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31.
Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera 总被引:5,自引:2,他引:3
Nucleotide sequences from a 434-bp region of the 16S rRNA gene were
analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps,
sawflies) to examine the patterns of variation within the gene fragment and
the taxonomic levels for which it shows maximum utility in phylogeny
estimation. A hierarchical approach was adopted in the study through
comparison of levels of sequence variation among taxa at different
taxonomic levels. As previously reported for many holometabolous insects,
the 16S data reported here for Hymenoptera are highly AT-rich and exhibit
strong site-to-site variation in substitution rate. More precise estimates
of the shape parameter (alpha) of the gamma distribution and the proportion
of invariant sites were obtained in this study by employing a reference
phylogeny and utilizing maximum-likelihood estimation. The effectiveness of
this approach to recovering expected phylogenies of selected hymenopteran
taxa has been tested against the use of maximum parsimony. This study finds
that the 16S gene is most informative for phylogenetic analysis at two
different levels: among closely related species or populations, and among
tribes, subfamilies, and families. Maximization of the phylogenetic signal
extracted from the 16S gene at higher taxonomic levels may require
consideration of the base composition bias and the site-to-site rate
variation in a maximum-likelihood framework.
相似文献
32.
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.
相似文献
33.
Deshpande NV Sabaté M Ligthart JM Kutryk MJ Serruys PW 《International journal of cardiovascular interventions》1998,1(1):45-48
Jailing of a side-branch is a known complication of stent implantation, and makes access to the side-branch difficult, especially if the stent is of the self-expanding type. Although plain balloon angioplasty is feasible for the jailed side-branches, the use of newer devices (a stent, Rotablation or atherectomy) has not been described. We describe a novel way of treating a side-branch jailed by a self-expanding stent by using stent implantation through the strut of a self-expanding stent. 相似文献
34.
A plasmid library of Acinetobacter calcoaceticus HindIII fragments was
constructed, and clones that complemented an Escherichia coli pabA mutant
were selected. Plasmids containing a 3.9-kb fragment of A. calcoaceticus
DNA that also complemented E. coli trpD and trpC-(trpF+) mutants were
obtained. We infer that complementation of E. coli pabA mutants was the
result of the expression of the amphibolic anthranilate-
synthase/p-aminobenzoate-synthase glutamine-amidotransferase gene and that
the plasmid insert carried the entire trpGDC gene cluster. In E. coli
minicells, the plasmid insert directed the synthesis of polypeptides of
44,000, 33,000, and 20,000 daltons, molecular masses that are consistent
with the reported molecular masses of phosphoribosylanthranilate
transferase, indoleglycerol-phosphate synthase, and anthranilate-synthase
component II, respectively. A 3,105- bp nucleotide sequence was determined.
Comparison of the A. calcoaceticus trpGDC sequences with other known trp
gene sequences has allowed insight into (1) the evolution of the amphibolic
trpG gene, (2) varied strategies for coordinate expression of trp genes,
and (3) mechanisms of gene fusions in the trp operon.
相似文献
35.
Phenotypes of Epstein-Barr virus LMP1 deletion mutants indicate transmembrane and amino-terminal cytoplasmic domains necessary for effects in B-lymphoma cells. 总被引:1,自引:0,他引:1 下载免费PDF全文
The Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) has previously been shown to cause EBV-negative B-lymphoma cells to grow in large clumps and to alter expression of surface activation and adhesion molecules (D. Wang, D. Liebowitz, F. Wang, C. Gregory, A. Rickinson, R. Larson, T. Springer, and E. Kieff, J. Virol. 62:1473-4184, 1988; F. Wang, C. Gregory, C. Sample, M. Rowe, D. Liebowitz, R. Murray, A. Rickinson, and E. Kieff, J. Virol. 64:2309-2318, 1990). In order to identify functional elements in the amino-terminal cytoplasmic domain and the first four transmembrane domains which were previously shown to be essential for LMP1 activity, three smaller deletion mutants were constructed and tested for their activity in B-lymphoma cells. The results of the present study indicate that the amino-terminal cytoplasmic domain, the first transmembrane domain, and the third and fourth transmembrane domains each contribute to LMP1's effects on B lymphocytes. 相似文献
36.
A search was made for the lymphoid cell type(s) which are the source of immune RNA (I-RNA) capable of transferring tumor-specific cell-mediated cytotoxicity (CMC). Hartley guinea pigs were immunized with syngeneic murine fibrosarcomas (BP-10 or BP-11) induced by 3,4-benzo(a)pyrene in C3H/HeJ mice, and the I-RNA was extracted individually from their spleens, lymph nodes, and peritoneal exudate (PE) cells. All three I-RNA preparations were able to convert normal C3H/HeJ mouse lymphocytes to effector cells significantly cytolytic to the specific syngeneic mouse tumor in vitro. Furthermore, lymphocytes and macrophages were purified from the spleens, lymph nodes, and PE cells of tumor-immunized guinea pigs. I-RNA was extracted from these purified cell populations and also from the pooled guinea pig lymphoid tissues. Normal C3H/HeJ lymphocytes were incubated with each type of I-RNA and tested in vitro for CMC against the specific tumor cells. Significant CMC against BP-10 targets was observed with mouse lymphocytes incubated with I-RNA extracted from pooled lymphoid tissues of BP-10 tumor-immunized guinea pigs. There was a reduced but still significant CMC when mouse lymphocytes were incubated with I-RNA extracted from purified guinea pig lymphocytes, whereas there was a markedly increased CMC when the I-RNA was extracted from purified guinea pig macrophages. As indicated by sucrose density gradient analysis, the lesser effectiveness of lymphocyte I-RNA was not due to RNA degradation resulting from lymphocyte purification or I-RNA extraction. Treatment of all types of I-RNA with RNase abrogated the transfer of CMC, whereas treatment of I-RNA with DNase or pronase did not. RNA extracted from the lymphoid tissues of guinea pigs immunized with complete Freund's adjuvant without tumor was ineffective. Mouse lymphocytes incubated with BP-10 macrophage I-RNA destroyed BP-10 but not BP-11 tumor cells, whereas lymphocytes incubated with BP-11 macrophage I-RNA killed BP-11 but not BP-10 tumor cells, thus indicating tumor specificity of the immunity transferred by macrophage I-RNA. Our results suggest that macrophages are the principal source of I-RNA capable of transferring tumor-specific CMC. 相似文献
37.
Burn injury initiates a shift in superantigen-induced T cell responses and host survival 总被引:4,自引:0,他引:4
Zang Y Dolan SM Ni Choileain N Kriynovich SJ Murphy TJ Sayles P Mannick JA Lederer JA 《Journal of immunology (Baltimore, Md. : 1950)》2004,172(8):4883-4892
Severe injury induces a temporal shift in immune reactivity that can cause serious complications or even death. We previously reported that mice exposed to bacterial superantigen (SAg) early after injury undergo a strong SAg response with lethal consequences. This study compares the early and late effects of burn injury on SAg reactivity in vivo to establish how injury influences adaptive immune responses. We found that mice challenged with ordinarily sublethal doses of staphylococcal enterotoxin A or staphylococcal enterotoxin B at 1 day after burn injury exhibited high mortality, whereas no mortality occurred at 7 days after injury. This shift in mortality correlated with higher Th2-type cytokines (IL-4 and IL-10) being expressed by CD4(+) and CD8(+) T cells from burn as opposed to sham mice at 7 days after injury. Lymph node cells from burn-injured mice also produced higher levels of Th2-type cytokines at 7 days after injury. The results of cell-mixing studies using CD4(+) and CD8(+) T cells mixed with APCs from sham or burn mice suggested that changes in both T cells and APCs are involved in the altered SAg response. Finally, the biological significance of altered SAg reactivity following injury was shown by demonstrating that blocking IL-10 activity in vivo caused higher SAg-induced mortality at 7 days after injury. These findings support the idea that injury promotes a Th2-type shift in adaptive immune reactivity. Although prior studies link this counterinflammatory-type response to lowered resistance to infection, the present results suggest it may sometimes benefit the injured host. 相似文献
38.
Murphy TJ Ni Choileain N Zang Y Mannick JA Lederer JA 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(5):2957-2963
Major injury initiates a systemic inflammatory response that can be detrimental to the host. We have recently reported that burn injury primes innate immune cells for a progressive increase in TLR4 and TLR2 agonist-induced proinflammatory cytokine production and that this inflammatory phenotype is exaggerated in adaptive immune system-deficient (Rag1(-/-)) mice. The present study uses a series of adoptive transfer experiments to determine which adaptive immune cell type(s) has the capacity to control innate inflammatory responses after injury. We first compared the relative changes in TLR4- and TLR2-induced TNF-alpha, IL-1beta, and IL-6 production by spleen cell populations prepared from wild-type (WT), Rag1(-/-), CD4(-/-), or CD8(-/-) mice 7 days after sham or burn injury. Our findings indicated that splenocytes prepared from burn-injured CD8(-/-) mice displayed TLR-induced cytokine production levels similar to those in WT mice. In contrast, spleen cells from burn-injured CD4(-/-) mice produced cytokines at significantly higher levels, equivalent to those in Rag1(-/-) mice. Moreover, reconstitution of Rag1(-/-) or CD4(-/-) mice with WT CD4(+) T cells reduced postinjury cytokine production to WT levels. Additional separation of CD4(+) T cells into CD4(+)CD25(+) and CD4(+)CD25(-) subpopulations before their adoptive transfer into Rag1(-/-) mice showed that CD4(+)CD25(+) T cells were capable of reducing TLR-stimulated cytokine production levels to WT levels, whereas CD4(+)CD25(-) T cells had no regulatory effect. These findings suggest a previously unsuspected role for CD4(+)CD25(+) T regulatory cells in controlling host inflammatory responses after injury. 相似文献
39.
Two modes of gating during late Na+ channel currents in frog sartorius muscle 总被引:17,自引:6,他引:11 下载免费PDF全文
Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods. 相似文献
40.
JB Sutro 《The Journal of general physiology》1986,87(1):1-24
Veratridine bath-applied to frog muscle makes inactivation of INa incomplete during a depolarizing voltage-clamp pulse and leads to a persistent veratridine-induced Na tail current. During repetitive depolarizations, the size of successive tail currents grows to a plateau and then gradually decreases. When pulsing is stopped, the tail current declines to zero with a time constant of approximately 3 s. Higher rates of stimulation result in a faster build-up of the tail current and a larger maximum value. I propose that veratridine binds only to open channels and, when bound, prevents normal fast inactivation and rapid shutting of the channel on return to rest. Veratridine-modified channels are also subject to a "slow" inactivation during long depolarizations or extended pulse trains. At rest, veratridine unbinds with a time constant of approximately 3 s. Three tests confirm these hypotheses: (a) the time course of the development of veratridine-induced tail currents parallels a running time integral of gNa during the pulse; (b) inactivating prepulses reduce the ability to evoke tails, and the voltage dependence of this reduction parallels the voltage dependence of h infinity; (c) chloramine-T, N-bromoacetamide, and scorpion toxin, agents that decrease inactivation in Na channels, each greatly enhance the tail currents and alter the time course of the appearance of the tails as predicted by the hypothesis. Veratridine-modified channels shut during hyperpolarizations from -90 mV and reopen on repolarization to -90 mV, a process that resembles normal activation gating. Veratridine appears to bind more rapidly during larger depolarizations. 相似文献