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PubMed2007

Role of the intracellular domain of IL-7 receptor in T cell development.

Signals from the IL-7R are uniquely required for T cell development and maintenance, despite the resemblance of IL-7R to other cytokine receptors and the apparent sharing of common signaling pathways. This unique requirement could either reflect unique expression of IL-7R or IL-7, or it could indicate that the IL-7R delivers unique signals. To determine whether the IL-7R provided unique signals, we exchanged its intracellular domain with that of other cytokine receptors: IL-4R, IL-9R, and prolactin receptor (PRLR). Chimeric receptors were used to reconstitute development of IL-7R(-/-) hemopoietic progenitors by transducing the receptors in retroviral vectors. Whereas IL-7R(-/-) thymocytes are arrested at the double-negative stage, IL-4R, IL-9R, or PRLR all imparted some progression to the double-positive stage. IL-4R and PRLR gave only small numbers of thymocytes, whereas IL-9R gave robust alphabeta T cell development and reconstitution of peripheral CD4 and CD8 cells, indicating that it can duplicate many of the functions of IL-7R. However, IL-9R failed to reconstitute rearrangement of the TCRgamma locus or development of gammadelta T cells. Thus, the IL-7R signals required in the alphabeta T cell lineage (such as survival and proliferation) are not unique to this receptor, whereas rearrangement of the TCRgamma locus may require a signal that is not shared by other receptors.

Animals↗

PubMed2001

Latent acquisition of timed responses in cerebellar cortex.

Evidence indicates that rabbit eyelid conditioning is mediated by plasticity in the interpositus cerebellar nucleus and in cerebellar cortex. Although the relative contributions of these sites are not fully characterized, evidence suggests that plasticity in the cerebellar cortex influences conditioned response amplitude and timing, whereas plasticity in the interpositus nucleus is necessary or permissive for conditioned response expression. Recent empirical and computational analyses suggest that, during training, plasticity is initially established in the cerebellar cortex, whereas conditioned response expression begins later as plasticity is induced in the interpositus nucleus. We used the dependence of response timing on the interstimulus interval (ISI) to test this latent learning hypothesis. Rabbits were initially trained using a tone conditioned stimulus (CS) with a relatively long ISI to a low-criterion threshold. The relative absence of plasticity in the interpositus nucleus was then examined via reversible disconnection of the cerebellar cortex. Later, to induce plasticity in the interpositus nucleus, subjects were trained to robust levels of conditioned response expression using a shorter ISI. Reversible disconnection of the cerebellar cortex at this time confirmed the presence of robust interpositus nucleus plasticity after the second phase. Subsequent probe trials with the long CS alone then revealed double-peaked responses whose peaks were appropriately timed to the two ISIs. The results are consistent with the hypothesis that temporally specific learning occurs first in the cerebellar cortex before the appearance of conditioned responses. This latent learning is expressed only after plasticity is induced in the interpositus nucleus.

Acoustic Stimulation↗

PubMed2026

Origin flexibility governs robust ssDNA engagement by the DnaA initiator.

In model bacteria, initiation of chromosome replication requires engagement of single-stranded DNA by oligomers of the DnaA-family initiator assembled within the origin DNA. Although arrays of double-strand motifs recognized by DnaA are a general feature of the origins, the DnaA-binding single-strand elements are elucidated in only a limited number of species, and the mechanical principles governing their recognition remain elusive. Using the Alphaproteobacterium Caulobacter crescentus, we identify a previously uncharacterized GA-rich single-stranded element in the origin that directly engages DnaA oligomers and is essential for robust initiation. This element is positioned at a subkilobase distance from the DnaA oligomerization region and is brought into proximity through dynamic structural rearrangements. Moreover, DnaA oligomers exhibit an unexpectedly broad yet constrained capacity to accommodate single-stranded sequence variation. These findings provide the molecular basis for origin plasticity, highlighting how origins can diverge while preserving initiation logic.

DNA, Single-Stranded↗

PubMed2004

Saccharomyces cerevisiae Hop1 zinc finger motif is the minimal region required for its function in vitro.

Saccharomyces cerevisiae meiosis-specific HOP1, which encodes a core component of synaptonemal complex, plays a key role in proper pairing of homologous chromosomes and processing of meiotic DNA double strand breaks. Isolation and analysis of hop1 mutants indicated that these functions require Cys(371) of Hop1 embedded in a region (residues 343-378) sharing homology to a zinc finger motif (ZnF). However, the precise biochemical function of Hop1, or its putative ZnF, in these processes is poorly understood. Our previous studies revealed that Hop1 is a DNA-binding protein, showed substantially higher binding affinity for G4 DNA, and enhances its formation. We report herein that ZnF appears to be sufficient for both zinc as well as DNA-binding activities. Molecular modeling studies suggested that Hop1 ZnF differs from the previously characterized natural ZnFs. The zinc-binding assay showed that the affinity for zinc is weaker for C371S ZnF mutant compared with the wild type (WT) ZnF. Analysis of CD spectra indicated that zinc and DNA induce substantial conformational changes in WT ZnF, but not in C371S ZnF mutant. The results from a number of different experimental approaches suggested that the DNA-binding properties of ZnF are similar to those of full-length Hop1 and that interaction with DNA rich in G residues is particularly robust. Significantly, WT ZnF by itself, but not C371S mutant, was able to bind duplex DNA and promote interstitial pairing of DNA double helices via the formation of guanine quartets. Together, these results implicate a direct role for Hop1 in pairing of homologous chromosomes during meiosis.

Amino Acid Sequence↗

PubMed2001

Neuronal and glial beta-secretase (BACE) protein expression in transgenic Tg2576 mice with amyloid plaque pathology.

We measured tissue distribution and expression pattern of the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE) in the brains of transgenic Tg2576 mice that show amyloid pathology. BACE protein was expressed at high levels in brain; at lower levels in heart and liver; and at very low levels in pancreas, kidney, and thymus and was almost absent in spleen and lung when assayed by Western blot analysis. We observed strictly neuronal expression of BACE protein in the brains of nontransgenic control mice, with the most robust immunocytochemical labeling present in the cerebral cortex, hippocampal formation, thalamus, and cholinergic basal forebrain nuclei. BACE protein levels did not differ significantly between control and transgenic mice or as a result of aging. However, in the aged, 17-month-old Tg2576 mice there was robust amyloid plaque formation, and BACE protein was also present in reactive astrocytes present near amyloid plaques, as shown by double immunofluorescent labeling and confocal laser scanning microscopy. The lack of astrocytic BACE immunoreactivity in young transgenic Tg2576 mice suggests that it is not the APP overexpression but rather the amyloid plaque formation that stimulates astrocytic BACE expression in Tg2576 mice. Our data also suggest that the neuronal overexpression of APP does not induce the overexpression of its metabolizing enzyme in neurons. Alternatively, the age-dependent accumulation of amyloid plaques in the Tg2576 mice does not require increased neuronal expression of BACE. Our data support the hypothesis that neurons are the primary source of beta-amyloid peptides in brain and that astrocytic beta-amyloid generation may contribute to amyloid plaque formation at later stages or under conditions when astrocytes are activated.

Alzheimer Disease↗

PubMed2004

Single-shot two-echo technique for simultaneous measurement of GABA and creatine in the human brain in vivo.

A single-shot, two-echo method for the simultaneous detection of multiple-quantum (MQ)-filtered gamma-aminobutyric acid (GABA) and creatine (Cr) was developed and demonstrated in the human brain in vivo at 3 Tesla. The simultaneously measured Cr singlet served as a navigator for the spectral phase of GABA and any frequency shift during measurements due to drift in the static magnetic field (B(0)) or subject movement, as well as an internal concentration reference. In addition, the use of a double-band frequency-selective MQ filter for C(3) and C(4) methylene protons of GABA provided a very robust measurement of GABA, with excellent suppression of overlapping metabolites such as Cr and glutathione (GSH) in each single scan. Contamination from overlapping macromolecules was also demonstrated to be negligible with this method. The GABA-to-Cr ratio was 0.09 +/- 0.03 (mean +/- SD, N = 17) and the estimated concentration of GABA in the frontoparietal region of the human brain in vivo was 0.66 +/- 0.19 micromol/g (mean +/- SD, N = 17) with the internal reference method, and 0.69 +/- 0.18 micromol/g (mean +/- SD, N = 17) with the external reference method. The observed pattern of GABA doublet was consistent among all subjects, with a frequency separation of approximately 13 Hz.

Adult↗

PubMed2001

A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher-order DNA architectures. Further, these two small unimolecular G-quadruplexes, which are distinct from each other and from parallel-stranded G-quadruplexes, provide novel targets for ligand recognition. Our results demonstrate that the double chain reversal loop connectivity identified previously by our laboratory within the Tetrahymena telomere d(T2G4)4 quadruplex, is a robust folding topology, since it has now also been observed within the d(G2T4G2CAG2GT4G2T) quadruplex. The identification of a G-(T-T) triad and a T-T-T triple, expands on the available recognition alignments for base triads and triples.

Aptamers, Nucleotide↗

PubMed2005

Retrograde study of projections from the tuberomammillary nucleus to the dorsal raphe and the locus coeruleus in the rat.

In the first series of experiments, a retrograde tracer, WGA-apo-HRP-gold (WG), was injected into the dorsal raphe (DR) or the locus coeruleus (LC) and adenosine deaminase immunostaining was subsequently performed for the tuberomammillary nucleus (TMN) in order to investigate projections from the TMN to the two brainstem monoaminergic nuclei. Following rostral DR injections, the majority of retrogradely labeled cells were located in the dorsomedial and ventrolateral subdivisions of the TMN. At middle DR levels, midline injections resulted in labeling mainly in the ventrolateral subdivision, whereas lateral wing injections produced labeling mostly in ventral and caudal TMN subdivisions. When injections were made in the caudal DR, only a few cells were observed along the rostro-caudal extent of the TMN. On the other hand, following rostral LC injections, labeled neurons were observed mainly in ventrolateral and ventral subdivisions of TMN. For principal LC injections, labeled cells were observed mostly in ventrolateral, ventral, and caudal TMN subdivisions, whereas for injections at caudal pole of LC, only a few cells were located along the rostro-caudal extent of the TMN. In the second series of experiments, an iontophoretic injection of fluorogold (FG) into the DR was paired with a pressure injection of WG into the LC to investigate the collateral distribution of TMN axonal fibers to DR and LC. Double-labeled cells were observed in ventrolateral, ventral, and caudal TMN subdivisions. The present study indicated that there exists a robust projection from the TMN to the DR or the LC and that some TMN neurons have axon collaterals projecting to both DR and LC. The TMN neurons with such axon collaterals might provide simultaneous, possibly more efficient, way of controlling the brainstem monoaminergic nuclei, thus influencing various sleep and arousal states of the animal.

Animals↗

PubMed2005

Covalently networked monolayer-protected nanoparticle films.

Covalently networked films of nanoparticles can be assembled on various substrates from functionalized monolayer-protected clusters (MPCs) via ester coupling reactions. Exposure of a specifically modified substrate to alternating solutions of 11-mercaptoundecanoic acid exchanged and 11-mercaptoundecanol exchanged MPCs, in the presence of ester coupling reagents, 1,3-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, results in the formation of a multilayer film with ester bridges between individual nanoparticles. These films can be grown in a controlled manner to various thicknesses and exhibit certain properties that are consistent with films having other types of interparticle connectivity, including chemical vapor response behavior and quantized double layer charging. Ester coupling of MPCs into assembled films is a straightforward and highly versatile approach that results in robust films that can endure harsher chemical environments than other types of films. The stability of these covalent films is assessed and compared to other more traditional MPC film assemblies.

Journal Article↗

PubMed2006

Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation.

The primary goal of proteomics is to gain a better understanding of biological function at the protein expression level. As the field matures, numerous technologies are being developed to aid in the identification, quantification and characterization of protein expression and post-translational modifications on a near-global scale. Stable isotope labeling by amino acids in cell culture is one such technique that has shown broad biological applications. While we have recently shown the application of this technology to a model of metastatic prostate cancer, we now report a substantial improvement in quantitative analysis using a linear ion-trap Fourier transform ion cyclotron resonance mass spectrometer (LTQ FT) and novel quantification software. This resulted in the quantification of nearly 1400 proteins, a greater than 3-fold increase in comparison to our earlier study. This dramatic increase in proteome coverage can be attributed to (1) use of a double-labeling strategy, (2) greater sensitivity, speed and mass accuracy provided by the LTQ FT mass spectrometer, and (3) more robust quantification software. Finally, by using a concatenated target/decoy protein database for our peptide searches, we now report these data in the context of an estimated false-positive rate of one percent.

Arginine↗

PubMed2002

Electrochemical analysis of nucleic acids at boron-doped diamond electrodes.

Highly conductive boron-doped diamond (BDD) electrodes are well suited for performing electrochemical measurements of nucleic acids in aqueous solution under diffusion-only control. The advantageous properties of this electrodic material in this context include reproducibility and the small background currents observed at very positive potentials, along with its robustness under extreme conditions so offering promising capabilities in future applications involving thermal heating or ultrasonic treatment. tRNA, single and double stranded DNA and 2'-deoxyguanosine 5'-monophosphate (dGMP) have been studied and well defined peaks were observed in all cases, directly assignable to the electro-oxidation of deoxyguanosine monophosphate.

Boron↗

PubMed2005

Saccharomyces cerevisiae Mre11 is a high-affinity G4 DNA-binding protein and a G-rich DNA-specific endonuclease: implications for replication of telomeric DNA.

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2' DNA and the resulting protein-DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn2+-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.

DNA↗

PubMed2006

Examining the effect of residential change on intimate partner violence risk.

OBJECTIVES: To examine the relationship between residential change and a woman's subsequent risk of intimate partner violence (IPV), whether by a past or a new offender, a relationship that has not been prospectively examined to date. DESIGN: A dynamic cohort of women who recently changed residence (movers) was compared with those who did not (non-movers) for 12-month risk of IPV by a past offender and of IPV by a new offender. PARTICIPANTS AND METHODS: Secondary analysis of a linked, longitudinal National Crime Victimization Survey dataset including 10 754 recent movers and 10 236 non-movers among women aged 18-44 years. RESULTS: The risk of IPV by either a past or a new offender was almost double for women who had recently moved compared with those who had not moved. This increased risk proved to be robust, as it persisted when the data were weighted and unweighted, and when the main effect was adjusted by measured covariates. CONCLUSIONS: The apparent increase in IPV risk after residential change may be a marker of a pre-existing problem or a precursor of subsequent problems. Unlike past research that has considered residential change after abuse or as a simultaneous exposure, this study focused solely on empirically measuring the risk of IPV after a recent move. This decision has important public health ramifications: determination of IPV exposure is not always possible, whereas soliciting a woman's history of residence may be more feasible. If transience puts a woman at greater risk for victimisation by an intimate partner, increased awareness may have a vital role in protecting women who move.

Adolescent↗

PubMed1993

Perception of male drinkers as a function of their alcoholic beverage preference.

The present study examined perception of a man's likelihood of driving after drinking and his ability to do so as a function of his choice of beverage. Perceptions of his social characteristics were also examined. The sample was composed of 200 volunteer undergraduate psychology students. Equal numbers of male and female subjects read one of four vignettes which varied only in the male protagonist's choice of beverage: beer, wine, shots of distilled spirits, or cola. After reading the story, subjects answered multiple-choice questions about the character, tapping social judgments, situational judgments and drinking and driving judgments. Subject drinking habits were also assessed. The most notable result was the consistently positive perception of the protagonist portrayed as an abstainer. The lack of a consistent alcoholic beverage distinction implies that the beer-spirits double standard is far from a clear-cut discrimination. Possible explanations for results are discussed. This study questions the robustness of the alcoholic beverage type bias and reflects the need for future research.

Adult↗

PubMed2001

PD-loop technology: PNA openers at work.

A concise survey of the emerging PD-loop technology is presented, which outlines several exemplary methods with robust DNA diagnostic potential: duplex DNA capture, topological DNA labeling, nondenaturing DNA sequencing and hybridization of molecular beacons to double-stranded DNA. Advantages of these new PNA-based assays over existing techniques for sequence-specific detection and manipulation of DNA duplexes are discussed. Future prospects for the further development of PD-loop technology are highlighted.

Base Sequence↗

PubMed2001

Fatty acid specificity of hormone-sensitive lipase. Implication in the selective hydrolysis of triacylglycerols.

The selective mobilization of fatty acids from white fat cells depends on their molecular structure, in particular the degree of unsaturation. The present study was designed to examine if the release of fatty acids by hormone-sensitive lipase (HSL) in vitro i) is influenced by the amount of unsaturation, ii) depends on the temperature, and iii) could explain the selective pattern of fatty acid mobilization and notably the preferential mobilization of certain highly unsaturated fatty acids. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 35 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation from 0 to 6 double bonds was measured. Fatty acid composition of in vitro released NEFA was compared with that of fat cell triacylglycerols (TAG), the ratio % NEFA/% TAG being defined as the relative hydrolysis. The relative hydrolysis of individual fatty acids differed widely, ranging from 0.44 (24:1n-9) to 1.49 (18:1n-7) with rat HSL, and from 0.38 (24:1n-9) to 1.67 (18:1n-7) with human HSL. No major difference was observed between rat and human HSL. The relative release was dependent on the number of double bonds according to chain length. The amount of fatty acid released by recombinant rat HSL was decreased but remained robust at 4 degrees C compared with 37 degrees C, and the relative hydrolysis of some individual fatty acids was affected. The relative hydrolysis of fatty acids moderately, weakly, and highly mobilized by adipose tissue in vivo was similar and close to unity in vitro. We conclude that i) the release of fatty acids by HSL is only slightly affected by their degree of unsaturation, ii) the ability of HSL to efficiently and selectively release fatty acids at low temperature could reflect a cold adaptability for poikilotherms or hibernators when endogenous lipids are needed, and iii) the selectivity of fatty acid hydrolysis by HSL does not fully account for the selective pattern of fatty acid mobilization, but could contribute to explain the preferential mobilization of some highly unsaturated fatty acids compared with others.

Animals↗

PubMed2001

Double-blind, placebo-controlled study of single-dose amphetamine formulations in ADHD.

OBJECTIVE: To compare the efficacy and time course of single morning doses of Adderall, extended-release, and immediate-release dextroamphetamine sulfate. METHOD: Thirty-five children with attention-deficit/hyperactivity disorder, combined type, were given Adderall, immediate-release dextroamphetamine, dextroamphetamine Spansules, and placebo in a randomized, double-blind, crossover study. Behavior ratings, locomotor activity measurements, and academic measures were obtained over a period of 8 weeks. RESULTS: All three drugs exhibited robust efficacy versus placebo on nearly all measures. The effects of dextroamphetamine Spansules were less robust in the morning, particularly compared with Adderall, but they lasted 3 to 6 hours longer, depending on the measure. Although parent behavior ratings and locomotor activity showed improvements up to 12 hours after single doses of all three drugs, the number of math problems attempted and completed correctly 4 hours after dosing were only robustly increased by Spansules. CONCLUSIONS: Both immediate-release amphetamines demonstrated earlier onset of effects, but dextroamphetamine Spansules showed more sustained effects that were present on a wider range of measures.

Amphetamines↗

PubMed2025

Impact of RNA extraction on respiratory microbiome analysis using third-generation sequencing.

BACKGROUND: The respiratory microbiome, which comprises bacteria, fungi, and viruses, plays a crucial role in respiratory health and disease. However, its study is limited by the low microbial biomass in respiratory samples and the dominance of host RNA. Metatranscriptomics offers comprehensive insights into active microbial communities and their interactions with the host but requires optimized RNA extraction protocols for robust and unbiased analysis. This study evaluated two RNA extraction kits—one employing chemical lysis (CL) and another combining chemical and mechanical lysis (CML)—to determine their effectiveness for metatranscriptomic analysis of respiratory samples. RESULTS: The CML protocol significantly increased double-stranded DNA (dsDNA) library yields, leading to higher sequencing read counts for both sample types (p < 0.0001). The read length was unaffected by the lysis protocol for the BAL and NPS samples. Taxonomic profiling revealed that CML enhanced the detection of robust microorganisms, such as gram-positive bacteria and fungi, without compromising viral detection. CONCLUSIONS: The CML protocol demonstrated superior recovery of genetic material, particularly for fungi and gram-positive bacteria, making it better suited for comprehensive metatranscriptomic analyses. These findings underscore the need for tailored RNA extraction strategies on the basis of sample type and research objectives. Optimized metatranscriptomic protocols are pivotal for advancing our understanding of the respiratory microbiome and its role in health and disease.

Microbiota↗