Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

The use of DNA fingerprinting to estimate annual survival rates in the Saker Falcon(Falco cherrug)

Summary DNA fingerprinting of nestlings ofFalco cherrug was used to determine indirectly the survival of the corresponding adult parent birds, which are difficult to catch in sufficient numbers. This approach is possible because Saker falcons show a high degree of site and mate tenacity. DNA profiles of nestlings from the same territory but from different years were compared. Three patterns of band-sharing coefficients between broods from the same territory were found: if band-sharing coefficients within and between broods from consecutive years were similar but significantly different from those of unrelated birds, it indicated that all young were full sibs and that neither adult was replaced between years. If band-sharing coefficients between broods at the same site indicated no relatedness across years and were equal to those of unrelated birds, then both breeding partners apparently had changed. If the band-sharing coefficients between broods of the same territory and consecutive years were significantly lower than those of full sibs, but higher than those of unrelated birds, the loss of one adult bird was indicated. The analysis of 32 broods (years 1993 to 1997) provided a minimal estimate for annual adult survival of 82% for a wild population of Saker Falcons in Kazakhstan.

---

Abschätzung der jährlichen Überlebensraten des Sakerfalken(Falco cherrug) mittels DNA-Fingerprinting

Zusammenfassung Um die Gefährdung und Populationsdynamik des Sakerfalken (Falco cherrug) beurteilen zu können, benötigen wir genaue Angaben zu Mortalität und Überlebensraten. Während es bei dieser Art relativ einfach ist, Nestlinge zu fangen, ist es nahezu unmöglich, eine ausreichend große Zahl an Altvögeln zu markieren, um durch Wiederfang oder Ringfundmeldungen die jährliche Überlebensrate zu ermitteln. Durch DNA-Fingerprinting von Jungfalken haben wir versucht, die minimale Überlebensrate von Altfalken indirekt zu bestimmen. Dieser Forschungsansatz wird dadurch möglich, daß die Sakerfalken eine hohe Philopatrie aufweisen und jedes Jahr im selben Revier brüten. Wenn man mehrere Jahre lang Blutproben der Jungvögel aus denselben Revieren sammelt, so kann man mittels DNA Fingerprinting indirekt ermitteln, ob die jeweiligen Altvögel identisch waren oder gewechselt haben: Vergleicht man die Band-Sharing-Koeffizienten (BSK) von Jungvögeln von zwei oder mehr Jahren aus demselben Revier, so ergeben sich drei Muster: Wenn die BSK-Werte innerhalb der Bruten und zwischen den Bruten identisch aber signifikant verschieden von denen nicht verwandter Vögel sind, so handelt es sich bei den Jungvögeln um Vollgeschwister; demnach sind die Altvögel identisch geblieben, d. h. sie haben von einem Jahr zum nächsten überlebt. Wenn die BSK-Werte zwischen zwei Bruten aus demselben Revier einen Wert annehmen, wie man ihn für unverwandte Tiere ermittelt, so müssen die Eltern gewechselt haben. Liegen die Werte zwischen zwei Bruten signifikant höher als die von nicht verwandten Tieren, aber niedriger als diejenigen von Vollgeschwistern, so ist vermutlich 1 Altvogel gewechselt worden. Die Analyse von 32 Bruten des Sakerfalken aus Kasachstan zeigt, daß die minimale jährliche Adultüberlebensrate bei 82% liegt.

     

Molecular phylogeography of the viperine snake Natrix maura (Serpentes: Colubridae): Evidence for strong intraspecific differentiation

The molecular phylogeography of the viperine snake, Natrix maura (Linnaeus, 1758), was investigated using complete sequences of the mitochondrial cytochrome b gene and genomic ISSR-PCR fingerprinting. In a total of 120 samples, 44 unique cytochrome b haplotypes were found which defined three major genetic lineages associated with samples from Morocco, Tunisia and Europe, respectively. The same lineages were supported by nuclear data. A possible fourth lineage exists in southern Spain. Genetic distances of cytochrome b sequences between the three main lineages were in the range of 3.9–5.6%, suggesting independent evolution since the early Pliocene. Distinction of the three major lineages at the subspecies or species level is discussed to account taxonomically for the high intraspecific variation in the viperine snake. A more detailed analysis of the European samples based on genetic diversity indices and a network reconstruction suggests a complex Pleistocene history for the viperine snake in Europe. Clear differentiation was found between populations south and north of the central Iberian mountain ranges, suggesting Pleistocene glacial refugia both in the southern and northern Iberian peninsula. In the south, genetic diversity was associated with the main river valleys, whereas northern haplotypes were more broadly distributed, indicating gene flow or postglacial range expansions. Unexpectedly high levels of genetic variation in southeastern France and northwestern Italy would be compatible with the hypothesis of a glacial refugium north of the Pyrenees or in Italy. However, due to the dependence of N. maura on warm climates, the assumption of a northern refugium seems unwarranted. We believe that further sampling in northern Spain is likely to reveal genetically diverse populations which could have served as sources for postglacial recolonization of France and Italy.     

Species distribution and <Emphasis Type="Italic">in vitro</Emphasis> antifungal susceptibility of oral yeast isolates from Tanzanian HIV-infected patients with primary and recurrent oropharyngeal candidiasis

Background  

In Tanzania, little is known on the species distribution and antifungal susceptibility profiles of yeast isolates from HIV-infected patients with primary and recurrent oropharyngeal candidiasis.     

Comprehensive characterization of heat shock protein 27 phosphorylation in human endothelial cells stimulated by the microbial dithiole thiolutin

Thiolutin is a sulfur-based microbial compound with known activity as an angiogenesis inhibitor. Relative to previously studied angiogenesis inhibitors, thiolutin is a remarkably potent inducer of heat shock protein 27 (Hsp27) phosphorylation. This phosphorylation requires p38 kinase but is independent of increased p38 phosphorylation. To elucidate how thiolutin regulates Hsp27 phosphorylation and ultimately angiogenesis, Hsp27 was immunoprecipitated using nonphosphorylated and phospho-Ser78 specific antibodies from lysates of thiolutin treated and untreated human umbilical vein endothelial cells and analyzed by LC-MS. Separate LC-MS analyses of Lys-C, Lys-C plus trypsin, and Lys-C plus Glu-C digests provided 100% sequence coverage, including the identification of a very large 13 kDa Lys-C fragment using a special sample handling procedure (4 M guanidine HCl) prior to the LC-MS analysis to improve the large peptide recovery. The analysis revealed a novel post-translational modification of Hsp27 involving truncation of the N-terminal Met and acetylation of the penultimate Thr. Analysis of a Glu-C fragment containing two phosphorylation sites, Ser78 and Ser82, and a tryptic fragment containing the other phosphorylation site, Ser15, enabled quantitative stoichiometry of Hsp27 phosphorylation by LC-MS. The strategy revealed details of Hsp27 phosphorylation, including significant di-phosphorylation at both Ser78 and Ser82, that would be difficult to obtain by traditional approaches because oligomerization of the hydrophobic N-terminal region of the molecule prevents efficient enzymatic cleavage. The combination of Western blotting, immunoprecipation, and LC-MS provides a quantitative analysis of thiolutin-stimulated Hsp27 phosphorylation and further defines the role of Hsp27 in the antiangiogenic activities of thiolutin and related dithiolethiones.     

Phylogeography of the blue tit ( Parus teneriffae -group) on the Canary Islands based on mitochondrial DNA sequence data and morphometrics

An analysis of the sequences of the mitochondrial cytochrome b gene (1005 bp) of the Parus teneriffae-group from the Canary Islands and North Africa revealed new insights into the phylogeography of this taxon. The origin of the radiation on the Canarian Archipelago was apparently one of the central islands—Tenerife or Gran Canaria. The populations on El Hierro (P. t. ombriosus) and La Palma (P. t. palmensis) represent distinct monophyletic lineages. Blue tits from Gran Canaria are genetically distinct from those of La Gomera and Tenerife (P. t. teneriffae), which supports the results of other studies and suggests the existence of an—until now—undescribed taxon there. In contrast, the populations on the eastern islands of Fuerteventura and Lanzarote (P. t. degener) could not be distinguished from North African blue tits (P. t. ultramarinus), and these populations should be subsumed under the subspecies ultramarinus. Taxonomic recommendations based on these results include the distinction of the northern European P. caeruleus from P. teneriffae, including blue tits from North Africa and the Canary Islands, the treatment of degener and ultramarinus as synonymous (P. teneriffae ultramarinus) and a new blue tit taxon on the island of Gran Canaria (P. t. hedwigii nov. ssp.), which is formally described. The genetic results are in parts supported by bioacoustic and morphological data.     

Triterpene saponins from Chenopodium quinoa Willd

Twenty triterpene saponins (1-20) have been isolated from different parts of Chenopodium quinoa (flowers, fruits, seed coats, and seeds) and their structures have been elucidated by analysis of chemical and spectroscopic data including 1D- and 2D-NMR. Four compounds (1-4) were identified: 3beta-[(O-beta-d-glucopyranosyl-(1-->3)-alpha-l-arabinopyranosyl)oxy]-23-oxo-olean-12-en-28-oic acid beta-d-glucopyranoside (1), 3beta-[(O-beta-d-glucopyranosyl-(1-->3)-alpha-l-arabinopyranosyl)oxy]-27-oxo-olean-12-en-28-oic acid beta-d-glucopyranoside (2), 3-O-alpha-l-arabinopyranosyl serjanic acid 28-O-beta-d-glucopyranosyl ester (3), and 3-O-beta-d-glucuronopyranosyl serjanic acid 28-O-beta-d-glucopyranosyl ester (4). The following known compounds have not previously been reported as saponin constituents from the flowers and the fruits of this plant: two bidesmosides of serjanic acid (5,6), four bidesmosides of oleanolic acid (7-10), five bidesmosides of phytolaccagenic acid (11-15), four bidesmosides of hederagenin (16-19), and one bidesmoside of 3beta,23,30-trihydroxy olean-12-en-28-oic acid (20). The cytotoxicity of these saponins and their aglycones was tested in HeLa cells. Induction of apoptosis in Caco-2 cells by bidesmosidic saponins 1-4 and their aglycones I-III was determined by flow cytometric DNA analysis. The saponins with an aldehyde group were most active. The relationships between structure and cytotoxic activity of saponins and their aglycones are discussed.