While induced FoxP3(+) T cells (iTreg cells) are promising cellular therapeutics to treat inflammatory diseases, a limitation in utilizing iTreg cells prepared in vitro is their low stability in inflammatory conditions. Progesterone (P4) is an immune regulatory nuclear hormone with a potent Treg induction activity. We reasoned that this function of progesterone would be utilized to generate iTreg cells with highly suppressive activity and improved stability in vivo. Here we generated iTreg cells with progesterone in vitro and found that progesterone generates iTreg cells that are highly stable in inflammatory conditions. Moreover, P4-induced iTreg cells highly express latency-associated peptide TGF-β1 and are efficient in regulating inflammation in multiple tissues, whereas control iTreg cells induced with TGF-β1 alone are less stable and ineffective in suppressing inflammation. The function of progesterone in inducing iTreg cells with improved regulatory activity is associated with the function of P4 in suppressing the mTOR pathway. Moreover, the function of progesterone in inducing FoxP3(+) T cells is decreased but not completely abolished on nuclear progesterone receptor-deficient T cells, suggesting that both nuclear and nonnuclear progesterone receptors are involved in mediating the function. We conclude that P4 can be utilized to generate iTreg cells with a high therapeutic potential in treatment of tissue inflammation.

Olfactory bulb (OB) glomeruli have long been considered functional units in the processing of odor information. Recently, it has been shown that axons from olfactory receptor neurons (ORNs) expressing the same odorant receptor gene converge onto two or a few topographically fixed glomeruli in the OB. The interactions between ORN axons, mitral/tufted cell dendrites, juxtaglomerular (JG) cells, and glial cells during the development of glomeruli is of great importance in light of this receptor gene glomerular topography in the primary olfactory projection. To explore the development of mammalian olfactory glomeruli, we investigated the relationships among radial glia (RG), astrocytes, ORNs, JG cells, mitral/tufted cell dendrites, and olfactory Schwann cells throughout embryonic and early postnatal development. Our results indicate that glomeruli are formed through an invariant sequence of cellular events: (1) pioneering ORN axons contact the rostral telencephalon at approximately E11-14, which coincides with the onset of morphologic changes in telencephalic RG; (2) at E15-16, RG branch and begin to form two plexuses, one located in the subventricular layer and the other superficial to the presumptive mitral cell layer; (3) at E17-18, ORN axons accumulate in a dense band superficial to the outer radial glia plexus; (4) at E19-20, processes from RG and astrocytes begin to ramify to form glial tufts, or glial glomeruli. Coincident with the formation of these glial glomeruli, ORN axons intermingle with the glial processes and form proto-glomeruli; (5) at E21 to P0, JG cells begin to migrate into position surrounding glomeruli, (6) and at P4, the apical tuft of mitral cells becomes restricted to a single glomerulus. Interestingly, glomerular development also occurs in a distinct rostral to caudal gradient. That is, glomeruli in the rostral OB develop earlier than those in the caudal OB, but the sequence of cellular events at any point in the bulb is invariant. These results demonstrate that glomeruli are formed in a specific spatiotemporal sequence beginning with ORN axon-glia contacts, then JG cell arrival, and finally mitral cell apical dendrite restriction.

The aim of this study was to analyse the electroencephalogram (EEG) background activity of Alzheimer's disease (AD) patients using multiscale entropy (MSE). MSE is a recently developed method that quantifies the regularity of a signal on different time scales. These time scales are inspected by means of several coarse-grained sequences formed from the analysed signals. We recorded the EEGs from 19 scalp electrodes in 11 AD patients and 11 age-matched controls and estimated the MSE profile for each epoch of the EEG recordings. The shape of the MSE profiles reveals the EEG complexity, and it suggests that the EEG contains information in deeper scales than the smallest one. Moreover, the results showed that the EEG background activity is less complex in AD patients than control subjects. We found significant differences between both subject groups at electrodes F3, F7, Fp1, Fp2, T5, T6, P3, P4, O1 and O2 (p-value < 0.01, Student's t-test). These findings indicate that the EEG complexity analysis performed on deeper time scales by MSE may be a useful tool in order to increase our knowledge of AD.

The goals of this work were to establish a reproducible and effective model of apoptosis in a cell line derived from advanced prostate cancer and to study the role of the caspase family of proteases in mediating apoptosis in this system. The study involved the use of the prostate cancer cell line LNCaP. Apoptosis was induced using the hydroxymethyl glutaryl CoA reductase inhibitor, lovastatin, and was evaluated by agarose gel electrophoresis of genomic DNA, morphological criteria, and terminal deoxynucleotidyl transferase-mediated nick end labeling. Caspases were studied by catalytic activity, mRNA induction, and protein processing. Lovastatin (30 microM) was an effective inducer of apoptosis, causing changes that were evident after 48 h and essentially complete after 96-120 h of treatment. These effects were prevented by the simultaneous addition of mevalonate (300 microM) to the culture medium. Lovastatin induced a proteolytic activity that was able to cleave the enzyme poly(ADP-ribose) polymerase and the substrate Z-DEVD-AFC, which is modeled after the P1-P4 amino acids of the poly(ADP-ribose) polymerase cleavage site. Caspase-7, but not caspase-3, underwent proteolytic activation during lovastatin-induced apoptosis, an effect prevented by mevalonate. Caspase-7 was the only detected interleukin 1beta converting enzyme family protease with DEVD cleavage activity that exhibited lovastatin-induced mRNA up-regulation. Again, mevalonate blocked this effect. Lovastatin-induced apoptosis also was prevented when the caspase inhibitors Z-DEVD-CH2F or Z-VAD-CH2F (100 microM) where added to the medium. These studies have identified lovastatin as a powerful inducer of apoptosis in the cell line LNCaP. Caspase activation was a necessary event for LNCaP cells to undergo apoptosis during treatment with lovastatin. Of the caspases tested, only caspase-7 underwent proteolytic activation after stimulation with lovastatin. Identification of caspase-7 as a potential mediator of lovastatin-induced apoptosis broadens our knowledge of the molecular events associated with programmed cell death in a cell line derived from prostatic epithelium.

A new method for quantifying irregularity of EEGs is proposed in this study. The entropy, an information measure, determines the uniformity of proportion distribution. The peakedness or flatness of the distribution of the EEG power spectrum, representing EEG rhythmicity, can be measured by the entropy, because the power spectrum consists of proportions of power at each frequency. The irregularity of the EEG was measured by the entropy of the power spectrum, called an irregularity index (II). The II was obtained from the power spectrum at F3, F4, C3, C4, P3, P4, O1 and O2 during rest and mental arithmetic in 10 normal subjects. Relative band powers of delta, theta, alpha and beta bands and alpha peak frequency were also obtained. EEGs during rest were significantly more irregular anteriorly than in the occipital areas. Alpha activity was also more irregular in the anterior region. A greater degree of EEG desynchronization during mental arithmetic was found over the left hemisphere and the right occipital area. The II was more sensitive to such desynchronization than alpha band power and alpha peak frequency. The differences in spectral structures between rest and mental arithmetic conditions, mainly over the left hemisphere, were also confirmed by the Kullback-Leibler information.

OBJECTIVE

The aim of this study was to analyse the regularity of the EEG background activity of Alzheimer's disease (AD) patients to test the hypothesis that the irregularity of the AD patients' EEG is lower than that of age-matched controls.

METHODS

We recorded the EEG from 19 scalp electrodes in 10 AD patients and 8 age-matched controls and estimated the Approximate Entropy (ApEn). ApEn is a non-linear statistic that can be used to quantify the irregularity of a time series. Larger values correspond to more complexity or irregularity. A spectral analysis was also performed.

RESULTS

ApEn was significantly lower in the AD patients at electrodes P3 and P4 (P < 0.01), indicating a decrease of irregularity. We obtained 70% sensitivity and 100% specificity at P3, and 80% sensitivity and 75% specificity at P4. Results seemed to be complementary to spectral analysis.

CONCLUSIONS

The decreased irregularity found in the EEG of AD patients in the parietal region leads us to think that EEG analysis with ApEn could be a useful tool to increase our insight into brain dysfunction in AD. However, caution should be applied due to the small sample size.

CONCLUSIONS

This article represents a first step in demonstrating the feasibility of ApEn for recognition of EEG changes in AD.

There is a current high demand for nontypable Haemophilus influenzae (NTHi) vaccines. Various options for the composition of such vaccines are possible. Decisions about the vaccine composition have to take into account the antigenic variability of NTHi, so even complex immunogens such as whole bacteria would preferentially have a tailor-made antigenic composition. We will present a summary of NTHi vaccine development, describing research efforts from SmithKline Beecham and other laboratories. Currently, major (P1, P2, P4, P5) and minor (P6, D15, TbpA/B, ellipsis) outer membrane proteins, LPS, adhesins (HMW, Hia, pili, P5) are being studied. Preclinical results with LPD, P5 (LB1) and OMP26 from our laboratories will be described including the use of animal models of otitis and lung infection.

It has been proposed that the cleaved form of protease-activated receptor 3 (PAR3) acts as a cofactor for thrombin cleavage and activation of PAR4 on murine platelets, but the molecular basis of this physiologically important effect remains elusive. X-ray crystal structures of murine thrombin bound to extracellular fragments of the murine receptors PAR3 ((38)SFNGGPQNTFEEFPLSDIE(56)) and PAR4 ((51)KSSDKPNPR downward arrow GYPGKFCANDSDTLELPASSQA(81), downward arrow = site of cleavage) have been solved at 2.0 and 3.5 A resolution, respectively. The cleaved form of PAR3, traced in the electron density maps from Gln-44 to Glu-56, makes extensive hydrophobic and electrostatic contacts with exosite I of thrombin through the fragment (47)FEEFPLSDIE(56). Occupancy of exosite I by PAR3 allosterically changes the conformation of the 60-loop and shifts the position of Trp-60d approximately 10 A with a resulting widening of the access to the active site. The PAR4 fragment, traced entirely in the electron density maps except for five C-terminal residues, clamps Trp-60d, Tyr-60a, and the aryl-binding site of thrombin with Pro-56 and Pro-58 at the P2 and P4 positions and engages the primary specificity pocket with Arg-59. The fragment then leaves the active site with Gly-60 and folds into a short helical turn that directs the backbone away from exosite I and over the autolysis loop. The structures demonstrate that thrombin activation of PAR4 may occur with exosite I available to bind cofactor molecules, like the cleaved form of PAR3, whose function is to promote substrate diffusion into the active site by allosterically changing the conformation of the 60-loop.

OBJECTIVE

This study examined distances covered at low (1-2 ms(-2)), moderate (2-3 ms(-2)) and high (>3 ms(-2)) acceleration (L(ACC), M(ACC) and H(ACC) respectively) and deceleration (L(DEC), M(DEC), and H(DEC) respectively) during competitive football games. Temporal and transient patterns of acceleration and deceleration were also examined.

METHODS

Observational, repeated measures.

METHODS

Thirty-six professional male professional footballers were monitored using a 10 Hz non-differential global positioning system (NdGPS). Match data was organised into six 15 min periods (P1: 1-15 min, P2: 16-30 min, P3: 31-45 min, P4: 46-60 min, P5: 61-75 min, and P6: 76-90 min) for analysis of temporal patterns, and into eighteen 5 min periods for analysis of transient patterns. ANOVA with Bonferroni post hoc tests were used to identify significant (p<0.05) differences between periods.

RESULTS

Distance covered at L(ACC), M(ACC), H(ACC), L(DEC), M(DEC), and H(DEC) was 424±75 m, 242±25 m, 178±38 m, 365±54 m, 210±23 m and 162±29 m respectively. Between period decrements ranged from 8.0% to 13.2% from P1 to P3, 9.2% to 16.3% from P4 to P6, and from 14.9% to 21.0% from P1 to P6. Following PEAK H(ACC) (148% of mean 5 min H(ACC)), H(ACC) at 5POST was 10.4% lower than mean (p<0.01).

CONCLUSIONS

Time-dependent reductions in distances covered suggest that acceleration and deceleration capability are acutely compromised during match play. Further, the occurrence of transient fatigue may be supported by the findings that HACC and HDEC performance following PEAK was approximately 10% lower than mean values.

Using quantitative RNase protection assays, we have monitored the appearance of mRNAs generated during lytic infection of tightly synchronized murine cells by the autonomous parvovirus minute virus of mice. Our results demonstrate that transcripts from the P4 promoter can be detected prior to those from the P39 promoter, providing direct evidence for a temporal order of expression between the two parvovirus promoters.

Sex differences have been reported in a variety of affective and neurodegenerative disorders that involve dysfunctional dopamine (DA) neurotransmission. In addition, there is evidence for differences in sensitivity to the abuse-related effects of psychostimulants across the menstrual cycle which may result from effects of ovarian hormones on DA function. The goal of the present study was to extend previous work examining menstrual cycle-related changes in DA D2 receptor availability in humans to drug-naive female cynomolgus monkeys (n=7) using the selective D2-like receptor ligand [(18)F]fluoroclebopride (FCP) and a high-resolution microPET P4 scanner. Menstrual cycle phase was characterized by daily vaginal swabs and measurements of serum progesterone levels. PET studies were conducted once during the luteal phase and once during the follicular phase. Regions of interest in the caudate nucleus, putamen, and cerebellum were defined on coregistered MRIs. Distribution volumes were calculated for FCP in each structure and the distribution volume ratio (DVR) for both brain regions relative to the cerebellum was used as a measure of D2 receptor availability. FCP DVRs were significantly higher in the luteal phase compared to the follicular phase in both the caudate nucleus (11.7% difference, p=0.02) and putamen (11.6% difference, p=0.03). These findings extend earlier work in humans and suggest that changes in DA receptor availability may be involved in the variation in symptoms of various neuropsychiatric disorders across the menstrual cycle, including differences in sensitivity to the abuse-related effects of stimulants.

The aquatic monocotyledonous plant Spirodela polyrhiza was labelled with [33P]Pi for short periods under non-equilibrium conditions. An InsP6 fraction was obtained and dissected by using enantiospecific (enzymic) and non-enantiospecific (chemical) means to determine the relative labelling of individual phosphate substituents on the inositol ring of InsP6. Phosphates in positions D-1, -2, -3, -4, -5 and -6 contained approx. 21%, 32-39%, 9-10%, 14-16%, 19-23% and 16-18% of the label respectively. We conclude from the foregoing, together with identities [described in the preceding paper, Brearley and Hanke (1996) Biochem. J. 314, 215-225] of inositol phosphates found in this plant at a developmental stage associated with massive accumulation of InsP6, that synthesis of InsP6 from myo-inositol proceeds according to the sequence Ins3P->>Ins(3,4)P2->>Ins(3,4,6)P3->>Ins(3,4,5,6)P4->>Ins(1,3,4,5,6 ) P5->>InsP6 in Spirodela polyrhiza. These results represent the first description of the synthetic sequence to InsP6 in the plant kingdom and the only comprehensive description of endogenous inositol phosphates in any plant tissue. The sequence described differs from that reported in the slime mould Dictyostelium discoideum.

Changes in the size and shape of the corpus callosum (CC)--and in number, size, and structure of callosal axons--between embryonic day 38 (E38) and postnatal day 150 (P150) were studied by light and electron microscope in 25 kittens. The development of the CC was divided into three phases: 1. Embryonic development (E38, 53, 58): At E38, only part of the body of the CC was formed. At E53 and E58, the CC was still very short, but its different parts (genu, body, and splenium) had formed. The cross-sectional callosal area (CCA) was 5.4 mm2 at E53 and 5.6 mm2 at E58. The CC contained 46.3 and 56.4 million axons at E53 and E58 respectively. Mean axon diameters were 0.26 micron at E53 and 0.27 micron at E58. 2. Early postnatal development (P4, 9, 15, 18, 21, 26): The CC at P4 was much longer than at E58 and still slightly elongated during this phase; CCA reached 8.55 mm2 at P4 and 8.88 mm2 at P26. There was a substantial axonal loss (66.8 million at P4 and 52.6 million at P26). From P15 onward, premyelinated and myelinated axons were seen. Mean axon diameter increased from 0.30 micron at P4 to 0.33 micron at P26. 3. Late postnatal development (P39, 57, 92, 107, 150). The CC grew dramatically in both length and thickness, the latter especially in the genu. CCA was 10.1 mm2 at P39 and 15.3 mm2 at P150. The number of axons still decreased (46.5 million at P39 and 31.9 million at P150). The growth of the CCA paralleled the increase of myelinated axons (0.5% at P26 and 29.6% at P150 and in the mean axon diameters (0.34 micron at P39 and 0.42 micron at P150). A number of axonal ultrastructural peculiarities (electron-dense bodies, large vacuoles, lamellated bodies, etc., including those mentioned below) were noticed; their frequency at different ages was estimated as the percent of total axons. Interestingly, accumulations of vesicles inside axons increased from 4.1% at E53 to 8.9% at P26, dropped to 0.2% at P39, and remained below 1% thereafter. Swollen mitochondria increased from 0.2% at E53 to 0.9% at P26 and dropped to 0.06% (on the average) from P39 onward. Accumulations of vesicles and swollen mitochondria increased during the phase of rapid axonal elimination; thus, they may indicate axonal retraction and/or degeneration. Microglia-gitter cells and astrocytes showing signs of phagocytosis were found during the embryonic and early postnatal development and may be involved in axon elimination.

It is well established that GABAA-mediated postsynaptic potentials are excitatory in many brain regions during embryonic and early postnatal life. The pre-Bötzinger complex (PBC) in the brainstem is an essential component of the respiratory rhythm-generating network, where GABAA-mediated inhibition plays a critical role in generating a stable respiratory rhythm in adult animals. In the present study, using the perforated patch technique, we investigated the maturation of GABAA receptor-mediated effects on rhythmically active PBC neurons and on the motor output in slice preparations from P0-15 neonatal mice. The reversal potential of GABAA receptor-mediated current (EGABA-A) switched from depolarizing to hyperpolarizing within the first postnatal week. EGABA-A was -13.7 +/- 9.8 mV at P0, then it changed to -44.8 +/- 7.0 mV at P2 and -71.5 +/- 6.8 mV at P4. Perfusion of bicarbonate-free saline has no detectable influence on EGABA-A, indicating that a lack of Cl- extrusion during P0-3 is mainly responsible for early GABAA-ergic excitation. At the network level, blockade of GABAA receptors with bicuculline did not significantly change the frequency of rhythmic bursts recorded from hypoglossal nerve roots before P3, whereas it increased the coefficient of variation. After P3, bicuculline increased burst frequency with little effect on the coefficient of variation. Thus, chloride-mediated inhibition, which appears in PBC neurons after P3, coincides with the appearance of GABAA-mediated modulation of the respiratory rhythm. GABAA receptor-activated inhibition may therefore be necessary for frequency modulation in the respiratory network beginning on the fourth postnatal day in the mouse brainstem.

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron (SMN) protein due to the functional loss of the SMN1 gene and the inability of its paralog, SMN2, to fully compensate due to reduced exon 7 splicing efficiency. Since SMA patients have at least one copy of SMN2, drug discovery campaigns have sought to identify SMN2 inducers. C5-substituted quinazolines increase SMN2 promoter activity in cell-based assays and a derivative, RG3039, has progressed to clinical testing. It is orally bioavailable, brain-penetrant and has been shown to be an inhibitor of the mRNA decapping enzyme, DcpS. Our pharmacological characterization of RG3039, reported here, demonstrates that RG3039 can extend survival and improve function in two SMA mouse models of varying disease severity (Taiwanese 5058 Hemi and 2B/- SMA mice), and positively impacts neuromuscular pathologies. In 2B/- SMA mice, RG3039 provided a >600% survival benefit (median 18 days to >112 days) when dosing began at P4, highlighting the importance of early intervention. We determined the minimum effective dose and the associated pharmacokinetic (PK) and exposure relationship of RG3039 and DcpS inhibition ex vivo. These data support the long PK half-life with extended pharmacodynamic outcome of RG3039 in 2B/- SMA mice. In motor neurons, RG3039 significantly increased both the average number of cells with gems and average number of gems per cell, which is used as an indirect measure of SMN levels. These studies contribute to dose selection and exposure estimates for the first studies with RG3039 in human subjects.

Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v)) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v) (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pH(v) under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v) dysregulation in a neo1(ts) mutant restored viability whereas cholesterol accumulation in human NPC1(-/-) fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.

During the early stages of pregnancy, fertilized embryos must attach to the uterine epithelium, invade into the underlying uterine stroma, and the stroma must then differentiate in a process termed decidualization in order for a successful pregnancy to be initiated. The steroid hormone progesterone (P4) is an integral mediator of these early pregnancy events, exerting its effects via the progesterone receptor (PR). Insights gained from the use of mouse models and genomic profiling has identified many of the key molecules enlisted by PR to execute the paradigm of early pregnancy. This review describes several of the molecules through which the PR exerts its pleiotropic effects including ligands, receptors, chaperones, signaling proteins and transcription factors. Understanding these molecules and their concatenation is of vital importance to our ability to clinically treat reproductive health problems like infertility and endometriosis.

The sequence logo for DNA binding sites of the bacteriophage P1 replication protein RepA shows unusually high sequence conservation ( approximately 2 bits) at a minor groove that faces RepA. However, B-form DNA can support only 1 bit of sequence conservation via contacts into the minor groove. The high conservation in RepA sites therefore implies a distorted DNA helix with direct or indirect contacts to the protein. Here I show that a high minor groove conservation signature also appears in sequence logos of sites for other replication origin binding proteins (Rts1, DnaA, P4 alpha, EBNA1, ORC) and promoter binding proteins (sigma(70), sigma(D) factors). This finding implies that DNA binding proteins generally use non-B-form DNA distortion such as base flipping to initiate replication and transcription.

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.

A key bottleneck in RNA structural studies is preparing milligram quantities of RNA, and current techniques have changed little in over a decade. To address this, we have developed an affinity tag-based purification method of RNA oligonucleotides. The tag is attached to the 3'-end of almost any desired RNA sequence, allowing for the rapid and specific removal of the RNA of interest directly from in vitro transcription reactions using an affinity column to which a specific RNA-binding protein has been attached. Following a wash, the RNA of interest is eluted by the addition of imidazole to the column, activating a mutant HdeltaV ribozyme incorporated into the tag. The affinity column can then be rapidly regenerated using conditions that release the protein-RNA tag interaction without denaturing the protein. To demonstrate that this method rapidly generates high-quality RNA, we have transcribed, purified, and generated diffraction-quality crystals of a mutant form of the Tetrahymena thermophila P4-P6 domain in a 48-h time period.

We have previously demonstrated that bacterial infection (Escherichia coli) in neonatal rats is associated with impaired memory in a fear-conditioning task in adulthood. This impairment, however, is only observed if a peripheral immune challenge (lipopolysaccharide; LPS) is administered around the time of learning. We used a brief separation/handling paradigm to determine if the adult memory impairment associated with neonatal-infection could be prevented. Naturally occurring variations in maternal care promote striking variations in offspring cognitive development, and handling paradigms are used to manipulate the quality and quantity of maternal care. Rats were injected on post natal (P) day 4 with E. coli or PBS, and half from each group were handled for 15 min/day from P4 to 20. All rats were then tested in adulthood. Neonatal handling of rats infected as neonates prevented the increase in microglial cell marker reactivity within the hippocampus, and the exaggerated brain IL-1beta production to LPS normally produced by the infection. Thus, these neural processes were now comparable to levels of non-infected PBS controls. Furthermore, handling completely prevented LPS-induced memory impairment in a context-fear task in adult rats infected as neonates. Finally, neonatal handling dramatically improved spatial learning and memory and decreased anxiety in rats treated early with PBS, but had no beneficial effect on these measures in rats infected as neonates. Taken together, these data suggest that maternal care may profoundly influence neuroinflammatory processes in adulthood, and that infection may also prevent maternal care influences on cognition later in life.

Addition of diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) to permeabilized G1-arrested baby hamster kidney cells resulted in the stimulation of DNA synthesis. No stimulation was observed in cells from exponentially growing cultures. The Ap4A-stimulated [3H]dTTP incorporation was inhibited by nalidixic acid, daunomycin, chloroquine diphosphate, EDTA, and N-ethylmaleimide. It was dose-dependent in regard to the amount of permeabilized cells and of Ap4A. Numerous replication eyes were formed in the DNA molecules of stimulated cells. Pulse-chase experiments showed that the synthesis of DNA was discontinuous, resulting in the appearance of approximately 4S Okazaki fragments and their ligation to high molecular weight DNA. These results strongly suggest that Ap4A stimulated the initiation of DNA synthesis in baby hamster kidney cells that had been arrested in G1 by serum deprival.

The anteroventral periventricular nucleus of the preoptic region (AVPV) represents a key site for hormonal feedback on gonadotropin secretion. It plays a critical role in the neural control of luteinizing hormone secretion and contains high densities of neurons that express receptors for estrogen and progesterone. In this study in situ hybridization was used to examine the expression of mRNAs encoding the estrogen (ER) and progesterone (PR) receptors in the AVPV during the estrous cycle. ER gene expression fluctuated during the cycle with the lowest levels of ER mRNA observed in animals killed on the afternoon of proestrus, and the highest levels present in animals killed during metestrus. This apparent inverse relationship between circulating levels of estradiol (E2) and ER mRNA levels in AVPV neurons was supported by the observation that treatment of ovariectomized rats with E2 suppressed expression of ER mRNA in the AVPV. The influence of progesterone (P4) on ER expression was less pronounced, but a significant increase in ER mRNA in the AVPV was detected 3 h after treatment with P4. In contrast, PR mRNA levels were highest in the AVPV during diestrus and lowest on the morning of proestrus suggesting that PR expression in the AVPV is regulated in a complex manner that may reflect the combined regulatory effects of E2 and P4. E2 treatment caused a dramatic induction of PR mRNA in the AVPV, but P4 did not affect PR mRNA expression acutely, although PR mRNA appears to be attenuated in the AVPV 27 h after P4 treatment. These findings suggest that ovarian steroid hormones regulate ER and PR gene expression in the AVPV during the estrous cycle, which may represent molecular events that contribute to cyclic changes in the responsiveness of AVPV neurons to steroid hormones.

In cerebral cortex of rats treated with increasing doses of LiCl, the relative concentrations of Ins(1)P, Ins(4)P and Ins(5)P (when InsP is a myo-inositol phosphate) are approx. 10:1:0.2 at all doses. In rats treated with LiCl followed by increasing doses of pilocarpine a similar relationship occurs. myo-Inositol-1-phosphatase (InsP1ase) from bovine brain hydrolyses Ins(1)P, Ins(4)P and Ins(5)P at comparable rates, and these substrates have similar Km values. The hydrolysis of Ins(4)P is inhibited by Li+ to a greater degree than is hydrolysis of Ins(1)P and Ins(5)P. D-Ins(1,4,5)P3 and D-Ins(1,4)P2 are neither substrates nor inhibitors of InsP1ase. A dialysed high-speed supernatant of rat brain showed a greater rate of hydrolysis of Ins(1)P than of D-Ins(1,4)P2 and a lower sensitivity of the bisphosphate hydrolysis to LiCl, as compared with the monophosphate. That enzyme preparation produced Ins(4)P at a greater rate than Ins(1)P when D-Ins(1,4)P2 was the substrate. The amount of D-Ins(3)P [i.e. L-Ins(1)P, possibly from D-Ins(1,3,4)P3] is only 11% of that of D-Ins(1)P on stimulation with pilocarpine in the presence of Li+. DL-Ins(1,4)P2 was hydrolysed by InsP1ase to the extent of about 50%; both Ins(4)P and Ins(1)P are products, the former being produced more rapidly than the latter; apparently L-Ins(1,4)P2 is a substrate for InsP1ase. Li+, but not Ins(2)P, inhibited the hydrolysis of L-Ins(1,4)P2. The following were neither substrates nor inhibitors of InsP1ase; Ins(1,6)P2, Ins(1,2)P2, Ins(1,2,5,6)P4, Ins(1,2,4,5,6)P5, Ins(1,3,4,5,6)P5 and phytic acid. myo-Inositol 1,2-cyclic phosphate was neither substrate nor inhibitor of InsP1ase. We conclude that the 10-fold greater tissue contents of Ins(1)P relative to Ins(4)P in both stimulated and non-stimulated rat brain in vivo are the consequence of a much larger amount of PtdIns metabolism than polyphosphoinositide metabolism under these conditions.