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Substrate supply, fine roots, and temperature control proteolytic enzyme activity in temperate forest soils

Temperature and substrate availability constrain the activity of the extracellular enzymes that decompose and release nutrients from soil organic matter (SOM). Proteolytic enzymes are the primary class of enzymes involved in the depolymerization of nitrogen (N) from proteinaceous components of SOM, and their activity affects the rate of N cycling in forest soils. The objectives of this study were to determine whether and how temperature and substrate availability affect the activity of proteolytic enzymes in temperate forest soils, and whether the activity of proteolytic enzymes and other enzymes involved in the acquisition of N (i.e., chitinolytic and ligninolytic enzymes) differs between trees species that form associations with either ectomycorrhizal or arbuscular mycorrhizal fungi. Temperature limitation of proteolytic enzyme activity was observed only early in the growing season when soil temperatures in the field were near 4 degrees C. Substrate limitation to proteolytic activity persisted well into the growing season. Ligninolytic enzyme activity was higher in soils dominated by ectomycorrhizal associated tree species. In contrast, the activity of proteolytic and chitinolytic enzymes did not differ, but there were differences between mycorrhizal association in the control of roots on enzyme activity. Roots of ectomycorrhizal species but not those of arbuscular mycorrhizal species exerted significant control over proteolytic, chitinolytic, and ligninolytic enzyme activity; the absence of ectomycorrhizal fine roots reduced the activity of all three enzymes. These results suggest that climate warming in the absence of increases in substrate availability may have a modest effect on soil-N cycling, and that global changes that alter belowground carbon allocation by trees are likely to have a larger effect on nitrogen cycling in stands dominated by ectomycorrhizal fungi.

Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB

We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature‐dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 degrees C. At 0 degrees C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N‐ and C‐ domains of FepA. However, colicin B penetration into the cell at 37 degrees C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. We did not detect TonB‐dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 degrees C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N‐domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.

Ethionine-Induced ATP Depletion Represses mTOR Signaling in the Absence of Increases in AMP-Activated Protein Kinase Activity in the Rat Liver

Administration of ethionine to female rats caused a rapid and severe decline in liver ATP and inhibited hepatic protein synthesis in association with hypophosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 70-kDa ribosomal protein S6 kinase (S6K1), two key regulatory proteins involved in initiation of mRNA translation. Phosphorylation of both regulatory proteins is mediated through a signaling pathway that involves the mammalian target of rapamycin (mTOR). Recent studies indicate that AMP-activated protein kinase (AMPK) plays a role in the cellular response to environmental stresses, which deplete ATP, and suppresses protein synthesis through downregulated mTOR signaling. We investigated the possible involvement of AMPK in the ethionine-induced inhibition of protein synthesis. The administration of ethionine surprisingly decreased AMPK activity compared with controls despite ATP depletion. We conclude that inhibition of protein synthesis by ethionine is due to AMPK-independent inhibition of mTOR signaling following ATP depletion.

Time-dependent effects of short-term training on muscle metabolism during the early phase of exercise

In this study, we investigated the hypothesis that the metabolic adaptations observed during steady-state exercise soon after the onset of training would be displayed during the nonsteady period of moderate exercise and would occur in the absence of increases in peak aerobic power (Vo2peak) and in muscle oxidative potential. Nine untrained males [age = 20.8 +/- 0.70 (SE) yr] performed a cycle task at 62% Vo2peak before (Pre-T) and after (Post-T) training for 2 h/day for 5 days at task intensity. Tissue samples extracted from the vastus lateralis at 0 min (before exercise) and at 10, 60, and 180 s of exercise, indicated that at Pre-T, reductions (P < 0.05) in phosphocreatine and increases (P < 0.05) in creatine, inorganic phosphate, calculated free ADP, and free AMP occurred at 60 and 180 s but not at 10 s. At Post-T, the concentrations of all metabolites were blunted (P < 0.05) at 60 s. Training also reduced (P < 0.05) the increase in lactate and the lactate-to-pyruvate ratio observed during exercise at Pre-T. These adaptations occurred in the absence of change in Vo2peak (47.8 +/- 1.7 vs. 49.2 +/- 1.7 mlxkg(-1)xmin(-1)) and in the activities (molxkg protein(-1)xh(-1)) of succinic dehydrogenase (3.48 +/- 0.21 vs. 3.77 +/- 0.35) and citrate synthase (7.48 +/- 0.61 vs. 8.52 +/- 0.65) but not cytochrome oxidase (70.8 +/- 5.1 vs. 79.6 +/- 6.6 U/g protein; P < 0.05). It is concluded that the tighter metabolic control observed following short-term training is initially expressed during the nonsteady state, probably as a result of increases in oxidative phosphorylation that is not dependent on changes in Vo2peak while the role of oxidative potential remains uncertain.

Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists and Positive Allosteric Modulators Using the Parallel Oocyte Electrophysiology Test Station

Neuronal acetylcholine receptors (nAChRs) of the alpha7 subtype are ligand-gated ion channels that are widely distributed throughout the central nervous system and considered as attractive targets for the treatment of various neuropsychiatric and neurodegenerative diseases. Both agonists and positive allosteric modulators (PAMs) are being developed as means to enhance the function of alpha7 nAChRs. The in vitro characterization of alpha7 ligands, including agonists and PAMs, relies on multiple technologies, but only electrophysiological measurements assess the channel activity directly. Traditional electrophysiological approaches utilizing two-electrode voltage clamp or patch clamp in isolated cells have very low throughput to significantly impact drug discovery. Abbott (Abbott Park, IL) has developed a two-electrode voltage clamp-based system, the Parallel Oocyte Electrophysiology Test Station (POETs()), that allows for the investigation of ligand-gated ion channels such as alpha7 nAChRs in a higher-throughput manner. We describe the utility of this technology in the discovery of selective alpha7 agonists and PAMs. With alpha7 agonists, POETs experiments involved both single- and multiple-point concentration-response testing revealing diverse activation profiles (zero efficacy desensitizing, partial, and full agonists). In the characterization of alpha7 PAMs, POETs testing has served as a reliable primary or secondary screen identifying compounds that fall into distinct functional types depending on the manner in which current potentiation occurred. Type I PAMs (eg, genistein, NS1738, and 5-hydroxyindole) increase predominantly the peak amplitude response, type II PAMs affect the peak current and current decay (eg, PNU-120,596 and 4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), and anothertype slowing the current decay kinetics in the absence of increases in the peak current. In summary, POETs technology allows for significant impact on higher throughput in the testing of alpha7 agonists and PAMs and for identification of compounds with unique profiles that could prove valuable in identifying an optimum in vitro profile in the development of therapeutics for which the alpha7 subtype is considered.

Allantoin in Human Plasma, Serum, and Nasal-Lining Fluids as a Biomarker of Oxidative Stress: Avoiding Artifacts and Establishing Realin vivoConcentrations

Urate is the terminal product of purine metabolism in primates, including humans. Urate is also an efficient scavenger of oxidizing species and is thought to be an important antioxidant in human body fluids. Allantoin, the major oxidation product of urate, has been suggested as a candidate biomarker of oxidative stress because it is not produced metabolically. Although urate is converted to allantoin under strongly alkaline pH, such conditions have been used in the past to facilitate extraction of allantoin. We evolved a method for the determination of allantoin concentrations in human plasma and serum by gas chromatography-mass spectrometry without such artifact. With this method, we show that alkaline conditions do indeed cause breakdown of urate, leading to significant overestimation of allantoin concentration in human samples. By using our alternative method, serum samples from 98 volunteers were analyzed, and allantoin levels were found to be significantly lower than was previously reported. The in vivo utility and sensitivity of our method was further evaluated in human nasal-lining fluids. We were able to demonstrate an ozone-induced increase in allantoin, in the absence of increases in either ascorbate or glutathione oxidation products.

Vasomotion and insulin‐mediated capillary recruitment – part of the explanation?

Vasomotion and insulin‐mediated capillary recruitment – part of the explanation?

Fluoresceination of FepA during colicin B killing: effects of temperature, toxin and TonB.

We studied the reactivity of 35 genetically engineered Cys sulphydryl groups at different locations in Escherichia coli FepA. Modification of surface loop residues by fluorescein maleimide (FM) was strongly temperature-dependent in vivo, whereas reactivity at other sites was much less affected. Control reactions with bovine serum albumin showed that the temperature dependence of loop residue reactivity was unusually high, indicating that conformational changes in multiple loops (L2, L3, L4, L5, L7, L8, L10) transform the receptor to a more accessible form at 37 degrees C. At 0 degrees C colicin B binding impaired or blocked labelling at 8 of 10 surface loop sites, presumably by steric hindrance. Overall, colicin B adsorption decreased the reactivity of more than half of the 35 sites, in both the N- and C- domains of FepA. However, colicin B penetration into the cell at 37 degrees C did not augment the chemical modification of any residues in FepA. The FM modification patterns were similarly unaffected by the tonB locus. FepA was expressed at lower levels in a tonB host strain, but when we accounted for this decrease its FM labelling was comparable whether TonB was present or absent. Thus we did not detect TonB-dependent structural changes in FepA, either alone or when it interacted with colicin B at 37 degrees C. The only changes in chemical modification were reductions from steric hindrance when the bacteriocin bound to the receptor protein. The absence of increases in the reactivity of N-domain residues argues against the idea that the colicin B polypeptide traverses the FepA channel.

Outcome After Aortic Valve Replacement for Low-Flow/Low-Gradient Aortic Stenosis Without Contractile Reserve on Dobutamine Stress Echocardiography

This study investigated whether aortic valve replacement (AVR) is associated with improved survival in patients with severe low-flow/low-gradient aortic stenosis (LF/LGAS) without contractile reserve (CR) on dobutamine stress echocardiography (DSE). Patients with LF/LGAS without CR have a high mortality rate with conservative therapy. The benefit of AVR in this subset of patients remains controversial. Eighty-one consecutive patients with symptomatic calcified LF/LGAS (valve area <or=1 cm(2), left ventricular ejection fraction <or=40%, mean pressure gradient [MPG] <or=40 mm Hg) without CR on DSE were enrolled. Absence of CR was defined as the absence of increase in stroke volume of >or=20% compared with the baseline value. Multivariable analysis and propensity scores were used to compare survival according to whether or not AVR was performed (n = 55). Five-year survival was higher in AVR patients compared with medically managed patients (54 +/- 7% vs. 13 +/- 7%, p = 0.001) despite a high operative mortality of 22% (n = 12). An AVR was independently associated with lower 5-year mortality (adjusted hazard ratio from 0.16 to 5.21 varying with time [95% confidence interval: 0.12-3.16 to 0.21-8.50], p = 0.00026). In 42 propensity-matched patients, 5-year survival was markedly improved by AVR (65 +/- 11% vs. 11 +/- 7%, p = 0.019). Associated bypass surgery (p = 0.007) and MPG <or=20 mm Hg (p = 0.035) were independently predictive of operative mortality. Late survival after AVR (excluding operative death) was 69 +/- 8% at 5 years. In patients with LF/LGAS without CR on DSE, AVR is associated with better outcome compared with medical management. Surgery should not be withheld from this subset of patients solely on the basis of lack of CR on DSE.

Dosage des hormones thyroïdiennes et surrénaliennes dans le liquide amniotique: aide au diagnostic

Ultrasound scanning is able to detect foetal goiter due either to an hypothyroidy either to an hyperthyroidy, or clitoris hypertrophia resulting from adrenal hyperplasia in female, during the second half of pregnancy. The diagnosis of these rare diseases is of interest because the treatment can be started during pregnancy. An amniotic fluid punction can be discussed and its biochemical analysis may be of interest even though very few commercial assays have been tested on amniotic fluid. Our aim was two investigate the practicability and the value of free thyroxin (FT4), thyrotropin (TSH), 17alpha hydroxyprogesterone (17-OHP) and delta 4 androstenedione (Delta4A) measurement on amniotic fluid using commercially available assays for serum. FT4 and TSH are detectable at low levels in amniotic fluid. FT4 significantly increases from 2.1 pmol/L to 4.2 pmol/L while TSH significantly decreases from 0.27 mU/L to 0.12 mU/L during the second half of pregnancy. An increase in amniotic fluid TSH concentration contributes to the diagnosis of foetal hypothyroidy while the measurement of amniotic fluid FT4 is not informative in case of foetal goiter. 17-OHP and Delta4A are present in amniotic fluid at the same level as in serum. 17-OHP significantly decreases from 1.9 ng/mL to 1 ng/mL during the second half of pregnancy while Delta4A significantly increases from 0.5 ng/mL to 0.8 ng/mL. Absence of increase in their concentrations excludes any severe adrenal hyperplasia.

St. Sauver et al. Respond to "Lower Urinary Tract Symptoms and Inflammation"

We appreciate the opportunity to respond to the erudite commentary by Drs. Freedland and Aronson (1) regarding our manuscript examining associations between C-reactive protein levels and development of lower urinary tract symptoms (LUTS) and other urologic outcomes (2). The authors correctly note that the associations we found of elevated C-reactive protein levels with irritative LUTS and peak fl ow rates were restricted to only the most rapid worsening of these outcomes. Because C-reactive protein is a very nonspecific marker of systemic inflammation, it may be that inflammation in the urogenital tract needs to be substantial before systemic inflammatory markers are affected; consequently, this possibility may be why we saw associations of only elevated C-reactive protein levels with the most rapid increases in irritative LUTS and the most rapid declines in peak flow rates. Therefore, we completely agree with the authors that better markers of inflammation that are more proximal to the urologic system could substantially enhance our understanding of the role of inflammation in the development of LUTS and prostatic enlargement. Additionally, as noted by the authors (1), inflammation found in prostate biopsies has been only weakly associated with either irritative or obstructive symptoms (3). It is clear that LUTS may develop even in the absence of increases in prostate volume, and it is possible that inflammation in other components ofthe urogenital system could play an important role in the development of LUTS. Unfortunately, prostate biopsies are invasive, and the question of how to measure inflammation in the bladder, the bladder neck, or even the urethra remains perplexing. The discovery of inflammatory markers that are tightly linked to inflammation in the urogenital tract but that could be tested from serum or urine would be extremely useful in furthering our understanding of how prostatic versus nonprostatic inflammation might contribute to the development of LUTS. Drs. Freedland and Aronson (1) also note that prospective trials of antiinflammatory medications to treat established LUTS or prostatic enlargement have been disappointing. However, the benefit of antiinflammatory agents might be realized in primary rather than secondary prevention of these conditions. It is possible that inflammation may play a role in the development of LUTS or prostatic enlargement, but, once these conditions are established, antiinflammatory agents might have little effect in alleviating these conditions. Intervening earlier in the pathogenic process might help prevent their development. Indeed, the authors note the recent study in which statins were associated with a reduction in prostate-specific antigen levels in healthy men, suggesting an effect on the prostate (4). Additionally, in our own cohort, we previously found that long-term nonsteroidal antiinflammatory drug use was associated with a decreased likelihood of developing LUTS and an enlarged prostate (5). Future studies could help determine whether inhibiting inflammatory processes before LUTS or prostatic enlargement is established might be useful in preventing these conditions.

OSSEOUS LESIONS IN THE METACARPO(TARSO)PHALANGEAL JOINT DIAGNOSED USING LOW‐FIELD MAGNETIC RESONANCE IMAGING IN STANDING HORSES

We report the use of low-field standing magnetic resonance imaging in the standing horse for the diagnosis of osseous lesions in the metacarpophalangeal (MCP) or metatarsophalangeal (MTP) joint that were not apparent using standard radiography. Thirteen horses were studied and all had thickening of the subchondral bone plate and abnormal signal intensity in the adjacent spongiosa in either the condyles of metacarpal/metatarsal III or the proximal phalanx or both. Abnormalities were characterized by diffuse decreased signal intensity on T1-weighting adjacent to the subchondral bone and within the spongiosa in at least two imaging planes; in the absence of increases in signal intensity in fat-suppressed images, this change was interpreted as bone sclerosis. Nine horses also had a diffuse decreased signal intensity on T2*-weighting in the same areas and five had a diffuse increase in signal intensity in fat-suppressed images in conjunction with a decrease in signal intensity on T1- and T2*-weighted images; the increase in signal intensity in fat-suppressed images was interpreted as fluid accumulation. Five horses had a focal area of change in signal intensity within the subchondral bone with apparent loss of definition between the subchondral bone and the articular cartilage. Eleven horses were available for follow up, of which eight were sound and three remained lame. We conclude that lameness originating from the MCP or MTP joint may be associated with osseous damage in horses of any signalment in the absence of radiographic changes.

Reproductive responses to photoperiod persist in olfactory bulbectomized Siberian hamsters ( Phodopus sungorus)

In reproductively photoperiodic Syrian hamsters, removal of the olfactory bulbs (OBx) leads to a marked and sustained increase in gonadotrophin secretion which prevents normal testicular regression in short photoperiods. In contrast, among reproductively nonphotoperiodic laboratory strains of rats and mice, bulbectomy unmasks reproductive responses to photoperiod. The role of the olfactory bulbs has been proposed to have opposite effects on responsiveness to photoperiod, depending on the photoperiodicity of the reproductive system; however, Syrian hamsters are the only reproductively photoperiodic rodent species for which the role of the olfactory bulb in reproductive endocrinology has been assessed. This experiment evaluated the role of the olfactory bulbs in the photoperiodic control of reproduction in Siberian hamsters ( Phodopus sungorus), an established model species for the study of neural substrates mediating seasonality. Relative to control hamsters housed in long days (15 h light/day), exposure of adult male hamsters to short days (9 h light/day) for 8 weeks led to a temporal expansion of the pattern of nocturnal locomotor activity, testicular regression, decreases in testosterone (T) production, and undetectable levels of plasma follicle-stimulating hormone (FSH). Bilateral olfactory bulbectomy failed to affect any of these responses to short days. The patterns of entrainment to long and short days suggests that pre-pineal mechanisms involved in photoperiodic timekeeping are functioning normally in OBx hamsters. The absence of increases in FSH following bulbectomy in long days is incompatible with the hypothesis that the olfactory bulbs provide tonic inhibition of the HPG axis in this species. In marked contrast to Syrian hamsters, the olfactory bulbs of Siberian hamsters play essentially no role in the modulation of tonic gonadotrophin production or gonadotrophin responses to photoperiod.

Predictor of increase in caregiver burden for disabled elderly at home

In order to classify the caregivers at high risk of increase in their burden early, linear discriminant analysis was performed to obtain an effective discriminant model for differentiation of the presence or absence of increase in caregiver burden. The data obtained by self-administered questionnaire from 193 caregivers of frail elderly from January to February of 2005 were used. The discriminant analysis yielded a statistically significant function explaining 35.0% (Rc = 0.59; d.f. = 6; p = 0.0001). The configuration indicated that the psychological predictors of change in caregiver burden with much perceived stress (1.47), high caregiver burden at baseline (1.28), emotional control (0.75), effort to achieve (−0.28), symptomatic depression (0.20) and “ikigai” (purpose in life) (0.18) made statistically significant contributions to the differentiation between no increase and increase in caregiver burden. The discriminant function showed a sensitivity of 86% and specificity of 81%, and successfully classified 83% of the caregivers. The function at baseline is a simple and useful method for screening of an increase in caregiver burden among caregivers for the frail elderly at home.

Fetal growth restriction and maternal cardiac function

Cardiac output rises in pregnancy, and most of this increase occurs in the first trimester. Both heart rate and stroke volume contribute to this increase, which, coupled with a decrease in mean arterial pressure, determines a reduction of maternal total vascular resistance (TVR) in physiological pregnancy. The absence of a ‚correct’ maternal cardiovascular compensatory response (absence of increase in cardiac output, heart rate, stroke volume, left ventricular mass and decrease in maternal TVR), in addition to abnormal trophoblastic invasion, might be one of the factors that could determine a reduced placental perfusion and, eventually, the development of fetal intrauterine growth restriction (IUGR). In fact, pregnancies complicated by IUGR appear to lack the stimulus to induce the hemodynamic changes typically present in physiological pregnancy such as the increase in preload, maternal heart rate, stroke volume, the enlargement of the left atrium and, above all, the reduction of TVR. It is difficult to e...

Angiogenesis inhibitor attenuates parathyroid hormone-induced anabolic effect

In vivo osteogenic responses to anabolic stimuli are expected to be accompanied by angiogenesis as well as in the process of remodeling of bone. Consequently, angiogenesis might play an important role in mediating bone forming stimulating effect of parathyroid hormone (PTH). To investigate this relationship, we used actively growing young Sprague–Dawley rats and CKD-732, one of the angiogenesis inhibitor (AI) to reveal the relationship of angiogenesis in the effect of PTH. The groups were divided as (1) vehicle [VEH group], (2) PTH(1–84) [PTH group], (3) AI alone [AI group], (4) PTH(1–84) + AI concomitance [PTH-AI group] and were treated for 6 weeks. The bone mineral density (BMD) of PTH group was higher than VEH group and the gain of bone mass was attenuated in PTH-AI group. The maximal failure load in PTH group was higher than VEH group, but it was definitely attenuated by concurrent use of AI. Moreover, the toughness showed similar significant deterioration in PTH-AI group. General bone turnover was also significantly decreased in PTH-AI group as shown by the absence of increase in osteocalcin and β-crosslaps and by decrease in metaphyseal length. The BMD or the biomechanic data of AI only group were similar to the VEH group, suggesting the minimal effect of AI itself on the normal modeling phase of the growing rats. In conclusion, the angiogenesis seemed to contribute to completing the anabolic effect of PTH especially for bone strength.

Factors Influencing the Incidence of Utilization Deficiencies and Other Patterns of Recall/Strategy‐Use Relations in a Strategic Memory Task

Factors that influence the incidence of utilization deficiencies and other recall/strategy-use patterns on a strategic memory task were evaluated in two hundred and fourteen 7- and 9-year-old children. Both utilization deficiencies and the incidence of children showing increases in both recall and strategy use over phases were more likely to be observed on phase transitions involving prompting or when different lists of items were included on consecutive trials; in contrast, increases in recall in the absence of increases in strategy use were more likely to be found between trials consisting of the same stimulus items. This research clearly indicates that patterns of recall/strategy-use relations, including utilization deficiencies, vary meaningfully with the age of the child and a variety of contextual factors.

Abstract for the Forthcoming International Federation of Placenta Associations Meetings (13th Meeting) in Kingston, Ontario, Canada

Neuroblastoma is the most common extra-cranial solid tumor in childhood; and patients in stage IV of the disease have a high propensity for tumor recurrence. Retinoid therapy has been utilized as a means to induce differentiation of tumor cells and to inhibit relapse. In this study, the expression of a common neuronal differentiation marker [neurofilament M (NF-M)] in human SK-N-SH neuroblastoma cells treated with 10 μM all-trans retinoic acid (ATRA) showed significantly increased expression in accordance with reduced cell number. This was accompanied by an increase in MitoSOX and DCFH2 oxidation that could be indicative of increased steady-state levels of reactive oxygen species (ROS) such as O2•− and H2O2, which correlated with increased levels of MnSOD activity and immuno-reactive protein. Furthermore PEG-catalase inhibited the DCFH2 oxidation signal to a greater extent in the ATRA-treated cells (relative to controls) at 96 h indicating that as the cells became more differentiated, steady-state levels of H2O2 increased in the absence of increases in peroxide-scavenging antioxidants (i.e., glutathione, glutathione peroxidase, and catalase). In addition, ATRA-induced stimulation of NF-M at 48 and 72 h was enhanced by decreasing SOD activity using siRNA directed at MnSOD. Finally, treatment with ATRA for 96 h in the presence of MnSOD siRNA or PEG-catalase inhibited ATRA induced increases in NF-M expression. These results provide strong support for the hypothesis that changes in steady-state levels of O2•− and H2O2 significantly contribute to the process of ATRA-induced differentiation in neuroblastoma, and suggest that retinoid therapy for neuroblastoma could potentially be enhanced by redox-based manipulations of superoxide metabolism to improve patient outcome.

Long-Term Potentiation in the Juvenile Superior Colliculus Requires Simultaneous Activation of NMDA Receptors and L-type Ca2+Channels and Reflects Addition of Newly Functional Synapses

The visual layers of the rodent superficial superior colliculus (sSC) have been the focus of many development studies of the molecular bases of retinotopic map formation, the role of early retinal waves in this process, and the development of glutamate synapses. Previous studies have documented long-term potentiation (LTP), believed to be critical to developmental synapse refinement, in the rodent sSC. However, the means of induction and the preparations used have varied widely, and thus cellular changes underlying this LTP remain ambiguous. Whole-cell and perforated patch clamping were used in this study to elucidate the cellular mechanism of electrically evoked LTP in the juvenile rat sSC. This LTP required relatively low-frequency stimulation (20 Hz) and simultaneous activation of NMDA receptors and L-type Ca2+ channels. Experiments focused on narrow-field vertical neurons, a documented excitatory cell type in the stratum griseum superficiale using bipolar stimulation in the stratum opticum. Strontium (Sr2+) replacement of calcium (Ca2+) was applied to study evoked quantal events before and after LTP induction at the same synapses. Paired-pulse ratio and coefficient of variance analyses examined presynaptic release. Increases in quantal frequency were invariably found in the absence of increases in quantal amplitude and probability of release. These data suggest that electrically stimulated LTP, in the juvenile sSC after eye opening, selectively involves the addition or stabilization of AMPA receptors at the large number of silent synapses known to appear in the sSC after eye opening.

Epigenetic Modification Can Regulate the Stem Cell State of Hematopoietic Cells.

The maintenance of undifferentiated state and ability for self-renewal constitutes key properties of hematopoietic stem cell (HSC). To explore the possibility that epigenetic modification contributes to regulation of HSC functions, we have studied whether distinctive epigenetic modification can be correlated to different functions of hematopoietic cells.As a first approach, the global DNA methylations in non-coding repetitive elements were examined using mIAP, mEtn, and mc.satellite regions as marker loci. Sequencing of sodium bisulfite-modified CpG islands in these loci showed that most primitive Lin-c-kit+CD34− cells displayed highest level of DNA methylation as compared to the progenitor-enriched Lin-c-kit+CD34+ cells or differentiated counterpart (Lin+ cells) in mc satellite regions (85.7%, 60 %, 65%, respectively) and mEtn regions (73%, 58%, 74%, respectively), but not in mIAP region. Interestingly, whereas most primitive Lin-c-kit+CD34− cells expressed highest level of methyl cytosine binding protein (MeCp2) or DNA methyl transferase 3 (DNMT3a, 3b), the Lin+ cells expressed bare to minimal level of these gene products despite their high maintenance of DNA methylation, suggesting a differential de-novo methylations between these cells.Similarly, the most primitive Lin-c-kit+CD34− cells exhibited highest level of total acetylated histone (Ac-H4) but these cells expressed also high levels of histone deacetylase (HDAC) as well as histone acetyl transferase (HAT). Subsequent pulse-labeling with C14-acetate demonstrated that immature bone marrow cells (lin-), but not mature Lin+ cells, exhibited active acetylation of H4 with higher turn-overs, thus showing active remodeling of chromatin structures in immature hematopoietic cells. Next, to explore whether alterations in epigenetic modification could influence HSC function, the effect of epigenetic blockers (5-Azacytidine or TSA) were examined for their influence on in-vivo self-renewing activity of transplanted HSCs. Thus, recipients that had been lethally irradiated and transplanted with congeneic HSCs were treated with blockers during first two weeks of recovery, and transplanted into secondary recipients 18 weeks later to determine CRU numbers regenerated. The result of this CRU assay revealed that HSCs had underwent 32-folds higher self-renewal with inhibition of HDAC (22 vs. 738 CRUs), and 11-folds higher self-renewal with inhibition of DNA methylation (22 vs. 248 CRUs) showing that self-renewing potential of “stimulated” HSC is regulated by epigenetic modification. Next, to see if stem cell fate can be also influenced by epigenetic reprogramming, the effects of epigenetic blockers during “stationary” phase of hematopoiesis were examined by treating donor mice with epigenetic blockers for 3 weeks before sacrifice. Limiting dilution transplantation of these marrows revealed that the CRU frequencies in the treated (5-Azacytidine + TSA) donor marrows were 7-folds higher compared to the un-treated donor marrow cells (1/22,9000 vs. 1/3000) in the absence of increase in total marrow cell numbers, suggesting de-novo generation of CRUs with epigenetic manipulations.Taken together, these results show that the epigenetic modification should be an important regulatory mechanism for self-renewal and fate decisions for normal HSCs in-vivo.