Blocked and unblocked 5' termini in reovirus genome RNA

Uniformly ³²P-labeled, double-stranded genome RNA isolated from purified reovirus contains two types of 5′-terminal sequences. One strand contains a phosphatase-resistant 5′-terminal structure, XpppG^*pCpU, which is also present in the viral mRNA. The 5′ blocking group, X, is removed by β-elimination indicating that it is a nucleoside containing free 2′,3′-hydroxyls. G^*pC is an alkaline-resistant, 2′-O-methylated sequence. The other strand contains a phosphatase-sensitive 5′ sequence, ppGpPupPyp. The results are discussed in relation to blocked 5′-terminal structures in other viral and cellular RNAs.     

Calcium dependency of pregnant rat myometrium: comparison of circular and longitudinal muscle

The purpose of this study was to determine if the changes in spontaneous contractions of circular uterine muscle during pregnancy were related to alterations in calcium (Ca) sensitivity or dependence. Circular muscle (CM) and longitudinal muscle (LM) segments from rats on Days 16-17 of gestation and at term were compared with respect to: sensitivity of potassium (K)-induced contractions to changes in extracellular Ca, and rate and magnitude of decrease of K- and acetylcholine (ACh)-induced contractions in Ca-free medium and in methoxyverapamil (D-600). The effects of low Ca and D-600 on spontaneous electrical activity of CM were also studied. Ca sensitivity was no different in CM and LM and did not change between Day 16 and term. There was no difference in the Ca-dependence of K- or ACh-induced contractions during this time. Potassium contractions declined more rapidly than ACh contractions in Ca-free media, especially in CM. Spontaneous action potentials in CM were Ca-dependent and disappeared in low Ca or D-600 on Days 16-17 and at term. Therefore the changes in contractions of CM during pregnancy are not related directly to Ca sensitivity or dependence, but indirectly via Ca modulation of the action potentials.     

Regulation of helper T cell responses to staphylococcal superantigens

Staphylococcal superantigens (sAgs) including toxic shock syndrome toxin-1 (TSST-1) and related enterotoxins are exoproteins with unique immunobiological properties. They bind to major histocompatibility complex (MHC) class II molecules of antigen-presenting cells outside the peptide groove, and induce massive proliferation of T cells bearing specific V beta determinants. This tri-molecular interaction leads to uncontrolled release of various proinflammatory cytokines especially interferon-gamma (IFN-gamma) and tumor necrosis factor-a (TNF-alpha), the key cytokines causing sAg-mediated shock. The effector T cells involved in this hyper-immune response are predominantly of the T helper-1 (Th1) phenotype. There is also some evidence that polarization to a Th2 response with the production of classical anti-inflammatory cytokines (such as interleukins IL-4 and IL-6) also occurs. Moreover, the emergence of a novel regulatory T cell (Tr1) subset, producing mainly IL-10 but little or no IL-2 and IL-4, has recently been described following repeated sAg stimulation. In this review, the current knowledge regarding the regulation of T helper cell subsets in response to staphylococcal sAgs is critically evaluated, and the role of various cytokines which directly influence T cell differentiation and polarization is summarized. Particular emphasis is directed towards pro-inflammatory as well as anti-inflammatory and regulatory effector functions during toxic shock. Based on this review, we propose that a delayed production of IL-10 by Tr1 cells may be the most prominent driving force in the down-regulation of the Th1 hyper-immune response, and the critical determinant for the eventual recovery of the host.     

Functions of the growth arrest specific 1 gene in the development of the mouse embryo

The growth arrest specific 1 (gas1) gene is highly expressed in quiescent mammalian cells (Schneider et al., 1988, Cell 54, 787-793). Overexpression of gas1 in normal and some cancer cell lines could inhibit G(0)/G(1) transition. Presently, we have examined the functions of this gene in the developing mouse embryo. The spatial-temporal expression patterns for gas1 were established in 8.5- to 14.5-day-old embryos by immunohistochemical staining and in situ hybridization. Gas1 was found heterogeneously expressed in most organ systems including the brain, heart, kidney, limb, lung, and gonad. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were investigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression could not prevent G(0)/G(1) progression. It was determined that gas1 could only induce growth arrest if p53 was also coexpressed. In contrast, gas1 overexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressing cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing heart and limb cells, we did not find these cells preferentially distributed at G0/G1, as compared with Gas1-negative cells. However, in the 12.5 day heart and limb, we did find significantly more Gas1-expressing cells distributed at G0/G1 phase than Gas1-negative cells. These results implied that Gas1 alone, during the early stages of development, could not inhibit cell growth. This inhibition was only established when the embryo grew older. We have overexpressed gas1 in subconfluent embryonic limb cells to determine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the interaction of gas1 with Ap-1, NFkappaB, and c-myc responsive elements tagged with a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-1, NFkappaB, and c-myc activities. In contrast, gas1 overexpression in 12.5 day limb cells enhanced AP-1 response while it inhibited NFkappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital tissues. We overexpressed gas1 in these tissues and discovered that it promoted interdigital cell death. Our in situ hybridization studies of limb sections and micromass cultures revealed that, during the early stages of chondrogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage started to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cells overexpressing gas1/GFP could not participate in cartilage formation, unlike cells that just express the GFP reporter. We speculated that the reason gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary between chondrogenic and nonchondrogenic forming regions.     

Interactions of EGF and ornithine decarboxylase activity in the regulation of gastric mucus synthesis in cigarette smoke exposed rats

Cigarette smoking has been shown to aggravate ulceration and delay ulcer healing. Smokers had a lower level of mucus in their stomachs. In the present study, we examined whether cigarette smoke or its extract reduced mucus production through the suppression of epidermal growth factor (EGF) associated with the reduction of polyamine biosynthesis both in vivo and in vitro. Ornithine decarboxylase (ODC) activities and mucus synthesis were determined in rat gastric mucosa and in human MKN-28 cells. Incubation of MKN-28 cells with EGF (0.01-1.00 ng/mL) significantly increased mucus synthesis in vitro, which was accompanied by an increase of ODC activity. Removal of salivary glands decreased the circulated EGF level and induced a significant reduction of mucus-secreting layer thickness in the gastric mucosa. Cigarette smoke or its extract markedly decreased mucus synthesis in vivo and in vitro, both of which could be completely reversed by intravenous administration of EGF (20 microg/kg) in rats or co-incubation with EGF (1 and 2 ng/mL) in MKN-28 cells. However, ODC activities, which were suppressed by cigarette smoke or its extract, were unaffected by intravenous administration of EGF in rats, or only partially reversed by co-incubation with EGF in MKN-28 cells. These findings indicate that both EGF and ODC activity represent two different entities in the modulation of cigarette smoking on gastric mucus synthesis. The action of EGF on mucus synthesis may only be partially if not dependent on ODC activity in the stomach.     

Proline-rich motifs of the Na+/H+ exchanger 2 isoform. Binding of Src homology domain 3 and role in apical targeting in epithelia

The NHE2 isoform of the Na+/H+ exchanger (NHE) displays two proline-rich sequences in its C-terminal region that resemble SH3 (Src homology 3)-binding domains. We investigated whether these regions (743PPSVTPAP750, termed Pro-1, and 786VPPKPPP792, termed Pro-2) can bind to SH3 domains and whether they are essential for NHE2 function and targeting. A fusion protein containing the Pro-1 region showed promiscuous binding to SH3 domains of several proteins in vitro, whereas a Pro-2 fusion bound preferentially to domains derived from kinases. In contrast, cytoplasmic regions of NHE1, NHE3, or NHE4 failed to interact. When expressed in antiporter-deficient cells, truncated NHE2 lacking both Pro-rich regions catalyzed Na+/H+ exchange, retained sensitivity to intracellular ATP, and was activated by hyperosmolarity, resembling full-length NHE2. The role of the Pro-rich regions in subcellular targeting was examined by transfection of epitope-tagged forms of NHE2 in porcine renal epithelial LLC-PK1 cells. Both full-length and Pro-2-truncated NHE2 localized almost exclusively to the apical membrane. By contrast, a mutant devoid of both Pro-1 and Pro-2 was preferentially sorted to the basolateral surface but also accumulated intracellularly. These observations indicate that the region encompassing Pro-1 is essential for appropriate subcellular targeting of NHE2.     

Assessing distributions of estimated drug shelf lives in stability analysis

A stability study is usually conducted to ensure that a drug product can meet the approved specifications prior to its expiration dating period (shelf life). Several approaches for determination of drug shelf life assuming random batches have been proposed. In this paper, we examine sampling distributions of the estimated shelf lives proposed by Shao and Chow (1994, Biometrics 50, 753-763). An application to some stability data from the pharmaceutical industry is presented.     

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.     

Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli

Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein. Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region. Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function. An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein. E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein. Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341. Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X. Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants. The possible implications of these results for the tertiary structure of the protein are discussed.