Two organ transplant recipients who received organs from a common donor and were diagnosed as having an Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disorder were studied to determine the mode of EBV transmission. The results of restriction fragment length polymorphism, polymerase chain reaction, and minisatellite DNA analyses demonstrate that both patients had a common strain of EBV and that this strain was transmitted from the donor's organs to both recipients. Posttransplant lymphoproliferative disorder resulted from the proliferation of EBV-immortalized B lymphocytes of the recipient, not those of the donor. 相似文献
Candida albicans is an opportunistic human fungal pathogen that causes systemic candidiasis as well as superficial mucosal candidiasis. In response to the host environment, C. albicans transitions between yeast and hyphal forms. In particular, hyphal growth is important in facilitating adhesion and invasion of host tissues, concomitant with the expression of various hypha-specific virulence factors. In previous work, we showed that the cyclic AMP (cAMP) signaling pathway plays a crucial role in morphogenic transitions and virulence of C. albicans by studying genes encoding adenylate cyclase-associated protein (CAP1) and high-affinity phosphodiesterase (PDE2) (Y. S. Bahn, J. Staab, and P. Sundstrom, Mol. Microbiol. 50:391-409, 2003; and Y. S. Bahn and P. Sundstrom, J. Bacteriol. 183:3211-3223, 2001). However, little is known about the downstream targets of the cAMP signaling pathway that are responsible for morphological transitions and the expression of virulence factors. Here, microarrays were probed with RNA from strains with hypoactive (cap1/cap1 null mutant), hyperactive (pde2/pde2 null mutant), and wild-type cAMP signaling pathways to provide insight into the molecular mechanisms of virulence that are regulated by cAMP and that are related to the morphogenesis of C. albicans. Genes controlling metabolic specialization, cell wall structure, ergosterol/lipid biosynthesis, and stress responses were modulated by cAMP during hypha formation. Phenotypic traits predicted to be regulated by cAMP from the profiling results correlated with the relative strengths of the mutants when tested for resistance to azoles and subjected to heat shock stress and oxidative/nitrosative stress. The results from this study provide important insights into the role of the cAMP signaling pathway not only in morphogenic transitions of C. albicans but also for adaptation to stress and for survival during host infections. 相似文献
IQGAP1 is a 190-kDa molecular scaffold containing several domains required
for interaction with numerous proteins. One domain is homologous to Ras
GTPase-activating protein (GAP) domains. However, instead of accelerating
hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD)
binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here
the crystal structure of the isolated IQGAP1 GRD. Despite low sequence
conservation, the overall structure of the GRD is very similar to the GAP
domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the
catalytic “arginine finger” seen in functional Ras GAPs, the GRD
has a conserved threonine residue. GRD residues 1099–1129 have no
structural equivalent in RasGAP and are seen to form an extension at one end
of the molecule. Because the sequence of these residues is highly conserved,
this region likely confers a functionality particular to IQGAP family GRDs. We
have used isothermal titration calorimetry to demonstrate that the isolated
GRD binds to active Cdc42. Assuming a mode of interaction similar to that
displayed in the Ras-RasGAP complex, we created an energy-minimized model of
Cdc42·GTP bound to the GRD. Residues of the GRD that contact Cdc42 map
to the surface of the GRD that displays the highest level of sequence
conservation. The model indicates that steric clash between threonine 1046
with the phosphate-binding loop and other subtle changes would likely disrupt
the proper geometry required for GTP hydrolysis.The small GTPase Ras functions as a binary switch in cell signaling
processes. When bound to GTP, Ras is able to interact with effector proteins,
including Raf kinase, and alter their activities. Ras signaling is terminated
when bound GTP is hydrolyzed to GDP and inorganic phosphate. The basal rate of
GTP hydrolysis on Ras is quite slow (∼1.2 × 10–4
s–1), but this rate of hydrolysis can be enhanced
∼105-fold by interaction with a GTPase-activating protein
(GAP)2
(1). Several RasGAPs have been
identified to date including p120 RasGAP and neurofibromin (NF1). The Rho
family of Ras-related small GTPases also function as binary switches in cell
signaling processes. Whereas the intrinsic rate of GTP hydrolysis on Rho
proteins is faster than Ras, this rate can also be stimulated by interaction
with a RhoGAP. Examination of the structures of the GAP domains of p120RasGAP
(2), neurofibromin
(3), SynGAP
(4), and the GAP domains from
the RhoGAPs p50 RhoGAP and the Bcr homology domain of phosphatidylinositol
3-kinase (5,
6) indicates that although
ostensibly different, these all-helical domains are structurally related
(7).IQGAP1 was discovered by chance during an attempt to isolate novel matrix
metalloproteinases (8).
Analysis reveals that the protein contains several discrete domains and motifs
including a region containing four isoleucine- and glutamine-rich motifs (IQ
repeats) and a region with sequence homology to the Ras-specific GAP domains
of p120RasGAP, NF1, and SynGAP
(2–4,
8). Subsequently, two homologs,
IQGAP2 and IQGAP3, have been discovered. The IQ repeats have been shown to
mediate binding to calmodulin and calmodulin-like proteins (e.g.
S100, myosin essential light chain), whereas the GAP-related domain (GRD) does
not appear to bind to Ras but instead is necessary for binding to the Rho
family GTPases Cdc42 and Rac1, primarily in their active forms
(9–11).
However, instead of accelerating hydrolysis of GTP, IQGAP1 preserves the
activated states of Cdc42 and Rac1 to the extent that overexpression of IQGAP1
in cells increases the levels of active GTPase
(12). Because IQGAP1
expression increases the level of activated Cdc42, initially there was some
confusion as to whether the protein might not represent a novel guanine
nucleotide exchange factor. However it now appears that IQGAP1 is an effector
of Cdc42 and Rac1 and preserves their activated states by tightly binding to
the GTPases and stabilizing them in a conformation not conducive to GTP
hydrolysis. IQGAP1 appears to be such an important effector for Cdc42 that
abrogation of binding to IQGAP1 not only reduces the levels of active Cdc42,
it also reduces membrane-localized Cdc42 and the cellular response to
bradykinin (12).A growing body of evidence implicates IQGAP1 in carcinogenesis. Expression
of IQGAP1 increases during the transition from a minimally to a highly
metastastic form of melanoma, and IQGAP1 has been found to be overexpressed in
ovarian, breast, lung, and colorectal cancers
(13–17).
In vitro, overexpressed IQGAP1 enhances cell motility and
invasiveness in a process that requires Cdc42 and Rac
(18). β-Catenin is one of
the many binding partners of IQGAP1 identified to date. IQGAP1 has been shown
to bind to β-catenin and interfere with β-catenin binding to
α-catenin, an interaction necessary for stable cell-cell adhesion
(19). Another study found that
IQGAP2 knock-out mice overexpress IQGAP1 and developage-dependent liver cancer
and apoptosis (20).To better understand how a protein domain homologous to others that
accelerate GTP hydrolysis can function as an effector and preserve the
GTP-bound state, we have determined the x-ray structure of the IQGAP1 GRD.
Despite low sequence identity, the GRD structure is quite similar to the GAP
domains of p120, neurofibromin, and SynGAP; however, unlike those domains, the
GRD possesses a conserved threonine in place of the catalytic arginine finger
and has a 31-residue insertion that projects from one end of the molecule.
Using the coordinates of Ras·GDP·AlF3 in complex with
the GAP domain of p120, we built a model of Cdc42·GTP bound to the GRD.
The model indicates that a steric clash between the conserved
Thr1046 and the phosphate-binding loop of Cdc42 and other subtle
changes within the active site would likely preclude nucleotide hydrolysis.
Sequence conservation mapped to the surface of the GRD indicates that the
surface with the highest degree of conservation overlaps with the surface that
makes contacts to Cdc42 in the model. 相似文献
For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq) to identify sex‐linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD‐seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non‐native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female‐linked and 19 putatively male‐linked sequences. Four female‐ and eight male‐linked markers amplified in all three life cycle stages. Using one female‐ and one male‐linked marker that were sex‐specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non‐native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD‐seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex‐linked markers in other haplodiplontic macroalgae for which genomes are lacking. 相似文献
A prominent trade-off in life history theory and evolution balances the costs of reproduction with those of basic somatic
needs. Hence, reproductive efforts may be reduced in environments where additional energy is required for somatic maintenance.
Here, we investigated male sperm stores in Atlantic mollies (Poecilia mexicana) from a sulfidic cave and several sulfidic and non-sulfidic surface habitats. We found significant differences among populations
in the number of sperm stripped per male, which was also correlated with differences in gonad weights. The largest sperm stores
were detected in males from non-sulfidic surface creeks, while males from a partially sulfidic surface system had lower sperm
counts, and males from completely sulfidic systems, surface as well as subterranean, had even fewer available sperm. We conclude
that the extreme environmental conditions in sulfidic habitats appear to constrain male sperm production, since hydrogen sulfide
as a naturally occurring toxin requires energy-demanding adaptations. Furthermore, we examined sperm counts of lab-reared
cave and surface mollies in response to energy limitation. Males from stock populations were placed under high and low food
treatments for a 2-week period and then stripped of sperm. Sperm counts of surface mollies tended to be reduced by low food
availability, whereas sperm counts of cave mollies did not significantly vary between food treatments, which likely points
towards a higher starvation resistance in cave mollies. 相似文献
A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters. 相似文献
The Avian Vampire Fly, Philornis downsi, has invaded the Galapagos Islands, where it causes high mortality of endemic and native landbird species, including most species of Darwin’s finches. Control methods are under development, but key information is missing about the reproductive biology of P. downsi and the behavior of flies in and near nests of their hosts. We used external and internal nest cameras to record the behavior of P. downsi adults within and outside nests of the Galapagos Flycatcher, Myiarchus magnirostris, throughout all stages of the nesting cycle. These recordings showed that P. downsi visited flycatcher nests throughout the day with higher fly activity during the nestling phase during vespertine hours. The observations also revealed that multiple P. downsi individuals can visit nests concurrently, and that there are some interactions among these flies within the nest. Fly visitation to nests occurred significantly more often while parent birds were away from the nest than in the nest, and this timing appears to be a strategy to avoid predation by parent birds. We report fly mating behavior outside the nest but not in the nest cavity. We discuss the relevance of these findings for the adaptive forces shaping P. downsi life history strategies as well as rearing and control measures.