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Testing for episodic-like memory in rats in the absence of time of day cues: replication of Babb and Crystal.

Two experiments were performed to look for evidence of episodic-like memory in rats. On each of a series of trials on an eight-arm radial maze, rats in two groups entered four open arms in Phase 1, with reward pellets on three arms and a favored reward (chocolate in Experiment 1 and cheese in Experiment 2) on the remaining arm. Phase 2 retention tests were given 30 min or 4 h after Phase 1, with all eight arms open. The four arms not entered in Phase 1 all contained reward pellets, and the three arms that contained pellets in Phase 1 were empty. In the replenish short group, the favored reward was replenished at the same location where it was found in Phase 1 at the 30 min retention interval but was absent (Experiment 1) or degraded (Experiment 2) at the 4 h retention interval. In the replenish long group, the favored reward was replenished at the 4 h retention interval but not at the 30 min retention interval. Over a number of daily trials that randomly mixed short and long delays, rats in both experiments learned to return earlier to the arm containing the favored reward at the retention interval when it was replenished than at the retention interval when it was absent or degraded. These results replicate earlier findings [Babb, S.J., Crystal, J.D., 2005, Discrimination of what, when, and where: implications for episodic-like memory in rats. Learn. Mot., 36, 177-189] and provide evidence of episodic-like memory in rats.

Analysis of Variance↗

DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication.

The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.

Bacterial Outer Membrane Proteins↗

Establishment of transcriptional competence in early and late S phase.

In animal cells, the process of DNA replication takes place in a programmed manner, with each gene region designated to replicate at a fixed time slot in S phase. Housekeeping genes undergo replication in the first half of S phase in all cell types, whereas the replication of many tissue specific genes is developmentally controlled, being late in most tissues but early in the tissue of expression. Here we employ nuclear DNA injection as an experimental system to test whether this phenomenon is due to differences in the ability to set up transcriptional competence during S phase. Our results show that, regardless of sequence, exogenous genes are a better template for transcription when injected into nuclei of cells in early as opposed to late S phase, and this expression state, once initiated, is preserved after cell division. DNA injected in late S phase is apparently repressed because it is packaged into chromatin containing deacetylated histones, and the same is true for late replicating chromosomal DNA. These findings suggest a mechanistic connection between replication timing and gene expression that might help to explain how epigenetic states can be maintained in vivo.

Acetylation↗

Replication and endoreplication in developing megakaryocytes in vitro.

To study the relation in time between replication and endoreplication and the relation between appearance of platelet-specific proteins and endoreplication in maturing megakaryocytes, peripheral blood mononuclear cells highly enriched in hematopoietic progenitors were cultured in liquid cultures and plasma clots in the presence of either interleukin-3 (IL-3) and stem cell factor (SCF) or medium conditioned by blood mononuclear cells stimulated by phytohemagglutinin (PHA). In plasma clots, megakaryocytic (MK) colonies appeared first on day 5 and reached a maximum by day 8, whereas the number of cells per colony increased until day 10, indicating that there was a single wave of MK colony formation. In liquid cultures, the first immunologically recognizable megakaryocytes appeared on day 5 and expressed GPIIb/IIIa and thrombospondin only, but all other platelet-specific protein markers appeared within 24 hours. Replating cells from liquid medium into plasma clots showed that 92 +/- 8% of day 6 GPIIb/IIIa-positive cells are capable of replicating. Their replicative potential decreased with age, however, so that between days 6 and 11, a linear correlation was noted between the logarithm of the percentage of megakaryocytes with replicative capacity and their age in culture. Replication ceased completely after day 10. In the presence of IL-3, polyploid megakaryocytes appeared at the same time that GPIIb/IIIa was expressed, and the megakaryocyte distribution into ploidy classes remained unchanged until day 20. In the presence of PHA-leukocyte conditioned medium (PHA-LCM), ploidy of megakaryocytes was shifted toward higher classes after day 6, and the process of endoreplication was completed by day 10. No changes in ploidy distribution were noted between days 10 and 20. These findings indicate that in the cohort of megakaryocytes derived from colony-forming units-megakaryocyte (CFU-MK), endoreplication can occur at an early stage of development, proceeds synchronously with replication, and is completed before the megakaryocytes exhaust their replicative potential.

Adult↗

In vivo analysis of cellular replication.

The number of previous cell replications that a metaphase cell has undergone in the presence of BrdUrd can be determined by the differential fluorescent patterns of metaphse chromosomes stained with Hoechst dye 33258. To examine if this technique could be applied to analyzing cell cycle kinetics in vivo, we infused Wistar rats with BrdUrd for 7.5-33 hr at concentrations of the nucleotide analog that did not inhibit cellular replication. Examination of the frequency of one, two, and three or more replication cycle cells as a function of BrdUrd infusion time indicates that cell replication times for rat bone marrow cells are relatively homogeneous. Analysis of this data with a computer simulation model produced a mean cell cycle duration of 9.2 hr, which is compatible with the fastest times obtained with radioisotope studies. These results support the potential of nonradioisotope analysis of cell replication in vivo.

Animals↗

Cell cycle parameters of adult rat hepatocytes in a defined medium. A note on the timing of nucleolar DNA replication.

Hepatocytes, isolated from adult (250-350 g) rats, attached and survived well in primary culture on highly diluted (less than 1 microgram/cm2) collagen gel in a synthetic medium without serum or hormones. About 20% of the cells "spontaneously" entered S phase during the first 4 days of culturing, and mitoses were easily demonstrated at the near physiological concentration (1.25 mM) of Ca++ prevailing in the medium. Cultures given 9 nM epidermal growth factor (EGF) and 20 nM insulin 20 h after inoculation showed vigorous DNA synthesis and mitotic activity. Autoradiography of such cells exposed to [3H]thymidine allowed the determination of the following cell cycle parameters: Lag period from EGF/insulin stimulation till onset of increased DNA synthesis, 17 h; rate of entry into S phase (kG1/S), 0.028/h; duration of S phase, 8.4 h; duration of G2 phase, 2.7 h. The peak DNA synthesis (pulse labelling index, 24%) and peak mitotic activity (mitotic index, 1.7%) occurred 35 and 43 h, respectively, after the stimulation with EGF/insulin. These values are comparable to those reported during the in vivo compensatory hyperplasia following partial hepatectomy of adult rats. A marked variation of the intranuclear [3H]thymidine pulse labelling pattern was noted: During the first 1.5 h of the S phase, the labelling was extranucleolar and during the last 1.5 h chiefly nucleolar. The cells survived well in the absence of glucocorticoid, whose effect on cell cycle parameters therefore could be studied. Dexamethasone (25-250 nM) did not appreciably affect the durations of S phase and G2 phase or the pattern of preferential extranucleolar and nucleolar DNA synthesis within the S phase.

Animals↗

Embryonic development in the pig up to the 64-cell stage, with reference to DNA replication and cell cycle times from the third cleavage division.

Preimplantation cleaving-stage embryos were recovered from Dutch Landrace (DL) and F1 Dutch Landrace x Great Yorkshire (DL x GY) gilts for which the time of insemination and ovulation were known. Embryonic cell counts were performed, usually after brief in vitro culture to estimate DNA synthesis. Special attention was given to the 4-cell stage. Beyond this stage, the mean cell cycle time was 14 hours for DL and 17 hours for F1 gilts. Generally, high indices of DNA synthesis were obtained (more than 60% of nuclei). There was prominent within and between gilt variability with regard to embryonic cell numbers. Gilts are especially heterogeneous with respect to the length of the 4-cell stage. The G 2 M phase of the 4-cell stage takes approximately 3.5 hours. Especially for F1 gilts, the age of the spermatozoa at the moment of ovulation was not related to the rate of cleavage and/or embryonic death. It is postulated that variability in the length of the 4-cell stage is reflected in genetic activation of the embryos at this stage and subsequently influences embryonic survival.

Journal Article↗

Construction of an optimal destructive sampling design for noncompartmental AUC estimation.

Based on toxicokinetic studies of a destructive sampling design, this work was aimed at selecting the number of time points, their locations, and the number of replicates per time point in order to obtain the most accurate and precise noncompartmental estimate of the area under the concentration-time curve (AUC). From a prior population pharmacokinetic model, the design is selected to minimize the scaled mean squared error of AUC. Designs are found for various sample sizes, number of time points, and a distribution of animals across time points from being very unbalanced to balanced. Their efficiencies are compared both theoretically and based on simulations. An algorithm has been implemented for this purpose using the symbolic resolution and numerical minimization capabilities of Mathematica and an example of its use is provided. This method provides efficient tools for constructing, validating, and comparing optimal sampling designs for destructive sampled toxicokinetic studies.

Algorithms↗

Alterations in the time of X chromosome replication induced by 5-azacytidine in a patient with 48,XXXY/47,XXY.

It has recently been shown that 5-azacytidine (5-azaC) can induce altered replication patterns of the late-replicating X chromosome in normal female cells. This has been demonstrated by bromodeoxyuridine labelling of cells late in the S phase. In the present study the same method was applied to the lymphocytes of a Klinefelter patient (48,XXXY/47,XXY). Significant 5-azaC-induced changes in the replication of the entire inactive X chromosome, from late to early, were found in the lymphocytes of this patient. These results indicate that hypomethylating agents can not only alter the replication of individual bands, but also change the gross replication schedule of multiple inactive X chromosomes in the presence of a Y chromosome.

Azacitidine↗

Timing of nucleolar DNA replication in Amoeba proteus.

Light- and electron-microscope autoradiography have been used to follow the incorporation of [3H]thymidine at different stages during the interphase of synchronously growing populations of Amoeba proteus. Two main patterns were found for tritiated thymidine incorporation, i.e. DNA synthesis. The major incorporation was in the central region of the nucleus, but a lesser degree of incorporation occurred in the nucleolar region. The bulk of this nucleolar DNA was found to be late replicating, i.e. it replicated during the G2 phase.

Amoeba↗

Effect of chromium picolinate on growth and serum and carcass traits of growing-finishing pigs.

Three experiments were conducted to evaluate chromium picolinate (CrPic) in growing-finishing pigs. Treatments were replicated four times within each experiment with three pigs per replicate in Exp. 1 and four pigs per replicate in Exp. 2 and 3. Average initial weights were 37.8, 30.5, and 22.4 kg in Exp. 1, 2, and 3, respectively. In Exp. 1, the basal corn-soybean meal diet (B, 120% NRC Lys) was supplemented with 0, 25, 50, 100, or 200 ppb of Cr from CrPic. Daily gain was increased (Cr cubic, P < .02) and serum cholesterol decreased (Cr cubic, P < .08) by addition of CrPic. In Exp. 2, the basal diet was supplemented with 0, 100, 200, 400, or 800 ppb of Cr from CrPic. Daily gain and ADFI were decreased (Cr linear, P < .05) by CrPic. Serum cholesterol also was decreased (Cr quadratic, P < .05) by CrPic. Longissimus muscle area (LMA) and percentage of muscling (MUS) were increased (Cr quadratic, P < .01) and 10th rib fat (TRF) was decreased (Cr quadratic, P < .01) by CrPic. In Exp. 3, pigs were allotted to the following treatments: 1) B, 2) B + 1,467 ppb of picolinate (Pic), 3) B + 200 ppb of Cr from CrCl3.6H2O, 4) B + 1,467 ppb of Pic + 200 ppb of Cr from CrCl3.6H2O, 5) B + 100 ppb of Cr from CrPic, or 6) B + 200 ppb of Cr from CrPic. Longissimus muscle area and MUS were increased (P < .01) and TRF decreased (P < .01) in pigs fed CrPic but not in pigs fed CrCl3.6H2O and(or) Pic.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

Automatic quantitative radiometric assay of bacterial metabolism.

In a two-compartment scintillation vial, suspensions of bacteria were cultured with 1 muCi of [U-14C] glucose and the released 14C02 was measured continuously, cumulatively, and automatically in a liquid-scintillation counter modified to maintain sample temperature at 37 degrees C. We could follow the metabolism of bacterial populations through their early phase of exponential growth with good precision. The data were obtained conveniently, with use of conventional reagents, glassware, and counting equipment. From analysis of the exponential portion of the curves for cumulative activity vs. time, we could measure cell replication rate precisely in units of time. The resulting values were demonstrably independent of some common experimental variables, including the number of bacteria in the inoculum and counting system sensitivity. Sensitivity of the bacteria to antibiotics was measured to within a few percent by noting the relative prolongation of replication time in the presence of those inhibitors. The digital data from the scintillation counter are susceptible to on- or off-line computer analysis, thus providing the prospect for a totally-automated analytical system. The method shows promise for the mechanized quantitative analysis of bacterial growth, and its inhibition.

Anti-Bacterial Agents↗

Properties of adeno-associated virus (type 1) replicated in rodent cells by murine adenovirus.

We report for the first time the replication of infectious adeno-associated virus type 1 (AAV-1) in rodent cells [primary mouse kidney (PMK) and mouse L929 cells] using murine adenovirus (MAV) as a helper virus and also the production of AAV-I virus antigen by herpes simplex virus type I (HSV-I) with its temperature-sensitive mutant ts 200 in mouse neuroblastoma (NB) cells. The infectious AAV virions produced by MAV on L cells had a buoyant density of 1.41 2ml in caesium chloride gradients.

Adenoviridae↗