Genetic structure of three Sorbus latifolia (Lam.) Pers. taxa endemic to northern Bavaria

The Franconian Alb (Bavaria, Germany) is rich in endemic Sorbus taxa, considered as apomictic microspecies and derived by hybridization between Sorbus aria aggregate and Sorbus torminalis (Sorbus latifolia aggregate). Molecular studies using the AFLP technique, neighbour joining, Bayesian clustering, principal coordinate analysis (PCo) and voucher studies were used to investigate genetic structure and origin of adult plants and cultivated offspring of three endemic S. latifolia taxa, namely Sorbus cordigastensis, Sorbus franconica and Sorbus adeana and probable parental species from the S. aria agg. and S. torminalis. The S. latifolia taxa, adults and progeny, showed low genetic variability and a more or less clonal structure, confirming an apomictic mode of reproduction. The investigated S. latifolia taxa were remarkably different among each other, confirming their status as microspecies. The AFLP data confirmed the hybrid origin of the S. latifolia taxa, they were 1.3–1.5 times more closely related to S. aria agg. than to S. torminalis. The S. aria agg. showed a complicate genetic structure and fell into four main groups, two intermediate groups besides Sorbus pannonica and Sorbus aria sensu stricto (S. aria s.str.). Some progeny of S. pannonica was more variable than expected and clustered partly with other groups indicating gene flow within S. aria agg. Different subgroups of the S. aria aggregate may be parental for the S. latifolia taxa, contributing to the remarkable genetic distances between them.     

Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage

Methylating agents are ubiquitous in the environment, and central in cancer therapy. The 1-methyladenine and 3-methylcytosine lesions in DNA/RNA contribute to the cytotoxicity of such agents. These lesions are directly reversed by ABH3 (hABH3) in humans and AlkB in Escherichia coli. Here, we report the structure of the hABH3 catalytic core in complex with iron and 2-oxoglutarate (2OG) at 1.5 A resolution and analyse key site-directed mutants. The hABH3 structure reveals the beta-strand jelly-roll fold that coordinates a catalytically active iron centre by a conserved His1-X-Asp/Glu-X(n)-His2 motif. This experimentally establishes hABH3 as a structural member of the Fe(II)/2OG-dependent dioxygenase superfamily, which couples substrate oxidation to conversion of 2OG into succinate and CO2. A positively charged DNA/RNA binding groove indicates a distinct nucleic acid binding conformation different from that predicted in the AlkB structure with three nucleotides. These results uncover previously unassigned key catalytic residues, identify a flexible hairpin involved in nucleotide flipping and ss/ds-DNA discrimination, and reveal self-hydroxylation of an active site leucine that may protect against uncoupled generation of dangerous oxygen radicals.     

Temporal relation between leukocyte accumulation in muscles and halted recovery 10-20 h after strength exercise.

Effects of normal strength exercise on leukocyte accumulation were examined in 10 well-trained male subjects (27.2 +/- 2.7 yr). The workout, consisting of five maximal sets of three repetitions of leg press exercise and five maximal sets of six repetitions of knee extension exercise, was performed with the dominant leg, and the other leg served as control. Repeated maximal isokinetic knee extensions at 60 degrees /s were performed to evaluate neuromuscular fatigue and recovery after the workout. Accumulation of leukocytes was assessed with 99mTc-labeled cells, and repeated images of the thighs were taken 1-24 h after the workout. Maximal force-generating capacity in the exercised leg was reduced by 17 +/- 2% (P < 0.01) after the workout. The course of recovery followed a biphasic pattern characterized by halted recovery 10-23 h after exercise. The presence of leukocytes was approximately 10% higher in the exercised than in the control thigh 10 h after exercise (P < 0.05). This difference increased to approximately 15% at 20 h after exercise (P < 0.05). The retarded recovery of maximal force-generating capacity 10-20 h after exercise, together with a significant infiltration of leukocytes in exercised muscle during the same time interval, shows a temporal relation between leukocyte infiltration and impaired recovery.     

Genetic,Structural, and Antigenic Analyses of Glycan Diversity in the O-Linked Protein Glycosylation Systems of Human Neisseria Species

Bacterial capsular polysaccharides and lipopolysaccharides are well-established ligands of innate and adaptive immune effectors and often exhibit structural and antigenic variability. Although many surface-localized glycoproteins have been identified in bacterial pathogens and symbionts, it not clear if and how selection impacts associated glycoform structure. Here, a systematic approach was devised to correlate gene repertoire with protein-associated glycoform structure in Neisseria species important to human health and disease. By manipulating the protein glycosylation (pgl) gene content and assessing the glycan structure by mass spectrometry and reactivity with monoclonal antibodies, it was established that protein-associated glycans are antigenically variable and that at least nine distinct glycoforms can be expressed in vitro. These studies also revealed that in addition to Neisseria gonorrhoeae strain N400, one other gonococcal strain and isolates of Neisseria meningitidis and Neisseria lactamica exhibit broad-spectrum O-linked protein glycosylation. Although a strong correlation between pgl gene content, glycoform expression, and serological profile was observed, there were significant exceptions, particularly with regard to levels of microheterogeneity. This work provides a technological platform for molecular serotyping of neisserial protein glycans and for elucidating pgl gene evolution.It is now well established that protein glycosylation based on both N- and O-linked modifications occurs in bacterial species. In N-linked systems exemplified by the system in Campylobacter jejuni, large numbers of proteins that are translocated to the periplasm are glycosylated based on the presence of sequon elements and asparagine-targeting oligosaccharyltransferases related to those that operate in eukaryotes (21, 36, 69, 73). Two O-linked systems associated with covalent modification of type IV pilin subunits in pathogenic Neisseria species and in selected strains of Pseudomonas aeruginosa have been particularly well characterized (2, 16, 46-48, 54). The latter systems are remarkably similar to the N-linked system characterized in C. jejuni in that oligosaccharides are synthesized cytoplasmically as lipid-linked precursors that are then flipped into the periplasm. Protein-targeting oligosaccharyltransferases structurally related to the WaaL family of O-antigen ligases then transfer the oligosaccharides to protein substrates (2, 18, 49). The similarities between these N- and O-linked systems are perhaps best illustrated by genetic and functional interactions between components of the C. jejuni oligosaccharide biosynthetic machinery and elements of the neisserial pilin glycosylation pathway (2, 18). In contrast, the mechanisms operating in other bacterial O-linked systems are not completely understood yet, and there appears to be considerable diversity in the mechanisms of oligosaccharide synthesis, transfer of the glycan to the protein, and the cellular compartment in which glycan addition takes place. Prime examples of this diversity include the glycosylation of major subunits of S-layers (53), flagella (40), and type IV pili, as well as nonpilus adhesins, such as autotransporters (7, 51) and a family of serine-rich proteins identified in Gram-positive species (72). Recently, the pilin glycosylation system in the Gram-negative species Neisseria gonorrhoeae (the etiological agent of gonorrhea) was shown to be a general O-linked system in which a large set of structurally distinct periplasmic proteins undergo glycosylation (64). Likewise, a general O-linked glycosylation system targeting periplasmic and surface-exposed proteins has been documented in Bacteroides fragilis (19). In addition, an increasing number of lipoproteins in Mycobacterium tuberculosis have been found to be O glycosylated, and current evidence suggests that a single glycosylation pathway operates with these proteins (50).The large number of bacterial protein glycosylation systems strongly suggests that these systems are advantageous and affect fitness. In fact, mutants with mutations in the general glycosylation systems of C. jejuni and B. fragilis are defective in mucosal colonization, although the fundamental basis for the observations is unclear (19, 23). In some cases, defects in protein stability and trafficking have been documented. Examples of the latter have been reported for the Aida and Ag43 autotransporter adhesins of Escherichia coli and the serine-rich Fap1 streptococcal adhesin (11, 35, 72). In these cases, the glycosylation status appears to influence protein integrity along with intracellular or membrane trafficking events.Glycosylation may also influence protein structure and function or activity at the extracellular level. In the context of host-symbiont and host-pathogen interactions, bacterial cell surface polysaccharides and glycolipid glycans are well-established targets of both innate and adaptive immune responses (13, 61). However, the potential influence of protein-linked carbohydrate on immune recognition and signaling is only beginning to be investigated. Given the well-established effect of conjugating protein to carbohydrate on glycan-related immunogenicity, glycoproteins could be predicted to promote a robust T-cell-dependent antibody response directed toward glycan epitopes. In line with this, immunization of mice with O-glycosylated type IV pilin from P. aeruginosa strain 1244 (which bears a single repeat unit of the O antigen, the dominant component of its lipopolysaccharide) resulted in protection against challenge with immunological specificity for the O-polysaccharide (27). In addition, structural heterogeneity of carbohydrate modifications has been shown to affect the serospecificity of Campylobacter flagellins (41). With regard to innate immunity, the N-linked protein glycans of C. jejuni have been shown to influence interleukin-6 production by human dendritic cells via interaction with the macrophage galactose-type lectin (MGL) (62). Also, flagellin glycosylation of the phytopathogenic bacteria Pseudomonas syringae pv. glycinea and P. syringae pv. tomato appears to play an important role in hypersensitive cell death in nonhost plants and in host cell recognition (56, 57). Similarly, the flagellin glycosylation status in P. aeruginosa influences proinflammatory responses in human cell cultures (63).Studies of O-linked flagellar glycosylation in P. aeruginosa, C. jejuni, and a number of Gram-positive species have revealed considerable variability in genomic glycosylation islands (40). In addition to differences in gene content, some genes localized in these loci are subject to phase (on-off) variation involving slipped-strand mispairing events. Similar findings have been obtained for the O-linked glycosylation system in N. gonorrhoeae and a related system in Neisseria meningitidis (2, 4, 29, 48). These observations strongly suggest that protein-associated glycans are positively selected. However, attempts to elucidate the evolutionary processes impacting these systems are complicated by difficulties in connecting genotype with phenotype. For example, predicting enzymatic activities of components involved in glycan biosynthesis based on the sequence alone is notoriously difficult. Therefore, glycosylation-related functions are characterized best by using purified components in in vitro assays. Moreover, despite recent advances in mass spectrometric (MS) and nuclear magnetic resonance (NMR) technologies, glycoprotein structural analysis is still arduous, particularly when proteins are expressed at low levels. Thus, current methodologies are not optimized for studies of large numbers of strains and mutants.The broad-spectrum O-linked protein glycosylation system of N. gonorrhoeae is particularly well characterized with regard to the genetics of oligosaccharide biosynthesis, modification, and transfer to protein via the PglO/PglL oligosaccharyltransferase. As shown using strain N400, combined genetic and MS analyses, including interspecies complementation, have revealed that this system (designated the pgl [protein glycosylation] system) is remarkably similar to the N-linked system of C. jejuni with respect to the use of a peptide-proximal 2,4-diacetamido-2,4,6-trideoxyhexose (DATDH) sugar and related biosynthetic pathways for generating lipid-linked glycan substrates (2, 18, 39). The lipid-linked DATDH sugar can be further converted successively into hexose (Hex)-DATDH disaccharide and Hex-Hex-DATDH trisaccharide forms by the PglA and PglE glycosyltransferases, respectively (2). The hexoses in both the di- and trisaccharide forms can also undergo O acetylation by the PglI enzyme (2, 70). As pglA, pglE, and pglI are each predicted to be subject to phase variation in some backgrounds, strains have the potential to express five distinct glycoforms (2, 4, 29, 48, 70). A similar system operates in N. meningitidis strain c311, although to date only pilin and the AniA nitrite reductase proteins have been shown to be glycosylated (37). Pioneering analyses of pilin from this strain identified a trisaccharide with a terminal alpha-1-4-linked digalactose moiety attached to DATDH (54). Interestingly, nearly one-half of N. meningitidis isolates are reported to have a unique allele of pglB designated pglB2 associated with synthesis of a proximal glyceramido-acetamido trideoxyhexose (GATDH) rather than DATDH (10). In addition, some strains of both N. gonorrhoeae and N. meningitidis have been reported to contain additional genes predicted to encode glycosyltransferases linked to the core locus that includes the pglF, pglB, pglC, and pglD genes (32, 48). Thus, it appears that the number of protein-associated glycans may be far greater than currently perceived. The genus Neisseria also includes a number of related species that colonize humans, including Neisseria lactamica, which is closely related to N. gonorrhoeae and N. meningitidis but is rarely associated with disease (24), as well as other, more divergent commensal species. An examination of recently available genome sequences of these nonpathogenic species revealed that they contain open reading frames (ORFs) whose products share high levels of amino acid identity with many of the protein glycosylation components found in N. gonorrhoeae and N. meningitidis and with many of the N. gonorrhoeae proteins targeted for glycosylation. However, protein glycosylation has not been documented in any of these species yet.Here, we developed a systematic approach for elucidating intra- and interstrain glycan diversity and its genetic basis in neisserial O-linked glycans by employing serotyping, mass spectrometric analyses, and genetically defined recombinant backgrounds. We then used these tools to demonstrate that protein-associated glycans are antigenically variable and that isolates of N. meningitidis and N. lactamica also exhibit broad-spectrum O-linked protein glycosylation.     

Interferon-gamma elicits a G-protein-dependent Ca2+ signal in human neutrophils after depletion of intracellular Ca2+ stores

Interferon-gamma (IFN-gamma) has multiple effects on Ca2+ signalling in polymorphonuclear neutrophils (PMNs), including evoked cytosolic Ca2+ transients, increased capacitative calcium influx and increased sequestration of Ca2+ in intracellular stores. The present study was conducted to elucidate the mechanism behind the Ca2+ transients. As observed before, the IFN-gamma-evoked Ca2+ signals were apparent when extracellular Ca2+ was removed. A new finding was that the proportion of responding cells and the extent of calcium release increased with increasing time in EGTA buffer. As assessed by N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated Ca2+ release, the intracellular stores were depleted during this incubation period, and the extent of depletion correlated well with the appearance of IFN-gamma-induced Ca2+ signals. This store dependence of the IFN-gamma-induced Ca2+ signals was confirmed by the appearance of IFN-gamma-evoked Ca2+ signals in the presence of extracellular Ca2+ after store depletion by thapsigargin. The appearance of IFN-gamma-mediated Ca2+-signals in the presence of EGTA indicates that IFN-gamma stimulates Ca2+ release from intracellular stores. This was confirmed by the inability of the calcium transportation blocker La3+ to abolish the IFN-gamma response and the total abrogation of the response by the phospholipase C inhibitor U73122. Although these latter results imply a role for inositol 1,4,5-trisphosphate(IP3) in IFN-gamma signalling, comparison of IFN-gamma-evoked responses with fMLP responses revealed clear differences that suggest different signal-transduction pathways. However, responses to fMLP and IFN-gamma were both depressed by pertussis toxin, and the IFN-gamma responses were, in addition, inhibited by the tyrosine kinase inhibitor genistein. Further evidence of the involvement of tyrosine kinase was a slight stimulatory effect of the protein tyrosine phosphatase inhibitor sodium orthovanadate. The PI-3K activity was of minor importance. In conclusion, we present evidence of a novel signal-transduction mechanism for IFN-gamma in PMNs, dependent on tyrosine kinase activity, a pertussis toxin-sensitive G protein and phospholipase C activity.     

Fibronectin promotes calcium signaling by interferon-gamma in human neutrophils via G-protein and sphingosine kinase-dependent mechanisms

A common intracellular signal activating polymorphonuclear leukocytes (PMN) in inflammation is a change in cytosolic calcium concentration. Previously, we have shown that interferon-γ (IFN-γ) induces transient calcium signals in PMN, but only after intracellular calcium store depletion. Using a digital imaging system, we show that adhesion of PMN is critical for IFN-γ-induced calcium signals, and with PMN attached to the optimal coating, the calcium signals are evoked even in presence of extracellular calcium, that is, non-depleted calcium stores. Adhesion to fibronectin, pure or extracted from plasma by gelatin, improved the IFN-γ responses compared with serum, plasma, or vitronectin coats. In accordance with previous observations, IFN-γ-induced calcium signals in fibronectin adherent cells were totally abolished by the G-protein inhibitor pertussis toxin and were also inhibited by the sphingosine kinase inhibitors dimethylsphingosine (DMS) and N-acetylsphingosine (N-Ac-Sp). PMN contact with fibronectin alone, measured in cells sedimenting onto a fibronectin-coated surface or by addition of fibronectin to glass-adherent cells, evoked transient calcium signals. However, PMN in suspension did not respond to the addition of fibronectin or arginine-glycine-aspartate (RGD). The fibronectin-induced calcium signals were also clearly depressed by pertussis toxin and by the sphingosine kinase inhibitors DMS, dihydrosphingosine (DHS), and N-Ac-Sp. When the product of sphingosine kinase activity, sphingosine I-phosphate (S1-P), was added to the cells, similar calcium signals were induced, which were dependent on a pertussis toxin-sensitive G-protein activity. Finally, addition of S1-P to the cells prior to stimulation with IFN-γ partly mimicked the priming effect of fibronectin. In conclusion, fibronectin contact evokes by itself a calcium signal in PMN and further promotes calcium signaling by IFN-γ. We suggest that fibronectin might activate sphingosine kinase, and that the sphingosine 1-phosphate thereby generated induces a calcium signal via a G-protein-dependent mechanism. Apparently, sphingosine kinase activity is also involved in IFN-γ induced calcium signals.     

Mitochondrial DNA sequence evolution and phylogeny of the Atlantic Alcidae,including the extinct great auk (Pinguinus impennis)

The Atlantic auk assemblage includes four extant species, razorbill (Alca torda), dovekie (Alle alle), common murre (Uria aalge), and thick-billed murre (U. lomvia), and one recently extinct species, the flightless great auk (Pinguinus impennis). To determine the phylogenetic relationships among the species, a contiguous 4.2-kb region of the mitochondrial genome from the extant species was amplified using PCR. This region included one ribosomal RNA gene, four transfer RNA genes, two protein-coding genes, the control region, and intergenic spacers. Sets of PCR primers for amplifying the same region from great auk were designed from sequences of the extant species. The authenticity of the great auk sequence was ascertained by alternative amplifications, cloning, and separate analyses in an independent laboratory. Phylogenetic analyses of the entire assemblage, made possible by the great auk sequence, fully resolved the phylogenetic relationships and split it into two primary lineages, Uria versus Alle, Alca, and Pinguinus. A sister group relationship was identified between Alca and Pinguinus to the exclusion of ALLE: Phylogenetically, the flightless great auk originated late relative to other divergences within the assemblage. This suggests that three highly divergent species in terms of adaptive specializations, Alca, Alle, and Pinguinus, evolved from a single lineage in the Atlantic Ocean, in a process similar to the initial adaptive radiation of alcids in the Pacific Ocean.     

Diurnal and Seasonal Variations of Serum Enzyme Activity in Cattle and Sheep

In order to test the variation of enzyme activity in serum of cattle and sheep during the day, blood samples were taken at three hrs. interval from 6 a.m. to 9 p.m. The following enzymes were assayed: Aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (AlAT = GPT), total lactate dehydrogenase (LDH), and a-hydroxybutyrate dehydrogenase (HBD). The variation between animals contributed by far to the greatest part of the total variation in clinical healthy animals. The time-of-day-dependant variation was less than 3 %, except for alanine aminotransferase. During the first two weeks of spring pasture serum aspartate and alanine aminotransferase levels were significantly raised in both cows and ewes, compared with serum levels of the same animals on indoor feeding. No such increase occurred in total lactate dehydrogenase.