity or antitoxin proteins. A genetic screen for Tsi2 mutants deficient in Tse2 interaction revealed an acidic patch distal to the Tsi2 homodimer interface that mediates toxin interaction and immunity. Consistent with this finding, we observed that destabilization of the Tsi2 dimer does not impact Tse2 interaction. The molecular insights into Tsi2 structure and function garnered from this study shed light on the mechanisms of T6 effector secretion, and indicate that the Tse2–Tsi2 effector–immunity pair has features distinguishing it from previously characterized toxin–immunity and toxin–antitoxin systems.     

Apoptosis of circulating tumor cells in prostate cancer patients.

BACKGROUND: The prescence of circulating tumor cells (CTCs) in the peripheral blood of cancer patients and their frequency has been correlated with disease status. METHODS: In this study, CTCs were characterized by flow cytometry and fluorescence microscopy after immunomagnetic enrichment from 7.5-ml blood samples collected from patients with prostate cancer in evacuated blood-draw tubes that contained an anticoagulant and a preservative. Events were classified as tumor cell candidates if they expressed cytokeratin, lacked CD45, and stained with the nucleic acid dye 4,6-diamidino-2-phenylindole. RESULTS: In the blood of prostate cancer patients, only few of these events were intact cells. Other CTC events appeared as damaged cells or cell fragments by microscopy. By flow cytometry, these events stained variably with 4,6-diamidino-2-phenylindole and frequently expressed the apoptosis-induced, caspase-cleaved cytokeratin 18. Similar patterns of cell disintegration were observed when cells of the prostate line LNCaP were exposed to paclitaxel before spiking the cells into normal blood samples. CONCLUSIONS: The different observed stages of tumor cell degradation or apoptosis varied greatly between patients and were not found in blood of normal donors. Enumeration of CTCs and identification of CTCs undergoing apoptosis may provide relevant information to evaluate the response to therapy in cancer patients.     

The CD45 tyrosine phosphatase regulates specific pools of antigen receptor-associated p59fyn and CD4-associated p56lck tyrosine in human T-cells.

A newly isolated T-cell line (CB1) derived from a T-acute lymphoblastic leukaemia (T-ALL) patient contained cells (40% of total) which did not express the CD45 phosphotyrosine phosphatase. The cells were sorted into CD45- and CD45+ populations and shown to be clonal in origin. T-cell receptor (TCR) cross-linking or coligation of the TCR with its CD4/CD8 co-receptors induced tyrosine phosphorylation and calcium signals in CD45+ but not in CD45- cells. Unexpectedly, whole cell p56lck and p59fyn tyrosine kinase activities were not reduced in CD45- compared to CD45+ cells. A novel technique was therefore developed to isolated specific pools of aggregated receptors expressed at the cell surface, together with their associated tyrosine kinases. Using this technique it was shown that cell surface CD4-p56lck kinase activity was 78% lower in CD45- than in CD45+ cells. Phosphorylation of TCR zeta- and gamma-chains occurred in TCR immunocomplexes from CD45+ but not CD45- cells, despite comparable levels of p59fyn and TCR proteins. Furthermore, TCR-associated tyrosine kinase activity towards an exogenous substrate was 84% lower in CD45- than in CD45+ cells. Addition of recombinant p59fyn to TCR immunocomplexes isolated from CD45-cells restored the phosphorylation of the TCR zeta- and gamma-chains. Our results demonstrate that CD45 selectively regulates the pools of p59fyn and p56lck kinases which are associated with the TCR and CD4 at the cell surface. Activation by CD45 of these receptor-associated kinase pools correlates with the ability of the TCR and its coreceptors to couple to intracellular signalling pathways.     

Disruption of the coenzyme binding site and dimer interface revealed in the crystal structure of mitochondrial aldehyde dehydrogenase "Asian" variant

Mitochondrial aldehyde dehydrogenase (ALDH2) is the major enzyme that oxidizes ethanol-derived acetaldehyde. A nearly inactive form of the enzyme, ALDH2*2, is found in about 40% of the East Asian population. This variant enzyme is defined by a glutamate to lysine substitution at residue 487 located within the oligomerization domain. ALDH2*2 has an increased Km for its coenzyme, NAD+, and a decreased kcat, which lead to low activity in vivo. Here we report the 2.1 A crystal structure of ALDH2*2. The structure shows a large disordered region located at the dimer interface that includes much of the coenzyme binding cleft and a loop of residues that form the base of the active site. As a consequence of these structural changes, the variant enzyme exhibits rigid body rotations of its catalytic and coenzyme-binding domains relative to the oligomerization domain. These structural perturbations are the direct result of the inability of lysine 487 to form important stabilizing hydrogen bonds with arginines 264 and 475. Thus, the elevated Km for coenzyme exhibited by this variant probably reflects the energetic penalty for reestablishing this site for productive coenzyme binding, whereas the structural alterations near the active site are consistent with the lowered Vmax.     

Design of an MRI-compatible robotic stereotactic device for minimally invasive interventions in the breast

The objective of this work was to develop a robotic device to perform biopsy and therapeutic interventions in the breast with real-time magnetic resonance imaging (MRI) guidance. The device was designed to allow for (i) stabilization of the breast by compression, (ii) definition of the interventional probe trajectory by setting the height and pitch of a probe insertion apparatus, and (iii) positioning of an interventional probe by setting the depth of insertion. The apparatus is fitted with five computer-controlled degrees of freedom for delivering an interventional procedure. The entire device is constructed of MR compatible materials, i.e. nonmagnetic and non-conductive, to eliminate artifacts and distortion of the MR images. The apparatus is remotely controlled by means of ultrasonic motors and a graphical user interface, providing real-time MR-guided planning and monitoring of the operation. Joint motion measurements found probe placement in less than 50 s and sub-millimeter repeatability of the probe tip for same-direction point-to-point movements. However, backlash in the rotation joint may incur probe tip positional errors of up to 5 mm at a distance of 40 mm from the rotation axis, which may occur for women with large breasts. The imprecision caused by this backlash becomes negligible as the probe tip nears the rotation axis. Real-time MR-guidance will allow the physician to correct this error Compatibility of the device within the MR environment was successfully tested on a 4 Tesla MR human scanner     

Correlations between net auxin and secondary xylem development in young Populus deltoides

Stems of cottonwood ( Populs deltoides Bartr. ex Marsh.) plants grown under different conditions were examined to determine the relation between net endogenous auxin yields and the acropetal advance of the primary-secondary vascular transition zone (TZ). In all treatments, the internode yielding maximum net auxin activity, as determined by the Avena curvature bioassay, closely corresponded with the internode in which the TZ occurred. Under short-day (SD) dormancy-inducing conditions, auxin yield declined steadily while the maximum auxin peak and the TZ shifted toward younger internodes. Auxin yields from these plants were extremely low after 5 weeks of SD compared with those from long-day (LD) plants. The only consistent auxin yield was obtained from internodes subtending young leaves beneath the apical bud. Plants placed in SD for 3 weeks and then returned to LD conditions showed an immediate increase in auxin yield in the stem, and the progressive acropetal advance of the TZ under SD was reversed. Therefore, within 7 LD the positions of the TZ and peak auxin yield corresponded to those observed before the imposition of SD Fully dormant plants placed in LD showed a dramatic rise in auxin yields during the first 2 weeks of renewed growth. Although low levels of auxin were found in the newly developing shoots after 6 LD, yields increased rapidly after 9 and 14 LD. The position of the TZ corresponded with the peak of net auxin activity after 9 and 14 LD.     

Haemosporidian Parasites of Antelopes and Other Vertebrates from Gabon,Central Africa

Re-examination, using molecular tools, of the diversity of haemosporidian parasites (among which the agents of human malaria are the best known) has generally led to rearrangements of traditional classifications. In this study, we explored the diversity of haemosporidian parasites infecting vertebrate species (particularly mammals, birds and reptiles) living in the forests of Gabon (Central Africa), by analyzing a collection of 492 bushmeat samples. We found that samples from five mammalian species (four duiker and one pangolin species), one bird and one turtle species were infected by haemosporidian parasites. In duikers (from which most of the infected specimens were obtained), we demonstrated the existence of at least two distinct parasite lineages related to Polychromophilus species (i.e., bat haemosporidian parasites) and to sauropsid Plasmodium (from birds and lizards). Molecular screening of sylvatic mosquitoes captured during a longitudinal survey revealed the presence of these haemosporidian parasite lineages also in several Anopheles species, suggesting a potential role in their transmission. Our results show that, differently from what was previously thought, several independent clades of haemosporidian parasites (family Plasmodiidae) infect mammals and are transmitted by anopheline mosquitoes.