The utility of the morphological variation of pollen for resolving the evolutionary history of Billia (subfam. Hippocastanoideae,Sapindaceae)

In this study, we examined the utility of pollen morphology for resolving questions about the evolutionary history of Billia, which is a poorly known genus of Neotropical trees. Billia has been traditionally circumscribed with two species and treated as sister to Aesculus L. However, the number of species in Billia is uncertain, because the genus exhibits abundant morphological diversity but little discontinuous variation. Therefore, Billia may be monotypic and highly polymorphic, or it may have two species with blurred boundaries due to incipient speciation and/or hybridization. Moreover, one recent molecular phylogenetic study shows Billia nested withinAesculus. Our work sought to address the following questions: (i) Are there discontinuities in the pollen of Billia that may suggest species boundaries? (ii) Does the pollen of Billia show evidence for inter-specific hybridization? (iii) Do the exine morphology and size of pollen in Billia differ from those in Aesculus? Our results from scanning electron microscopy showed that pollen exine morphology is not taxonomically informative in Billia but that there are significant differences in pollen size between red- and white-flowered individuals. Thus, our pollen data support the utility of flower color in Billia for species delimitation. Our assessments of pollen viability do not support hybridization in the genus, but cannot be used to rule it out. Finally, pollen exine morphology may lend some support to an evolutionary origin ofBillia within eastern North American Aesculus. In contrast, data on pollen size suggest that Billia may belong in a topological position outside of Aesculus.     

Structural and serological relatedness of the O-antigens of Proteus penneri 1 and 4 from a novel Proteus serogroup O72.

O-specific polysaccharides (O-antigens) of the lipopolysaccharides (LPS) of Proteus penneri strains 1 and 4 were studied using sugar analysis, (1)H and (13)C NMR spectroscopy, including 2D COSY, H-detected (1)H,(13)C HMQC, and rotating-frame NOE spectroscopy (ROESY). The following structures of the tetrasaccharide (strain 1) and pentasaccharide (strain 4) repeating units of the polysaccharides were established: [reaction: see text]. In the polysaccharide of P. penneri strain 4, glycosylation with the lateral Glc residue (75%) and O-acetylation of the lateral GalNAc residue (55%) are nonstoichiometric. This polysaccharide contains also other, minor O-acetyl groups, whose positions were not determined. The structural similarity of the O-specific polysaccharides was consistent with the close serological relatedness of the LPS, which was demonstrated by immunochemical studies with O-antisera against P. penneri 1 and 4. Based on these data, it was proposed to classify P. penneri strains 1 and 4 into a new Proteus serogroup, O72, as two subgroups, O72a and O72a,b, respectively. Serological cross-reactivity of P. penneri 1 O-antiserum with the LPS of P. penneri 40 and 41 was substantiated by the presence of an epitope(s) on the LPS core region shared by all P. penneri strains studied.     

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.     

Structure of the O-polysaccharide from Proteus myxofaciens. Classification of the bacterium into a new Proteus-O-serogroup.

The O-polysaccharide (O-antigen) was obtained from the lipopolysaccharide of Proteus myxofaciens, a Proteus strain producing copious amounts of slime, which was isolated from the gypsy moth larvae. The structure of the polysaccharide was studied by chemical analysis and 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY and H-detected 1H,13C HMQC experiments. It was found that the polysaccharide contains an amide of glucuronic acid (GlcA) with an unusual alpha-linked amino acid, Nepsilon-[(R)-1-carboxyethyl]-l-lysine (2S,8R-alaninolysine, 2S,8R-AlaLys), and has a linear tetrasaccharide repeating unit of the following structure: This structure is unique among known bacterial polysaccharide structures. On the basis of these and serological data, it is proposed that P. myxofaciens be classified into a new Proteus serogroup, O60, of which this strain is the single representative. Structural and serological relatedness of P. myxofaciens to other AlaLys-containing O-antigens of Proteus and Providencia is discussed.     

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.