Tumor necrosis factor-alpha stimulates gastric epithelial cell proliferation

TNF-alpha is a cytokine produced during gastric mucosal injury. We examined whether TNF-alpha could promote mucosal repair by stimulation of epithelial cell proliferation and explored further the underlying mechanisms in a rat gastric mucosal epithelial cell line (RGM-1). TNF-alpha treatment (1-10 ng/ml) for 12 or 24 h significantly increased cell proliferation but did not induce apoptosis in RGM-1 cells. TNF-alpha treatment significantly increased cytosolic phospholipase A(2) and cyclooxygenase-2 (COX-2) protein expression and PGE(2) level but did not affect the protein levels of EGF, basic fibroblast growth factor, and COX-1 in RGM-1 cells. The mRNA of TNF receptor (TNF-R) 2 but not of TNF-R1 was also increased. Dexamethasone dose dependently inhibited the stimulatory effect of TNF-alpha on cell proliferation, which was associated with a significant decrease in cellular COX-2 expression and PGE(2) level. A selective COX-2 inhibitor 3-(3-fluorophenyl)-4-[4-(methylsulfonyl)phenyl]-5,5-dimethyl-(5)H-furan-2-one (DFU) by itself had no effect on basal cell proliferation but significantly reduced the stimulatory effect of TNF-alpha on RMG-1 cells. Combination of dexamethasone and DFU did not produce an additive effect. PGE(2) significantly reversed the depressive action of dexamethasone on cell proliferation. These results suggest that TNF-alpha plays a regulatory role in epithelial cell repair in the gastric mucosa via the TNF-alpha receptor and activation of the arachidonic acid/PG pathway.     

Medullary taste responses are modulated by the bed nucleus of the stria terminalis

Previous studies have shown a modulatory influence of limbic forebrain areas, such as the central nucleus of the amygdala and lateral hypothalamus, on the activity of taste-responsive cells in the nucleus of the solitary tract (NST). The bed nucleus of the stria terminalis (BST), which receives gustatory afferent information, also sends descending axons to the NST. The present studies were designed to investigate the role of the BST in the modulation of NST gustatory activity. Extracellular action potentials were recorded from 101 taste-responsive cells in the NST of urethane-anesthetized hamsters and analyzed for a change in excitability following bilateral electrical stimulation of the BST. The response of NST taste cells to stimulation of the BST was predominately inhibitory. Orthodromic inhibitory responses were observed in 29 of 101 (28.7%) NST taste-responsive cells, with four cells inhibited bilaterally. An increase in excitability was observed in seven of the 101 (6.9%) NST taste cells. Of the 34 cells showing these responses, 25 were modulated by the ipsilateral BST and 15 by the contralateral; four were inhibited bilaterally and two inhibited ipsilaterally and excited contralaterally. The duration of inhibitory responses (mean = 177.9 ms) was significantly longer than that of excitatory responses (35.4 ms). Application of subthreshold electrical stimulation to the BST during taste trials inhibited or excited the taste responses of every BST-responsive NST cell tested with this protocol. NST neurons that were most responsive to sucrose, NaCl, citric acid or quinine hydrochloride were all affected by BST stimulation, although citric acid-best cells were significantly more often modulated and NaCl-best less often modulated than expected by chance. These results combine with excitatory and inhibitory modulation of NST neurons by the insular cortex, lateral hypothalamus and central nucleus of the amygdala to demonstrate extensive centrifugal modulation of brainstem gustatory neurons.     

Iron-sulfur cluster biogenesis in chloroplasts. Involvement of the scaffold protein CpIscA

The chloroplast contains many iron (Fe)-sulfur (S) proteins for the processes of photosynthesis and nitrogen and S assimilation. Although isolated chloroplasts are known to be able to synthesize their own Fe-S clusters, the machinery involved is largely unknown. Recently, a cysteine desulfurase was reported in Arabidopsis (Arabidopsis thaliana; AtCpNifS) that likely provides the S for Fe-S clusters. Here, we describe an additional putative component of the plastid Fe-S cluster assembly machinery in Arabidopsis: CpIscA, which has homology to bacterial IscA and SufA proteins that have a scaffold function during Fe-S cluster formation. CpIscA mRNA was shown to be expressed in all tissues tested, with higher expression level in green, photosynthetic tissues. The plastid localization of CpIscA was confirmed by green fluorescent protein fusions, in vitro import, and immunoblotting experiments. CpIscA was cloned and purified after expression in Escherichia coli. Addition of CpIscA significantly enhanced CpNifS-mediated in vitro reconstitution of the 2Fe-2S cluster in apo-ferredoxin. During incubation with CpNifS in a reconstitution mix, CpIscA was shown to acquire a transient Fe-S cluster. The Fe-S cluster could subsequently be transferred by CpIscA to apo-ferredoxin. We propose that the CpIscA protein serves as a scaffold in chloroplast Fe-S cluster assembly.     

Mechanical modulation of molecular signals which regulate anabolic and catabolic activity in bone tissue

Identifying the molecular mechanisms that regulate bone's adaptive response to alterations in load bearing may potentiate the discovery of interventions to curb osteoporosis. Adult female mice (BALB/cByJ) were subjected to catabolic (disuse) and anabolic (45 Hz, 0.3g vibration for 10 min/day) signals, and changes in the mRNA levels of thirteen genes were compared to altered indices of bone formation. Age-matched mice served as controls. Following 4 days of disuse, significant (P = 0.05) decreases in mRNA levels were measured for several genes, including collagen type I (-55%), osteonectin (-44%), osterix (-36%), and MMP-2 (-36%) all of which, after 21 days, had normalized to control levels. In contrast, expression of several genes in the vibrated group, which failed to show significant changes at 4 days, demonstrated significant increases after 21 days, including inducible nitric oxide synthase (iNOS) (39%, P = 0.07), MMP-2 (54%), and receptor activator of the nuclear factor kB ligand (RANKL) (32%). Correlations of gene expression patterns across experimental conditions and time points allowed the functional clustering of responsive genes into two distinct groups. Each cluster's specific regulatory role (formation vs. resorption) was reinforced by the 60% suppression of formation rates caused by disuse, and the 55% increase in formation rates stimulated by mechanical signals (P < 0.05). These data confirm the complexity of the bone remodeling process, both in terms of the number of genes involved, their interaction and coordination of resorptive and formative activity, and the temporal sensitivity of the processes. More detailed spatial and temporal correlations between altered mRNA levels and tissue plasticity may further delineate the molecules responsible for the control of bone mass and morphology.     

PIKE/nuclear PI 3-kinase signaling in preventing programmed cell death

PI 3-kinase enhancer (PIKE) is a nuclear GTPase that enhances PI 3-kinase (PI3K) activity. Nerve growth factor (NGF) treatment leads to PIKE activation by triggering the nuclear translocation of PLC-gamma1, which acts as a physiological guanine nucleotide exchange factor (GEF) for PIKE. PI3K occurs in the nuclei of a broad range of cell types, and various stimuli elicit PI3K nuclear translocation. While cytoplasmic PI3K has been well characterized, little is known about the biological function of nuclear PI3K. Surprisingly, nuclei from 30 min NGF-treated PC12 cells are resistant to DNA fragmentation initiated by the activated cell-free apoptosome, and both PIKE and nuclear PI3K are sufficient and necessary for this effect. Moreover, pretreatment of the control nucleus with PI(3,4,5)P3 alone mimics the anti-apoptotic activity of NGF by selectively preventing apoptosis, for which nuclear Akt is required but not sufficient. Recently, a nuclear PI(3,4,5)P3 receptor, nucleophosmin/B23, has been identified from NGF-treated PC12 nuclear extract. PI(3,4,5)P3/B23 complex mediates the anti-apoptotic effects of NGF by inhibiting DNA fragmentation activity of caspase-activated DNase (CAD). Thus, PI(3,4,5)P3/B23 complex and nuclear Akt effectors might coordinately mediate PIKE/nuclear PI3K signaling in promoting cell survival by NGF.     

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.     

Effects of paeoniflorin on the cerebral infarction, behavioral and cognitive impairments at the chronic stage of transient middle cerebral artery occlusion in rats

In the present study, the effects of paeoniflorin (PF), a characteristic monoterpene glucoside isolated from Paeoniae Radix, on cerebral infarction, neurological symptoms, tongue protrusion (TP) and performance in the water maze were examined at the chronic stage (4 weeks) of transient cerebral ischemia using a rat middle cerebral artery occlusion (MCAO) model. One-day (10 mg/kg, twice, s.c.) or seven-day (2.5-10 mg/kg, twice a day, s.c.) injection of PF significantly reduced the infarct volume as well as ameliorated the deficits in neurological symptoms caused by transient MCAO at chronic stage. Transient MCAO also induced impairments in TP and performance in the water maze. Treatment with PF was able to reverse or alleviate these impairments. These results indicate that PF may be effective for treatment of stroke.     

RNA silencing suppressor p21 of Beet yellows virus forms an RNA binding octameric ring structure

Many plant viruses encode proteins that suppress the antiviral RNA silencing response mounted by the host. The suppressors p19 from tombusvirus and p21 from Beet yellows virus appear to block silencing by directly binding siRNA, a critical mediator in the process. Here, we report the crystal structure of p21, which reveals an octameric ring architecture with a large central cavity of approximately 90 A diameter. The all alpha-helical p21 monomer consists of N- and C-terminal domains that associate with their neighboring counterparts through symmetric head-to-head and tail-to-tail interactions. A putative RNA binding surface is identified in the conserved, positive-charged inner surface of the ring. In contrast to the specific p19-siRNA duplex interaction, p21 is a general nucleic acid binding protein, interacting with 21 nt or longer single- and double-stranded RNAs in vitro. This study reveals an RNA binding structure adopted by the p21 silencing suppressor.