Real-time single-molecule fluorescence detection using confocal and near-field scanning optical microscopy has been applied to elucidate the nature of the “on–off” blinking observed in the Ser-65 → Thr (S65T) mutant of the green fluorescent protein (GFP). Fluorescence time traces as a function of the excitation intensity, with a time resolution of 100 μs and observation times up to 65 s, reveal the existence of a nonemissive state responsible for the long dark intervals in the GFP. We find that excitation intensity has a dramatic effect on the blinking. Whereas the time during which the fluorescence is on becomes shorter as the intensity is increased, the off-times are independent of excitation intensity. Statistical analysis of the on- and off-times renders a characteristic off-time of 1.6 ± 0.2 s and allows us to calculate a transition yield of ≈0.5 × 10−5 from the emissive to the nonemissive state. The saturation excitation intensity at which on- and off-times are equal is ≈1.5 kW/cm2. On the basis of the single-molecule data we calculate an absorption cross section of 6.5 × 10−17 cm2 for the S65T mutant. These results have important implications for the use of the GFP to follow dynamic processes in time at the single-molecular level.
Archeological analysis of faunal remains and of lithic and bone tools has suggested that hunting of medium to large mammals was a major element of Neanderthal subsistence. Plant foods are almost invisible in the archeological record, and it is impossible to estimate accurately their dietary importance. However, stable isotope (δ13C and δ15N) analysis of mammal bone collagen provides a direct measure of diet and has been applied to two Neanderthals and various faunal species from Vindija Cave, Croatia. The isotope evidence overwhelmingly points to the Neanderthals behaving as top-level carnivores, obtaining almost all of their dietary protein from animal sources. Earlier Neanderthals in France and Belgium have yielded similar results, and a pattern of European Neanderthal adaptation as carnivores is emerging. These data reinforce current taphonomic assessments of associated faunal elements and make it unlikely that the Neanderthals were acquiring animal protein principally through scavenging. Instead, these findings portray them as effective predators.
Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of Ptet promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2S-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range...
Birkett, Michael A.; Campbell, Colin A. M.; Chamberlain, Keith; Guerrieri, Emilio; Hick, Alastair J.; Martin, Janet L.; Matthes, Michaela; Napier, Johnathan A.; Pettersson, Jan; Pickett, John A.; Poppy, Guy M.; Pow, Eleanor M.; Pye, Barry J.; Smart, Lesle
Fonte: The National Academy of SciencesPublicador: The National Academy of Sciences
cis-Jasmone, or (Z)-jasmone, is well known as a component of plant volatiles, and its release can be induced by damage, for example during insect herbivory. Using the olfactory system of the lettuce aphid to investigate volatiles from plants avoided by this insect, (Z)-jasmone was found to be electrophysiologically active and also to be repellent in laboratory choice tests. In field studies, repellency from traps was demonstrated for the damson-hop aphid, and with cereal aphids numbers were reduced in plots of winter wheat treated with (Z)-jasmone. In contrast, attractant activity was found in laboratory and wind tunnel tests for insects acting antagonistically to aphids, namely the seven-spot ladybird and an aphid parasitoid. When applied in the vapor phase to intact bean plants, (Z)-jasmone induced the production of volatile compounds, including the monoterpene (E)-β-ocimene, which affect plant defense, for example by stimulating the activity of parasitic insects. These plants were more attractive to the aphid parasitoid in the wind tunnel when tested 48 h after exposure to (Z)-jasmone had ceased. This possible signaling role of (Z)-jasmone is qualitatively different from that of the biosynthetically related methyl jasmonate and gives a long-lasting effect after removal of the stimulus. Differential display was used to compare mRNA populations in bean leaves exposed to the vapor of (Z)-jasmone and methyl jasmonate. One differentially displayed fragment was cloned and shown by Northern blotting to be up-regulated in leaf tissue by (Z)-jasmone. This sequence was identified by homology as being derived from a gene encoding an α-tubulin isoform.
We report a strategy (called “tethering”) to discover low molecular weight ligands (≈250 Da) that bind weakly to targeted sites on proteins through an intermediary disulfide tether. A native or engineered cysteine in a protein is allowed to react reversibly with a small library of disulfide-containing molecules (≈1,200 compounds) at concentrations typically used in drug screening (10 to 200 μM). The cysteine-captured ligands, which are readily identified by MS, are among the most stable complexes, even though in the absence of the covalent tether the ligands may bind very weakly. This method was applied to generate a potent inhibitor for thymidylate synthase, an essential enzyme in pyrimidine metabolism with therapeutic applications in cancer and infectious diseases. The affinity of the untethered ligand (Ki≈1 mM) was improved 3,000-fold by synthesis of a small set of analogs with the aid of crystallographic structures of the tethered complex. Such site-directed ligand discovery allows one to nucleate drug design from a spatially targeted lead fragment.
A significant proportion of familial breast cancers cannot be explained by mutations in the BRCA1 or BRCA2 genes. We applied a strategy to identify predisposition loci for breast cancer by using mathematical models to identify early somatic genetic deletions in tumor tissues followed by targeted linkage analysis. Comparative genomic hybridization was used to study 61 breast tumors from 37 breast cancer families with no identified BRCA1 or BRCA2 mutations. Branching and phylogenetic tree models predicted that loss of 13q was one of the earliest genetic events in hereditary cancers. In a Swedish family with five breast cancer cases, all analyzed tumors showed distinct 13q deletions, with the minimal region of loss at 13q21-q22. Genotyping revealed segregation of a shared 13q21 germ-line haplotype in the family. Targeted linkage analysis was carried out in a set of 77 Finnish, Icelandic, and Swedish breast cancer families with no detected BRCA1 and BRCA2 mutations. A maximum parametric two-point logarithm of odds score of 2.76 was obtained for a marker at 13q21 (D13S1308, θ = 0.10). The multipoint logarithm of odds score under heterogeneity was 3.46. The results were further evaluated by simulation to assess the probability of obtaining significant evidence in favor of linkage by chance as well as to take into account the possible influence of the BRCA2 locus...
Poly(ADP-ribose) polymerase (PARP) is implicated in the
maintenance of genomic integrity, given that inhibition or depletion of
this enzyme increases genomic instability in cells exposed to genotoxic
agents. We previously showed that immortalized fibroblasts derived from
PARP−/− mice exhibit an unstable tetraploid population,
and partial chromosomal gains and losses in PARP−/− mice
and immortalized fibroblasts are accompanied by changes in the
expression of p53, Rb, and c-Jun, as well as other proteins. A
tetraploid population has also now been detected in primary fibroblasts
derived from PARP−/− mice. Oligonucleotide microarray
analysis was applied to characterize more comprehensively the
differences in gene expression between asynchronously dividing primary
fibroblasts derived from PARP−/− mice and their wild-type
littermates. Of the 11,000 genes monitored, 91 differentially expressed
genes were identified. The loss of PARP results in down-regulation of
the expression of several genes involved in regulation of cell cycle
progression or mitosis, DNA replication, or chromosomal processing or
assembly. PARP deficiency also up-regulates genes that encode
extracellular matrix or cytoskeletal proteins that are implicated in
cancer initiation or progression or in normal or premature aging. These
results provide insight into the mechanism by which PARP deficiency
impairs mitotic function...
IL-1β and its endogenous receptor antagonist (IL-1Ra)
are rapidly induced by seizures in the rodent hippocampus. Exogenously
applied IL-1β prolongs seizures in an IL-1R type I-mediated manner.
This effect depends on N-methyl-d-aspartate
receptor activation. We report here that intrahippocampal application
of recombinant IL-1Ra or its selective endogenous
overexpression in astrocytes under the control of glial acidic
fibrillary protein promoter potently inhibits motor and
electroencephalographic seizures induced by bicuculline methiodide in
mice. Accordingly, transgenic mice show a reduced seizure-related
c-fos mRNA expression in various forebrain areas
compared with their wild-type littermates. Recombinant IL-1Ra was
ineffective in mice deficient in IL-1R type I, having per
se a delayed onset to generalized convulsions. These results
demonstrate that IL-1Ra mediates potent anticonvulsant effects acting
on IL-1R type I and suggest that the balance between brain IL-1β and
IL-1Ra represents a crucial mechanism to control seizure
The miniaturization process applied to rubredoxins generated a
class of peptide-based metalloprotein models, named METP
transfer protein). The crystal structure
of Desulfovibrio vulgaris rubredoxin was selected as a
template for the construction of a tetrahedral
(Sγ-Cys)4 iron-binding site. Analysis of the
structure showed that a sphere of 17 Å in diameter, centered on the
metal, circumscribes two unconnected approximately C2
symmetry related β-hairpins, each containing the
-Cys-(Aaa)2-Cys- sequence. These observations provided a
starting point for the design of an undecapeptide, which self assembles
in the presence of tetrahedrally coordinating metal ions. The METP
peptide was synthesized in good yield by standard methodologies.
Successful assembly of the METP peptide with Co(II), Zn(II),
Fe(II/III), in the expected 2:1 stoichiometry, was proven by
UV-visible and circular dichroism spectroscopies. UV-visible analysis
of the metal complexes indicated the four Cys ligands tetrahedrally
arrange around the metal ion, as designed. Circular dichroism
measurements on both the free and metal-bound forms revealed that the
metal coordination drives the peptide chain to fold into a turned
conformation. NMR characterization of the Zn(II)-METP complex fully
supported the structure of the designed model. These results prove that
METP reproduces the main features of rubredoxin.
A new approach to the study of DNA/protein interactions has been
opened through the recent advances in the manipulation of single DNA
molecules. These allow the behavior of individual molecular motors to
be studied under load and compared with bulk measurements. One example
of such a motor is the DNA polymerase, which replicates DNA. We
measured the replication rate by a single enzyme of a stretched single
strand of DNA. The marked difference between the elasticity of single-
and double-stranded DNA allows for the monitoring of replication in
real time. We have found that the rate of replication depends strongly
on the stretching force applied to the template. In particular, by
varying the load we determined that the biochemical steps limiting
replication are coupled to movement. The replication rate increases at
low forces, decreases at forces greater than 4 pN, and ceases when the
single-stranded DNA substrate is under a load greater than ≈20 pN.
The decay of the replication rate follows an Arrhenius law and
indicates that multiple bases on the template strand are involved in
the rate-limiting step of each cycle. This observation is
consistent with the induced-fit mechanism for error detection
We present a coupled two-way clustering approach to gene microarray
data analysis. The main idea is to identify subsets of the genes and
samples, such that when one of these is used to cluster the other,
stable and significant partitions emerge. The search for such subsets
is a computationally complex task. We present an algorithm, based on
iterative clustering, that performs such a search. This analysis is
especially suitable for gene microarray data, where the contributions
of a variety of biological mechanisms to the gene expression levels are
entangled in a large body of experimental data. The method was applied
to two gene microarray data sets, on colon cancer and leukemia. By
identifying relevant subsets of the data and focusing on them we were
able to discover partitions and correlations that were masked and
hidden when the full dataset was used in the analysis. Some of these
partitions have clear biological interpretation; others can serve to
identify possible directions for future research.
Gene knockout technology has provided a powerful tool for functional analyses of genes expressed preferentially in a particular tissue. Given marked similarities between human and mouse skin, such studies with epidermally expressed genes have often provided valuable insights into human genetic skin disorders. Efficient silencing of a specified gene in a temporally regulated and epidermal-specific fashion could extend functional analyses to broadly expressed genes and increase the categories of human skin disorders to which parallels could be drawn. We have generated transgenic mice expressing Cre and a fusion protein between Cre recombinase and the tamoxifen responsive hormone-binding domain of the estrogen receptor (CreERtam) under the control of the human keratin 14 (K14) promoter. This promoter is strongly active in dividing cells of epidermis and some other stratified squamous epithelia. With K14–Cre, transgenic embryos recombine genetically introduced loxP sequences efficiently and selectively in the genomes of keratinocytes that reside in embryonic day 14.5 skin, tongue, and esophagus. With K14–CreERtam, postnatal transgenic mice show no Cre activity until tamoxifen is administered. If orally administered, tamoxifen activates keratinocyte-specific CreERtam...
Although extensive effort has been applied toward
understanding the mechanism by which enediynes cleave DNA, a continuous
assay for this phenomenon is still lacking. In fact, with the exception
of assays for DNase, continuous assays for most DNA cleavage events are
unavailable. This article describes the application of “molecular
break lights” (a single-stranded oligonucleotide that adopts a
stem-and-loop structure and carries a 5′-fluorescent moiety, a
3′-nonfluorescent quenching moiety, and an appropriate cleavage site
within the stem) to develop the first continuous assay for cleavage of
DNA by enediynes. Furthermore, the generality of this approach is
demonstrated by using the described assay to directly compare the DNA
cleavage by naturally occurring enediynes [calicheamicin and
esperamicin), non-enediyne small molecule agents (bleomycin,
methidiumpropyl–EDTA–Fe(II), and EDTA–Fe(II]), as well as the
restriction endonuclease BamHI. Given the simplicity,
speed, and sensitivity of this approach, the described methodology
could easily be extended to a high throughput format and become a new
method of choice in modern drug discovery to screen for novel
protein-based or small molecule-derived DNA cleavage agents.
Mathematical models have been developed to explore the population
dynamics of viral diseases among wildlife. However, assessing the
predictions stemming from these models with wildlife databases adequate
in size and temporal duration is uncommon. An epizootic of raccoon
rabies that began in the mid-Atlantic region of the United States in
the late 1970s has developed into one of the largest and most extensive
in the history of wildlife rabies. We analyzed the dynamics of local
epizootics at the county level by examining a database spanning more
than 20 years and including 35,387 rabid raccoons. The size, number,
and periodicity of rabies epizootics among raccoons were compared with
predictions derived from a susceptible, exposed, infectious, and
recovered model of raccoon rabies [Coyne, J., Smith, G. & McAllister,
F. E. (1989) Am. J. Vet. Res. 50,
2148–2154]. After our methods for defining epizootics were applied to
solutions of the model, the time series revealed recurrent epizootics
in some counties, with a median first epizootic period of 48 months.
Successive epizootics declined in size and the epizootic period
progressively decreased. Our reanalysis of the model predicted the
initial-epizootic period of 4–5 years...
The hair follicle is an epidermal derivative that undergoes cycles
of growth, involution, and rest. The hair cycle has well-orchestrated
kinetics regulated by interactions between mesenchymal and epithelial
cells, although the intracellular signals remain unclear. We previously
established keratinocyte-specific Stat3-disrupted mice, by which we
demonstrated that signal transducer and activator of transcription 3
(Stat3) is required for wound healing and anagen progression in the
hair cycle. Growth factor-dependent migration of Stat3-disrupted
keratinocytes was severely impaired, suggesting that not only wound
healing but also telogen-to-anagen progression required organized
keratinocyte migration in response to mesenchymal stimuli. In the
present study, to examine whether Stat3 activation in keratinocytes is
a prerequisite for hair cycle progression, we applied methods for
experimental anagen induction to Stat3-disrupted mice. It was
demonstrated that anagen was successfully induced in Stat3-disrupted as
well as wild-type mice by chemical or mechanical stimulation, i.e., by
topical application of phorbol 12-myristate 13-acetate (PMA) or by hair
plucking, respectively. This result indicated that anagen in these
methods occurred in the absence of Stat3. Furthermore...
Fluorescence correlation microscopy (FCM) was applied to characterize fusion proteins of the green fluorescent protein (GFP) on the cellular as well as molecular level within seconds in an integrated instrument. FCM combines the inherent sensitivity and high spatial resolution of fluorescence correlation spectroscopy with fluorescence imaging and micropositioning, thereby providing a spectrum of molecular information in the cellular context. Signatures of characteristic parameters derived from the autocorrelation functions served to distinguish a GFP fusion protein of the epidermal growth factor receptor from GFP fluorescence in the endoplasmic reticulum and cytoplasm. Diffusion constants measured for free transiently expressed GFP reproduced values reported previously with other techniques. The accessible concentration range extends from millions to only a few thousand molecules per cell, with single molecule detectability in the femtoliter detection volume. The detailed molecular characterization offered by FCM is fully compatible with automation in sample identification and detection, offering new possibilities for highly integrated high-throughput screening.
We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside/RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS–MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408...
HIV infection is accompanied by an early immune dysfunction limiting host control of virus and likely contributing to difficulties in achieving a successful vaccine against HIV. We report here that the HIV Tat protein is strongly immunosuppressive, both immediately after immunization of mice with soluble protein (sTat), and in seroconverting humans, and propose that Tat-induced suppression cripples immune surveillance to HIV infection. We show that macrophages are sensitive to sTat stimulation at concentrations 1,000-fold lower (500 pM) than T cells, and this stimulation is accompanied by the immunosuppressive induction of Fas ligand on the macrophage. T cell proliferative defects induced by sTat in vitro can be completely (at lower concentrations of sTat) or partially (at higher concentrations) reversed by antagonists to Fas/Fas ligand interaction. We further report a method to preserve immunogenicity while inactivating Tat immunosuppression through oxidation, which advances the use of oxidized Tat as a component of an anti-HIV vaccine. These observations define additional methods to study the immunosuppressive functions of sTat that now may be rapidly applied to primary isolates from individuals with differing clinical courses. Our findings have immediate relevance for vaccine development...
This report describes the integration of laser-scanning fluorometric cytometry and nonseparation ligand-binding techniques to provide new assay methods adaptable to miniaturization and high-throughput screening. Receptor-bound, cyanine dye-labeled ligands, [Cy]ligands, were discriminated from those free in solution by measuring the accumulated fluorescence associated with a receptor-containing particle. To illustrate the various binding formats accommodated by this technique, saturation- and competition-binding analyses were performed with [Cy]ligands and their cognate receptors expressed in CHO cells or as fusion proteins coated on polystyrene microspheres. We have successfully applied this technique to the analysis of G protein-coupled receptors, cytokine receptors, and SH2 domains. Multiparameter readouts from ligands labeled separately with Cy5 and Cy5.5 demonstrate the simultaneous analysis of two target receptors in a single well. In addition, laser-scanning cytometry has been used to assay enzymes such as phosphatases and in the development of single-step fluorescent immunoassays.
This study stemmed from the observation that the brain of human as well as nonhuman primates is capable of forming and memorizing remarkably accurate internal representations of the dynamics of the arm. These dynamics establish a functional relation between applied force and ensuing arm motion, a relation that generally is quite complex and nonlinear. Current evidence shows that the motor control system is capable of adapting to perturbing forces that depend on motion variables such as position, velocity, and acceleration. The experiments we report here were aimed at establishing whether or not the motor system also may adapt to forces that depend explicitly on time rather than on motion variables. Surprisingly, the experiments suggest a negative answer. When asked to compensate for a predictable and repeated time-varying pattern of disturbing forces, subjects learned to counteract the disturbance by producing forces that did not depend on time but on the velocity and the position of the arm. We conclude from this evidence that time and time-dependent dynamics are not explicitly represented within the neural structures that are responsible for motor adaptation. Although our findings are not sufficient to rule out the presence of a timing structure within the central nervous system...