Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR

A sensitive, labor-saving, and easily automatable nonradioactive procedure named APEX-FCS (amplified probe extension detected by fluorescence correlation spectroscopy) has been established to detect specific in vitro amplification of pathogen genomic sequences. As an example, Mycobacterium tuberculosis genomic DNA was subjected to PCR amplification with the Stoffel fragment of Thermus aquaticus DNA polymerase in the presence of nanomolar concentrations of a rhodamine-labeled probe (third primer), binding to the target in between the micromolar amplification primers. The probe becomes extended only when specific amplification occurs. Its low concentration avoids false-positives due to unspecific hybridization under PCR conditions. With increasing portion of extended probe molecules, the probe’s average translational diffusion properties gradually change over the course of the reaction, reflecting amplification kinetics. Following PCR, this change from a stage of high to a stage of low mobility can directly be monitored during a 30-s measurement using a fluorescence correlation spectroscopy device. Quantitation down to 10 target molecules in a background of 2.5 μg unspecific DNA without post-PCR probe manipulations could be achieved with different primer/probe combinations. The assay holds the promise to concurrently perform amplification, probe hybridization, and specific detection without opening the reaction chamber, if sealable foils are used.

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na+/glucose cotransporter. The Na+/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na+/Cl−/γ-aminobutyric acid, Na+/iodide and H+/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.

cAMP receptor protein–cAMP plays a crucial role in glucose–lactose diauxie by activating the major glucose transporter gene in Escherichia coli

The inhibition of β-galactosidase expression in a medium containing both glucose and lactose is a typical example of the glucose effect in Escherichia coli. We studied the glucose effect in the lacL8UV5 promoter mutant, which is independent of cAMP and cAMP receptor protein (CRP). A strong inhibition of β-galactosidase expression by glucose and a diauxic growth were observed when the lacL8UV5 cells were grown on a glucose–lactose medium. The addition of isopropyl β-d-thiogalactoside to the culture medium eliminated the glucose effect. Disruption of the crr gene or overproduction of LacY also eliminated the glucose effect. These results are fully consistent with our previous finding that the glucose effect in wild-type cells growing in a glucose–lactose medium is not due to the reduction of CRP–cAMP levels but is due to the inducer exclusion. We found that the glucose effect in the lacL8UV5 cells was no longer observed when either the crp or the cya gene was disrupted. Evidence suggested that CRP–cAMP may not enhance directly the lac repressor action in vivo. Northern blot analysis revealed that the mRNA for ptsG, a major glucose transporter gene, was markedly reduced in a Δcrp or Δcya background. The constitutive expression of the ptsG gene by the introduction of a multicopy plasmid restored the glucose effect in Δcya or Δcrp cells. We conclude that CRP–cAMP plays a crucial role in inducer exclusion, which is responsible for the glucose–lactose diauxie, by activating the expression of the ptsG gene.

Network formation of lipid membranes: Triggering structural transitions by chain melting

Phospholipids when dispersed in excess water generally form vesicular membrane structures. Cryo-transmission and freeze-fracture electron microscopy are combined here with calorimetry and viscometry to demonstrate the reversible conversion of phosphatidylglycerol aqueous vesicle suspensions to a three-dimensional structure that consists of extended bilayer networks. Thermodynamic analysis indicates that the structural transitions arise from two effects: (i) the enhanced membrane elasticity accompanying the lipid state fluctuations on chain melting and (ii) solvent-associated interactions (including electrostatics) that favor a change in membrane curvature. The material properties of the hydrogels and their reversible formation offer the possibility of future applications, for example in drug delivery, the design of structural switches, or for understanding vesicle fusion or fission processes.

Cleavage planes in frog eggs are altered by strong magnetic fields

Early cleavages of Xenopus embryos were oriented in strong, static magnetic fields. Third-cleavage planes, normally horizontal, were seen to orient to a vertical plane parallel with a vertical magnetic field. Second cleavages, normally vertical, could also be oriented by applying a horizontal magnetic field. We argue that these changes in cleavage-furrow geometries result from changes in the orientation of the mitotic apparatus. We hypothesize that the magnetic field acts directly on the microtubules of the mitotic apparatus. Considerations of the length of the astral microtubules, their diamagnetic anisotropy, and flexural rigidity predict the required field strength for an effect that agrees with the data. This observation provides a clear example of a static magnetic-field effect on a fundamental cellular process, cell division.

Jun NH2-terminal kinase is constitutively activated in T cells transformed by the intracellular parasite Theileria parva

When T cells become infected by the parasite Theileria parva, they acquire a transformed phenotype and no longer require antigen-specific stimulation or exogenous growth factors. This is accompanied by constitutive interleukin 2 (IL-2) and IL-2 receptor expression. Transformation can be reversed entirely by elimination of the parasites using the specific drug BW720c. Extracellular signal-regulated kinase and jun NH2-terminal kinase (JNK) are members of the mitogen-activated protein kinase family, which play a central role in the regulation of cellular differentiation and proliferation and also participate in the regulation of IL-2 and IL-2 receptor gene expression. T. parva was found to induce an unorthodox pattern of mitogen-activated protein kinase expression in infected T cells. JNK-1 and JNK-2 are constitutively active in a parasite-dependent manner, but have altered properties. In contrast, extracellular signal-regulated kinase-2 is not activated even though its activation pathway is functionally intact. Different components of the T cell receptor (TCR)-dependent signal transduction pathways also were examined. The TCRζ or CD3ɛ chains were found not to be phosphorylated and T. parva-transformed T cells were resistant to inhibitors that block the early steps of T cell activation. Compounds that inhibit the progression of T cells to proliferation, however, were inhibitory. Our data provide the first example, to our knowledge, for parasite-mediated JNK activation, and our findings strongly suggest that T. parva not only lifts the requirement for antigenic stimulation but also entirely bypasses early TCR-dependent signal transduction pathways to induce continuous proliferation.

Expression of hepatocyte nuclear factor 6 in rat liver is sex-dependent and regulated by growth hormone

Growth hormone (GH) binding to its receptor modulates gene transcription by influencing the amount or activity of transcription factors. In the rat, GH exerts sexually dimorphic effects on liver gene transcription through its pattern of secretion which is intermittent in males and continuous in females. The expression of the CYP2C12 gene coding for the female-specific cytochrome P450 2C12 protein is dependent on the continuous exposure to GH. To identify the transcription factor(s) that mediate(s) this sex-dependent GH effect, we studied the interactions of the CYP2C12 promoter with liver nuclear proteins obtained from male and female rats and from hypophysectomized animals treated or not by continuous GH infusion. GH treatment induced the binding of a protein that we identified as hepatocyte nuclear factor (HNF) 6, the prototype of a novel class of homeodomain transcription factors. HNF-6 competed with HNF-3 for binding to the same site in the CYP2C12 promoter. This HNF-6/HNF-3 binding site conveyed both HNF-6- and HNF-3-stimulated transcription of a reporter gene construct in transient cotransfection experiments. Electrophoretic mobility shift assays showed more HNF-6 DNA-binding activity in female than in male liver nuclear extracts. Liver HNF-6 mRNA was barely detectable in the hypophysectomized rats and was restored to normal levels by GH treatment. This work provides an example of a homeodomain-containing transcription factor that is GH-regulated and also reports on the hormonal regulation of HNF-6.

Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.

Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots—many of them at subpicomole amounts—were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Statistical significance of protein structure prediction by threading

In this study, we estimate the statistical significance of structure prediction by threading. We introduce a single parameter ɛ that serves as a universal measure determining the probability that the best alignment is indeed a native-like analog. Parameter ɛ takes into account both length and composition of the query sequence and the number of decoys in threading simulation. It can be computed directly from the query sequence and potential of interactions, eliminating the need for sequence reshuffling and realignment. Although our theoretical analysis is general, here we compare its predictions with the results of gapless threading. Finally we estimate the number of decoys from which the native structure can be found by existing potentials of interactions. We discuss how this analysis can be extended to determine the optimal gap penalties for any sequence-structure alignment (threading) method, thus optimizing it to maximum possible performance.

Building a dictionary for genomes: Identification of presumptive regulatory sites by statistical analysis

The availability of complete genome sequences and mRNA expression data for all genes creates new opportunities and challenges for identifying DNA sequence motifs that control gene expression. An algorithm, “MobyDick,” is presented that decomposes a set of DNA sequences into the most probable dictionary of motifs or words. This method is applicable to any set of DNA sequences: for example, all upstream regions in a genome or all genes expressed under certain conditions. Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter ones of various lengths, eliminating the need for a separate set of reference data to define probabilities. We have built a dictionary with 1,200 words for the 6,000 upstream regulatory regions in the yeast genome; the 500 most significant words (some with as few as 10 copies in all of the upstream regions) match 114 of 443 experimentally determined sites (a significance level of 18 standard deviations). When analyzing all of the genes up-regulated during sporulation as a group, we find many motifs in addition to the few previously identified by analyzing the subclusters individually to the expression subclusters. Applying MobyDick to the genes derepressed when the general repressor Tup1 is deleted, we find known as well as putative binding sites for its regulatory partners.

Production, specificity, and functionality of monoclonal antibodies to specific peptide–major histocompatibility complex class II complexes formed by processing of exogenous protein

Several unanswered questions in T cell immunobiology relating to intracellular processing or in vivo antigen presentation could be approached if convenient, specific, and sensitive reagents were available for detecting the peptide–major histocompatibility complex (MHC) class I or class II ligands recognized by αβ T cell receptors. For this reason, we have developed a method using homogeneously loaded peptide–MHC class II complexes to generate and select specific mAb reactive with these structures using hen egg lysozyme (HEL) and I-Ak as a model system. mAbs specific for either HEL-(46–61)–Ak or HEL-(116–129)–Ak have been isolated. They cross-react with a small subset of I-Ak molecules loaded with self peptides but can nonetheless be used for flow cytometry, immunoprecipitation, Western blotting, and intracellular immunofluorescence to detect specific HEL peptide–MHC class II complexes formed by either peptide exposure or natural processing of native HEL. An example of the utility of these reagents is provided herein by using one of the anti-HEL-(46–61)–Ak specific mAbs to visualize intracellular compartments where I-Ak is loaded with HEL-derived peptides early after antigen administration. Other uses, especially for in vivo tracking of specific ligand-bearing antigen-presenting cells, are discussed.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Formation of temporal-feature maps by axonal propagation of synaptic learning

Computational maps are of central importance to a neuronal representation of the outside world. In a map, neighboring neurons respond to similar sensory features. A well studied example is the computational map of interaural time differences (ITDs), which is essential to sound localization in a variety of species and allows resolution of ITDs of the order of 10 μs. Nevertheless, it is unclear how such an orderly representation of temporal features arises. We address this problem by modeling the ontogenetic development of an ITD map in the laminar nucleus of the barn owl. We show how the owl's ITD map can emerge from a combined action of homosynaptic spike-based Hebbian learning and its propagation along the presynaptic axon. In spike-based Hebbian learning, synaptic strengths are modified according to the timing of pre- and postsynaptic action potentials. In unspecific axonal learning, a synapse's modification gives rise to a factor that propagates along the presynaptic axon and affects the properties of synapses at neighboring neurons. Our results indicate that both Hebbian learning and its presynaptic propagation are necessary for map formation in the laminar nucleus, but the latter can be orders of magnitude weaker than the former. We argue that the algorithm is important for the formation of computational maps, when, in particular, time plays a key role.