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PubMed1993

Genetic variability and evolution of the Schistosoma genome analysed by using random amplified polymorphic DNA markers.

The usefulness of random amplified polymorphic DNA markers (RAPD) was assayed in an attempt to discriminate among species, strains and individuals within the genus Schistosoma. Depending on the species, 40-50 arbitrary decamer oligonucleotides were used as primers to amplify total DNA by the polymerase chain reaction (PCR). An important polymorphism was observed among 5 species, allowing a phylogenetic tree to be outlined. These differences can be used for rapid and accurate identification. A limited but easily detectable polymorphism was revealed among 3 strains of a single species (Schistosoma mansoni). Minor differences were observed among individuals of a single strain. A RAPD marker allows sexual discrimination between individuals from the terminal spined-egg species group. Although a limited number of strains have been examined, the results already indicate clearly that RAPD markers constitute a powerful tool for the analysis of genetic variability. This new tool will considerably extend the information available from morphology, isozyme and limited restriction fragment length polymorphism data and opens the way to genetic analysis of these species.

Animals↗
PubMed1991

Lipopolysaccharide induces double-stranded DNA fragmentation in mouse thymus: protective effect of zinc pretreatment.

Intraperitoneal injection of female NAW/W1 mice with 5 mg of Salmonella typhimurium lipopolysaccharide/kg results in decreased body and thymus weight. Reduced thymic weight is accompanied by fragmentation of DNA into multimers of about 200 bp size. This effect is consistent with the induction of intranucleosomal cleavage of double-stranded DNA in thymus. Maximal fragmentation of DNA occurs between 18 and 24 h after treatment; by 48 h post lipopolysaccharide treatment, there is little evidence of thymic DNA fragmentation. Pretreatment of mice with Zn protects against lipopolysaccharide-induced DNA fragmentation. This effect is maximal at about 72 h after Zn treatment (24 h after lipopolysaccharide treatment) and persists until about 96 h after Zn treatment. At 72 h after pretreatment, the antagonism of thymic DNA fragmentation by Zn is dose-dependent. To examine the role of the acute phase inflammatory response elicited by lipopolysaccharide treatment in the production of changes in thymic weight and DNA integrity, the effects of treatment with casein, a well-characterized inducer of the acute phase inflammatory response in mice, were examined. In contrast to the effect of lipopolysaccharide, casein treatment did not produce a similar pattern of DNA fragmentation in thymus. Taken together, these data suggest that lipopolysaccharide induces DNA fragmentation in thymus by a mechanism which does not occur during the pathophysiological changes which accompany the casein-induced acute phase response. Further, the antagonism by Zn of lipopolysaccharide-induced fragmentation of thymic DNA is consistent with earlier findings that Zn can prevent dexamethasone-induced DNA fragmentation in vitro.

Acute-Phase Reaction↗
PubMed1989

Effects of nitroimidazoles on neuronal cells in vitro.

Peripheral neuropathy is a significant dose-limiting side effect of the nitroimidazole drugs in vivo. We have thus undertaken a study on the mechanisms of nitroimidazole neurotoxicity in the cultured neuronal cell lines, PC-12 (rat pheochromocytoma) and NB4183 (mouse neuroblastoma). Cells were differentiated with either nerve growth factor or dibutyryl cAMP and then were exposed to misonidazole and SR 2508. Cells underwent extensive morphological changes following exposure to nitroimidazole drugs, including loss of differentiated neurite projections. Loss of neurites appeared to correlate with changes in neurofilament proteins. Immunoblot analysis of the neurofilament proteins revealed a loss of the major parent proteins and the appearance of lower molecular weight (degradation) fragments. Our preliminary data in cultured neuronal cell lines suggest that nitroimidazoles cause disruption and degradation of the neurofilament lattice with subsequent degeneration of dendritic projections, and provide an in vitro model for studying the cellular and biochemical mechanisms of drug-induced neurotoxicity.

Animals↗
PubMed2006

Detection, characterization and identification of crucifer phytoalexins using high-performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry.

We have analyzed 23 crucifer phytoalexins (e.g. brassinin, dioxibrassinin, cyclobrassinin, brassicanals A and C) by HPLC with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI-MS) using both negative and positive ion modes. Positive ion mode ESI-MS appeared more sensitive than negative ion mode ESI-MS in detecting this group of compounds. A new HPLC separation method, new LC-MS and LC-MS(2) data and proposed fragmentation pathways, LC retention times, and UV spectra for selected compounds are reported.

Brassicaceae↗
PubMed2004

Sumo1 conjugates mitochondrial substrates and participates in mitochondrial fission.

Mitochondrial fission requires the evolutionarily conserved dynamin related protein (DRP1), which is recruited from the cytosol to the mitochondrial outer membrane to coordinate membrane scission. Currently, the mechanism of recruitment and assembly of DRP1 on the mitochondria is unclear. Here, we identify Ubc9 and Sumo1 as specific DRP1-interacting proteins and demonstrate that DRP1 is a Sumo1 substrate. In addition, a surprising number of Sumo1 conjugates were observed in the mitochondrial fractions, suggesting that sumoylation is a common mitochondrial modification. Video microscopy demonstrates that YFP:Sumo1 is often found at the site of mitochondrial fission and remains tightly associated to the tips of fragmented mitochondria. Consistent with this, fluorescence microscopy revealed that a portion of total cytosolic YFP:Sumo1 colocalizes with endogenous mitochondrial DRP1. Finally, transient transfection of Sumo1 dramatically increases the level of mitochondrial fragmentation. Analysis of endogenous DRP1 levels indicates that overexpression of Sumo1 specifically protects DRP1 from degradation, resulting in a more stable, active pool of DRP1, which at least partially accounts for the excess fragmentation. Together, these data are the first to identify a function for Sumo1 on the mitochondria and suggest a novel role for the participation of Sumo1 in mitochondrial fission.

Animals↗
PubMed2026

Predicting bloodstream infection by plasma cell-free metagenomic sequencing: a prospective cohort study.

BACKGROUND: Patients receiving myelosuppressive chemotherapy or haematopoietic cell transplantation are at high risk for life-threatening bloodstream infections. A novel pre-emptive treatment paradigm guided by pathogen detection before symptoms appear might reduce this risk, but no validated screening test is available. This study evaluated the sensitivity and specificity of plasma microbial cell-free DNA metagenomic sequencing (mcfDNA-Seq) for predicting bloodstream infections in children and adolescents receiving therapy for high-risk leukaemia. METHODS: In this prospective cohort study, between Aug 9, 2017, and Feb 28, 2022, leftover clinical plasma samples were prospectively collected up to once per day from patients who were younger than 25 years, receiving care for leukaemia at St Jude Children's Research Hospital (Memphis, TN, USA), and at high risk for life-threatening bloodstream infections. mcfDNA-Seq was used to identify pathogen DNA in blood samples obtained during the 7 days before to 1 day after bloodstream infection onset, and in control samples from the same population in the absence of fever or infection. The testing laboratory was masked to sample status. Primary outcomes were predictive sensitivity of mcfDNA-Seq for detecting the expected bloodstream infection pathogen during the 3 days preceding the day of bloodstream infection onset, with a prespecified favourable sensitivity of 50%, and predictive specificity of mcfDNA-Seq in control samples. Exploratory analyses comprised assessing sensitivity and specificity restricted to bacteria or common bloodstream infection pathogens, and after applying a data-derived DNA fragment concentration cutoff; estimating the predictive sensitivity on each of the 7 days before bloodstream infection onset; identifying clinical characteristics that affected predictive sensitivity or specificity; and examining the clinical relevance of additional organisms identified by mcfDNA-Seq during bloodstream infection episodes. Diagnostic sensitivity was also assessed on samples collected on the day of, or day after, diagnosis of bloodstream infection. This study is registered with ClinicalTrials.gov, NCT03226158. FINDINGS: 94 evaluable bloodstream infections occurred in 60 (38%) of 158 enrolled participants; 19 episodes were previously described in the pilot phase of this study. The predictive sensitivity of mcfDNA-Seq was 51·9% (95% CI 40·5-63·1) for all bloodstream infection episodes, 53·8% (42·2-65·2) for bacterial infection only, and 51·9% (40·5-63·1) when applying a DNA fragment concentration cutoff of 140 molecules per μL. Sensitivity was lowest at day -7 and increased daily until the day of diagnosis. Diagnostic sensitivity was 81·3% (95% CI 71·0-89·1) for all bloodstream infection episodes and 83·1% (72·9-90·7) for bacterial infections only. Predictive specificity was 82·7% (95% CI 76·0-88·2), but improved to 88·9% (83·0-93·3) for common bloodstream infection pathogens, and to 93·8% (88·9-97·0) when also applying the DNA fragment concentration cutoff. Predictive sensitivity was higher in participants with acute lymphoblastic leukaemia (adjusted odds ratio [aOR] 11·1 [1·7-74·2] vs those with acute myeloid leukaemia), and it was lower in polymicrobial infections (aOR 0·0 [0·0-0·2] vs monomicrobial Gram-positive infections). Clinical false-positive results were positively associated with gastrointestinal disturbance alone (p=0·037) or combined with recent administration of high-dose cytarabine (p=0·012). Additional organisms identified by mcfDNA-Seq that were not identified by blood culture were less likely than expected organisms to have an increasing DNA concentration during the days preceding bloodstream infection diagnosis. INTERPRETATION: mcfDNA-Seq can detect causative pathogens before the onset of some bloodstream infection episodes in profoundly immunocompromised patients. Predictive specificity might be improved by restricting results to a subgroup of relevant organisms, excluding patients with high risk of false-positive results, or applying a higher concentration cutoff. Clinical trials are needed to evaluate mcfDNA-Seq-guided pre-emptive therapy for preventing life-threatening bloodstream infections in patients with high risk. FUNDING: The National Cancer Institute, American Lebanese Syrian Associated Charities, St Jude Children's Research Hospital, and Karius.

Adolescent↗
PubMed2006

Species boundaries and phylogeography of the "Euscorpius carpathicus complex" (Scorpiones: Euscorpiidae) in Italy.

Euscorpius tergestinus (C.L. Koch, 1837), Euscorpius concinnus (C.L. Koch, 1837) and Euscorpius sicanus (C.L. Koch, 1837), three presumed closely related species belonging to the "carpathicus group", occur in the Italian peninsula with a largely parapatric distribution and some zones of range overlap. These areas of sympatry represent interesting opportunities to investigate species boundaries in natural populations. Here we report on a study exploring genetic variation in sympatric populations of the three species from central Tuscany. Additional collecting sites, from different localities across Italy, were also included in the analysis in order to explore the phylogeographic structure of the group. Species boundaries and evolutionary relationships were examined by sequence comparison of mitochondrial 16S rRNA and nuclear ITS-1 rRNA gene fragments. DNA sequence data show no evidence of genetic introgression between different evolutionary lineages from the area of range overlap, suggesting the absence of either past or ongoing inter-specific gene flow. It is therefore probable that reproductive barriers exist, preventing gene pools from amalgamating. Furthermore, our results support the recent morphological distinction of E. tergestinus, as traditionally classified, into two different species: E. tergestinus and E. concinnus. Both mitochondrial and nuclear sequence data clearly indicate that the two taxa represent well-supported and deeply divergent lineages. Euscorpius sicanus seems to represent a monophyletic taxon, but the high genetic variability observed within this taxon calls for future investigation. The present distribution patterns across the Italian peninsula were mainly interpreted as the consequence of climatic oscillations.

Animals↗
PubMed2006

Proteolytic mapping of the adeno-associated virus capsid.

The three-dimensional structures of the viral capsid of three AAV serotypes have previously been determined by X-ray crystallography or cryoelectron microscopy. These studies of AAV and similar studies of autonomous parvoviruses have yielded important structural information about the virions in a low-energy conformation. However, there is little information on the structural properties of AAV virions in solution under physiological conditions. We demonstrate that proteolytic digestion of AAV2 virions with trypsin results in cleavage at a specific site on the capsid surface while the capsid remains intact. The products of digestion were mapped using unique antibodies, protein sequencing, mass spectroscopy, and 3D structure modeling to a region on a surface loop that is common to all three AAV2 structural proteins. Empty AAV2 capsids could be distinguished from full (DNA-containing) capsids, having an increased susceptibility of VP2 to trypsin and being digested more rapidly by chymotrypsin. Proteolytic analysis utilizing trypsin or chymotrypsin was also capable of distinguishing AAV2 from AAV1 and AAV5, as seen by differential susceptibility and unique fragment patterns. These data demonstrate a novel approach for studying the structure of AAV capsids in solution and should be valuable in the testing and engineering of AAV vectors for gene transfer.

Amino Acid Sequence↗
PubMed1989

Morphological and electrophysiological studies of human hippocampal transplants in the anterior eye chamber of athymic nude rats.

Human fetal hippocampal tissue from normal women was obtained following elective abortion in the 8th to the 11th week of gestation. The hippocampal tissue was transplanted to the anterior chamber of the eye of adult athymic nude rats, where it was allowed to develop for up to 9 months before histological and electrophysiological evaluation. The transplants were revascularized from the host iris and many grew extensively in oculo. Large neurons were present in all transplants. Immunohistochemical studies revealed glutamic acid decarboxylase-containing terminals and clusters of gamma-aminobutyric acid-positive nerve cell bodies within the transplants, as well as scattered tyrosine hydroxylase-positive and acetylcholinesterase-containing fibers. Single neurons recorded extracellularly from transplants 4-9 months in oculo showed a slow spontaneous discharge, with both complex and single action potentials. Stimulation of the transplant surface evoked a small initial wave followed by a larger and longer-lasting field potential, similar to that seen in hippocampus in situ. A conditioning-testing paradigm was used to evaluate the presence of inhibitory circuitry in the hippocampal transplants. Significant suppression of the evoked test response was seen with interstimulus intervals ranging from 20 to 500 ms. Superfusion of enkephalin (100-300 nM) or penicillin (1600 U/ml) increased slow-wave activity, as did tetanic electrical stimulation. These treatments appeared to generate ictal-like activity, which in some cases persisted as interictal spikes. Illumination of the retina also increased neuronal activity, presumably by reflex activation of cholinergic afferents from the parasympathetic innervation of the iris. Taken together, our data suggest that fragments of hippocampus from aborted first trimester human fetuses, grafted to the eye chamber of rodent hosts, develop many organotypic histological and physiological features. This preparation may provide a unique means for the study of neurobiological properties of human brain in both normal and disease states.

Animals↗
PubMed1997

The active site region of the vitamin K-dependent carboxylase includes both the amino-terminal hydrophobic and carboxy-terminal hydrophilic domains of the protein.

In order to localize the active site of the vitamin K-dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino-terminal and hydrophilic carboxy-terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino-terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino-terminal fragment is seriously underestimated by SDS-PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy-terminal hydrophilic domain.

Amino Acid Sequence↗
PubMed1997

Importance of the linker in expression of single-chain Fv antibody fragments: optimisation of peptide sequence using phage display technology.

We have investigated the potential for enhancing the production of functional single-chain Fv antibody fragments (sFv), by altering the sequence of the linker that joins the variable domains of the molecule. To identify new functionally improved linkers we have used a phage display library containing a random sequence of six amino acids in the linker. Multiple rounds of panning on the antigen led to the selection of six different linker sequences that enhanced the binding of phage to the antigen. Five of the linkers also improved the secretion of soluble sFv by approximately five-fold. Analysis of the predominant linker sequence isolated showed that this improvement is not due to an increased affinity for the antigen, nor to alterations in the toxicity to bacteria. However the linker did affect the denaturation of the sFv in urea. It is therefore possible that the novel sequence helps in the refolding or secretion of the molecule.

Amino Acid Sequence↗
PubMed1999

Relation of beta3-adrenergic receptor gene mutation to total body fat but not percent body fat and insulin levels in Thais.

A Trp64Arg mutation in the beta3-adrenergic receptor gene has been implicated in the pathophysiology of non-insulin-dependent diabetes mellitus and obesity. However, the findings have been controversial due to the use of different populations and different methods for the estimation of body fat. In the present study, the prevalence of Trp64Arg mutation of the beta3-adrenergic receptor gene was determined and its relation to body fat as assessed by dual-energy x-ray absorptiometry (DEXA) was evaluated in Thai men and women. The effect on insulin sensitivity as assessed by the serum insulin to glucose ratio was also examined. The subjects were 76 men and 135 women aged 20 to 80 years. Body fat and its regional distribution were assessed by DEXA. Mutation in the beta3-adrenergic receptor gene was determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism. Data are expressed as the mean +/- SEM. Fifty-nine subjects (28.0%) had the Trp64Arg mutation in the beta3-adrenergic receptor gene; 54 (25.6%) were heterozygotes and five (2.4%) were homozygotes. The gene frequency of Trp64Arg mutation was 15.2% in these subjects. In women, Trp64Arg mutation was not associated with the difference in total body fat (Trp/Arg or Arg/Arg, 19.4 +/- 1.0 kg; Trp/Trp, 19.2 +/- 0.6 kg) or percent body fat (Trp/Arg or Arg/Arg, 34.6% +/- 1.2%; Trp/Trp, 34.3% +/- 0.6%). In contrast to the findings in women, men with Trp64Arg mutation had lower total body fat after controlling for age (Trp/Arg or Arg/Arg, 13.2 +/- 1.1 kg; Trp/Trp, 15.8 +/- 0.7 kg; P < .05). However, no difference was found in percent body fat (Trp/Arg or Arg/Arg, 20.9% +/- 1.3%; Trp/Trp, 23.3% +/- 0.7%). No difference in the fasting insulin resistance index (FIRI) was found between subjects with and without Trp64Arg mutation. The data suggest that Trp64Arg mutation of the beta3-adrenergic receptor is common in Thais and appears to exert effects on total body fat but not percent body fat in men. Trp64Arg mutation is not associated with insulin resistance as assessed by the FIRI in Thais.

Adipose Tissue↗
PubMed1999

Antiapoptotic marker Bcl-X(L), expression on Reed-Sternberg cells of Hodgkin's disease using a novel monoclonal marker, YTH-2H12.

Inhibitors of apoptosis may regulate tissue differentiation and promote cell survival in neoplasia. A new apoptosis inhibitor of the bcl-2 gene family, bcl-X(L), was recently found in some types human neoplasia but not in normal tissue. We investigated bcl-X(L) expression in 419 cases of normal and neoplastic lymphoid lesions using immunohistochemistry with the monoclonal antibody bcl-X(L) (YTH-2H12). Ninety-four percent (141/150) of classic Hodgkin's disease (HD) were positive for bcl-X(L) with strong intensity in most Reed-Sternberg (RS) cells. Forty-eight percent (38/80) of nodular lymphocyte predominance (LPHD) were positive. In the non-Hodgkin's lymphomas (NHL), bcl-X(L) was expressed in a low percentage of cases (< 20%), with the exception of follicle center lymphoma, grade III/III (78%). All reactive hyperplastic lesions were negative for bcl-X(L). RS cells, which expressed bcl-X(L), were not labeled by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). We found RS cells expressing bcl-X(L) were absent of DNA fragmentation (apoptosis). Our data provide evidence that bcl-X(L) is abnormally expressed in the RS cells of HD and some types of NHL raising speculation that inhibition of apoptosis may be important in the pathogenesis of lymphoma, specifically HD. In addition, the previously reported correlation between bcl-X(L) and Epstein-Barr virus expression in HD was not supported by this study.

Animals↗
PubMed2002

Induction of apoptosis via mitogen-activated protein kinase pathway by a K vitamin analog in rat hepatocytes.

BACKGROUND/AIMS: Compound 5 (Cpd 5), a vitamin K analog, inhibits rat hepatocyte DNA synthesis and hepatoma cell growth. The aim of this study was to determine if the inhibitory effect of Cpd 5 on cell growth was related to apoptosis. METHODS: Isolated rat hepatocytes were cultured with Cpd 5, and mitogen-activated signaling pathway and apoptosis pathway were investigated using Western blot analysis. RESULTS: When rat hepatocytes were cultured with Cpd 5 for 48 h, apoptosis was evident, which included characteristic morphological changes, DNA fragmentation, and the activation of caspase 3 (CPP 32)-like protease. Examination of upstream events of apoptosis pathway showed that the expression of Bax was induced and bcl-2 was inhibited by Cpd 5 treatment. Concomitant with the induction of apoptosis, Cpd 5 activated the extracellular signal-regulated kinase (ERK) signaling pathway. PD 98059, a mitogen-activated protein kinase kinase inhibitor, and glutathione, an anti-thiol-oxidant, not only blocked Cpd 5-induced ERK phosphorylation, but also antagonized the activation of CPP-32, the altered Bcl-2/Bax expression, and DNA fragmentation. CONCLUSIONS: The data suggest that the ERK signaling pathway may be involved in the regulation of rat hepatocyte apoptosis induced by Cpd 5.

Animals↗
PubMed1997

Galectin-3 inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven rat bone marrow cell proliferation and GM-CSF-induced gene transcription.

The expression of galectin-3 (formerly known as IgE-binding protein or Mac-2) in rat bone marrow (BM) was investigated by FACS, immunocytochemical and immunoblot analysis. The functional significance of rat recombinant galectin-3 on mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF)-driven proliferation of macrophage progenitors and gene transcription was further examined. Immunocytochemical analysis of in situ BM sections demonstrated galectin-3 in myelopoietic cells and surrounding stroma, whereas erythropoietic and lymphopoietic environments essentially lacked galectin-3 expression. FACS analysis demonstrated that incubation of freshly isolated BMC with lactose, a competing ligand for galectin-3 binding to glycoconjugates, decreased binding of antigalectin antibodies to cells primarily expressing the myeloid antigen recognized by mAb His-54. Similarly, lectin-mediated binding of exogenous galectin-3 to myeloid lineage cells was also demonstrated. Immunoblot analysis of BM eluates demonstrated galectin-3 both in the extracellular matrix and in a lactose elutable form, bound to the surface of BMC. [3H]Thymidine incorporation studies on BMC cultured in the presence of galectin-3 demonstrated suppression of GM-CSF-induced proliferation by galectin-3. In addition, differential display analysis of immediate early gene expression in BMC cultured in the presence of galectin-3 revealed a 76.2% inhibition of GM-CSF-induced gene transcription by galectin-3 assessed by the number of PCR-fragments generated. Our data suggest a role for galectin-3 in the organization of myelopoietic compartments in rat BM and regulation of the action of growth factors on myelopoietic precursor cells.

Animals↗
PubMed2003

Multi locus fingerprinting of Listeria monocytogenes by sequence-specific labeling of DNA probes combined with array hybridization.

We have developed an alternative multi locus sequence typing (MLST) approach that targets the variable genetic changes directly in a DNA array format. Our approach is based on DNA array hybridization in combination with sequence-specific labeling of oligonucleotide probes. Listeria monocytogenes was chosen for the development and evaluation of the assay. The genes hlyA, iap, flaA, inlA and actA were targeted. Twenty-nine suitable probe regions were identified within these genes. The DNA array results from 32 different strains were compared to serotype and amplified fragment length polymorphism data. This comparison showed that our DNA array method gave good discrimination between the strains analyzed. In conclusion, the DNA array-based MLST method is a promising tool for fingerprint bacteria.

Bacterial Typing Techniques↗
PubMed1998

High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra.

Injected-ion mobility and high-pressure ion mobility techniques have been used to examine the conformations of bradykinin, insulin chain A, and several other peptide ions in the gas phase. Under the experimental conditions employed, evidence for multimer formation in the mass spectra of peptides is minimal or absent altogether. However, ion mobility distributions show that aggregates of peptides (containing a single charge per monomer unit) are observed at the same mass-to-charge ratios as the singly charged parent ions. Collision cross sections for these clusters show that they have tightly packed roughly spherical conformations. We have bracketed the average density as 0.87 < p < 1.00 g cm-3. In some cases, specific stable aggregate forms within a cluster size can be distinguished indicating that some high order structures are favored in the gas phase. Multimer formation between different sizes of polyalanine peptides shows no evidence for size specificity in aggregate formation. Collisional and thermal excitation studies have been used to examine structural transitions and dissociation of the multimers. Aggregates appear to dissociate via loss of singly charged monomers. The observation that peptide multimers can be concealed in mass spectral data requires that fragmentation patterns and reactivity studies of singly charged monomers be undertaken with care.

Algorithms↗
PubMed2003

Lifecycle closure, lineage sorting, and hybridization revealed in a phylogenetic analysis of European oak gallwasps (Hymenoptera: Cynipidae: Cynipini) using mitochondrial sequence data.

Oak gallwasps are cyclically parthenogenetic insects that induce a wide diversity of highly complex species- and generation-specific galls on oaks and other Fagaceae. Phylogenetic relationships within oak gallwasps remain to be established, while sexual and parthenogenetic generations of many species remain unpaired. Previous work on oak gallwasps has revealed substantial intra-specific variation, particularly between regions known to represent discrete Pleistocene glacial refuges. Here we use statistical phylogenetic inference methods on sequence data for a fragment of the mitochondrial cytochrome b gene to reconstruct the relationships among 62 oak gallwasp species. For 16 of these we also include 23 additional cytochrome b haplotype sequences from different Pleistocene refuge areas to test the effect of intra-specific variation on inter-specific phylogeny reconstruction. The reconstructed phylogenies show good intra-generic resolution and identify several conserved clades, but fail to reconstruct either very recent or very ancient divergences. Nine of the 16 species represented by multiple haplotypes are not monophyletic. The apparent discordance between the recovered gene tree and the current taxonomic classification can be explained through: (a) collapsing of some species currently known only from either a sexual or a parthenogenetic generation into a single cyclically parthenogenetic entity; (b) sorting of ancestral polymorphism in diverging lineages, and (c) horizontal transfer of haplotypes, perhaps due to hybridization within glacial refuges. Our conclusions emphasise the need for careful intra-specific sampling when reconstructing phylogenies for radiations of closely related species and imply that for certain taxonomic groups full phylogenetic resolution (using molecular markers) may not be attainable.

Animals↗