A high-throughput microscale method to release N-linked oligosaccharides from glycoproteins for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

This report describes a convenient method for the rapid and efficient release of N-linked oligosaccharides from low microgram amounts of glycoproteins. A 96-well MultiScreen assay system containing a polyvinylidene difluoride (PVDF) membrane is employed to immobilize glycoproteins for subsequent enzymatic deglycosylation. Recombinant tissue-type plasminogen activator (rt-PA) is used to demonstrate the deglycosylation of 0.1-50 micrograms of a glycoprotein. This method enabled the recovery of a sufficient amount of N-linked oligosaccharides released enzymatically with peptide N-glycosidase F (PNGaseF) from as little as 0.5 microgram rt-PA for subsequent analysis by matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) mass spectrometry. The immobilization of rt-PA to the PVDF membrane did not sterically inhibit the PNGaseF-mediated release of oligosaccharides from rt-PA as determined by tryptic mapping experiments. Comparison of the oligosaccharides released from 50 micrograms of rt-PA by either the 96-well plate method or by a standard solution digestion procedure showed no significant differences in the profiles obtained by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both neutral and sialylated oligosaccharide standards spiked into wells were recovered equally as determined by HPAEC-PAD. One advantage of this approach is that reduction and alkylation can be performed on submicrogram amounts of glycoproteins with easy removal of reagents prior to PNGaseF digestion. In addition, this method allows 60 glycoprotein samples to be deglycosylated in 1 day with MALDI-TOF or HPAEC-PAD analysis being performed on the following day.      

Irradiation induces G2/M cell cycle arrest and apoptosis in p53-deficient lymphoblastic leukemia cells without affecting Bcl-2 and Bax expression

The tumor suppressor p53 has been implicated in gamma irradiation-induced apoptosis. To investigate possible consequences of wild-type p53 loss in leukemia, we studied the effect of a single dose of gamma irradiation upon p53-deficient human T-ALL (acute lymphoblastic leukemia) CCRF - CEM cells. Exposure to 3 - 96 Gy caused p53-independent cell death in a dose and time-dependent fashion. By electron microscopic and other criteria, this cell death was classified as apoptosis. At low to intermediate levels of irradiation, apoptosis was preceded by accumulation of cells in the G2/M phase of the cell division cycle. Expression of Bcl-2 and Bax were not detectably altered after irradiation. Expression of the temperature sensitive mouse p53 V135 mutant induced apoptosis on its own but only slightly increased the sensitivity of CCRF - CEM cells to gamma irradiation. Thus, in these, and perhaps other leukemia cells, a p53- and Bcl-2/Bax-independent mechanism is operative that efficiently senses irradiation effects and translates this signal into arrest in the G2/M phase of the cell cycle and subsequent apoptosis.     

Unexpected Listronotus bonariensis (Coleoptera: Curculionidae) mortality in the presence of parasitoids

Field enclosure and laboratory cage experiments designed to measure the impact of the parasitoid Microctonus hyperodae Loan on the Argentine stem weevil, Listronotus bonariensis (Kuschel) have shown that under high parasitoid pressure, there is significant weevil mortality without obvious parasitism. Parasitoid-exposed, but unparasitized portions of caged populations died at rates significantly higher than both the parasitized weevils from the same population and the unexposed controls. Unexpectedly, parasitized weevils had the lowest mortality rates (prior to larval parasitoid emergence). It was of ecological significance that this mortality effect was detected under field conditions using several years of empirically-collected population dynamics data. Under both field and laboratory conditions this mortality was found to occur within shorter time intervals than that required for full parasitoid larval development; thus it could not be attributed to the mortality that occurs after the emergence of prepupal parasitoids. The mechanism for this mortality remains undetermined, although it could possibly be linked to pseudoparasitism.     

South American Origins of Microctonus hyperodae Loan (Hymenoptera: Braconidae) Established in New Zealand as Defined by Morphometric Analysis

Eight South American geographic populations of the thelytokous parasitoid Microctonus hyperodae Loan (Hymenoptera: Braconidae) were released in New Zealand in 1991 to assist in the suppression of the pasture pest Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). With one exception, parasitoids from each South American geographic population were released in equal numbers at each New Zealand release site. It was postulated that the South American geographic population(s) best suited to the conditions encountered at each New Zealand release locality would eventually become prevalent there. A morphometric analysis of adult parasitoids of known South American origins, reported previously, allowed M. hyperodae derived from west of the Andes (i.e. two collection sites in Chile) to be distinguished from parasitoids derived from east of the Andes (i.e. three collection sites in Argentina and one each in Brazil and Uruguay). Parasitoids derived from a fourth site in Argentina (S. C. de Bariloche) could not be clearly discriminated from either the 'east of the Andes' or 'west of the Andes' categories. A morphometric analysis of M. hyperodae adults collected from five of the New Zealand release sites from 1992-1994 is presented in this contribution. The analysis indicated that parasitoids derived from east of the Andes were significantly more prevalent than expected. The possible reasons for the initial success in New Zealand of one or more east of the Andes populations include the greater fecundity of M. hyperodae collected in Uruguay and the likelihood that M. hyperodae from east of the Andes co-evolved more recently with the stock from which New Zealand's L. bonariensis was founded.     

Evidence of Host Discrimination by Microctonus hyperodae Loan (Hymenoptera: Braconidae, Euphorinae), a Parasitoid of the Argentine Stem Weevil Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae)

Microctonus hyperodae is a solitary endoparasitoid of the Argentine stem weevil, Listronotus bonariensis. Early investigation into the biology of the parasitoid indicated that there was no discrimination between parasitized and unparasitized hosts. However, dissection data from two experiments were analyzed according to three mathematical models. Model I was based on the Poisson distribution and assumed random selection of hosts. The host discrimination model (model II) assumed that parasitized hosts had reduced attractiveness to searching parasitoids. A competition model (model III) made the assumption that competition between the early immature parasitoid stages led to premature mortality, which was not accounted for in the dissection results. The dissection data indicated statistically significant departure from the Poisson model. Results from the laboratory experiment indicated that host discrimination explained the results more accurately than parasitoid larval competition. Models II and III both provided adequate fits to the field data, although the departure from the observed data was greatest for the competition model. Both intra- and inter-ecotypic host recognition were evident, and there was some indication that clonal recognition took place. This is the first evidence of host discrimination by a member of the genus Microctonus. It has provided further indication of the success of M. hyperodae as a biological control agent.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

HUWE1 interacts with PCNA to alleviate replication stress

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S‐phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT‐type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA. We show that HUWE1‐knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono‐ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.