Blackcurrants Reduce the Risk of Postmenopausal Osteoporosis: A Pilot Double-Blind, Randomized, Placebo-Controlled Clinical Trial.

Beneficial effects of blackcurrant supplementation on bone metabolism in mice has recently been demonstrated, but no studies are available in humans. The current study aimed to examine the dose-dependent effects of blackcurrant in preventing bone loss and the underlying mechanisms of action in adult women. Forty peri- and early postmenopausal women were randomly assigned into one of three treatment groups for 6 months: (1) a placebo (control group, n = 13); (2) 392 mg/day of blackcurrant powder (low blackcurrant, BC, group, n = 16); and (3) 784 mg/day of blackcurrant powder (high BC group, n = 11). The significance of differences in outcome variables was tested by repeated-measures ANOVA with treatment and time as between- and within-subject factors, respectively. Overall, blackcurrant supplementation decreased the loss of whole-body bone mineral density (BMD) compared to the control group (p \u3c 0.05), though the improvement of whole-body BMD remained significant only in the high BC group (p \u3c 0.05). Blackcurrant supplementation also led to a significant increase in serum amino-terminal propeptide of type 1 procollagen (P1NP), a marker of bone formation (p \u3c 0.05). These findings suggest that daily consumption of 784 mg of blackcurrant powder for six months mitigates the risk of postmenopausal bone loss, potentially through enhancing bone formation. Further studies of larger samples with various skeletal conditions are warranted to confirm these findings

Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought about by an environmental stressor, demonstrate important differences in aging- versus stress-induced protein damage, and challenge the widely held view that chemical chaperones are accumulated during hypertonic stress to protect protein structure/function

Cytoprotective immune responses to exercise, heat, and dehydration stresses in men

In response to stress, hsp70 (heat shock protein 70) has been observed to increase acutely, and remain elevated with peak expression typically around 24 hours post-stress. Additionally, cells expressing inducible hsp72 are more resilient in the face of in vitro heat shock (42°C, 1+ hr), as measured by early apoptosis marker annexin V. NFAT5 (Nuclear Factor of Activated T cells) has been shown to have a similar protective effect in response to osmolal stimuli. CD40L is, like NFAT5 a less studied protective factor, that has been investigated as a response to exercise stress in the context of cardiovascular risk. This study completed three sets of experiments examining each of these three protective proteins in up to six health, college-aged males ((mean ± SD) age 23 ± 4 years; body mass 82.96 ± kg; height 175.33 ± 9.69; running VO2max 82.96 ± 2.88 mL·min−1·kg−1) who completed five experimental days each. Prior to experimental day, subjects began passive dehydration after noon until the next morning. On experimental day subjects submaximally exercised for two hours in the environmental chamber (35°C, 32% r.h.) to additionally dehydrate to a final value of −4–5% body mass. Subjects rested and rehydrated for 60 minutes and then completed an exercise challenge (25 minutes run at 70% VO2max, 800 m maximal sprint, 5 minutes maximal self-paced box-lifting) in the heat. This study found, according to predefined hypotheses, that intracellular hsp72, extracellular hsp70, and NFAT5 were elevated acutely with stress with acute decrease during rehydration. Values stayed elevated through 24 hours following experimental day. In response to one hour heat shock, PBMCs exhibited less annexin V expression with timepoints where hsp72 levels were concurrently higher when compared among PBMC samples collected at various timepoints. CD40L decreased acutely with stress and increased during recovery, as seen with sCD40L in other studies. ^ Conclusive inferences from this studies are that hsp72 protects cells in response to acute stress and recovery periods, NFAT5 does so in response to hydration status, and surface CD40L appears to decrease with acute stress and increase during recovery to baseline levels.

Cytoprotective immune responses to exercise, heat, and dehydration stresses in men

In response to stress, hsp70 (heat shock protein 70) has been observed to increase acutely, and remain elevated with peak expression typically around 24 hours post-stress. Additionally, cells expressing inducible hsp72 are more resilient in the face of in vitro heat shock (42°C, 1+ hr), as measured by early apoptosis marker annexin V. NFAT5 (Nuclear Factor of Activated T cells) has been shown to have a similar protective effect in response to osmolal stimuli. CD40L is, like NFAT5 a less studied protective factor, that has been investigated as a response to exercise stress in the context of cardiovascular risk. This study completed three sets of experiments examining each of these three protective proteins in up to six health, college-aged males ((mean ± SD) age 23 ± 4 years; body mass 82.96 ± kg; height 175.33 ± 9.69; running VO2max 82.96 ± 2.88 mL·min−1·kg−1) who completed five experimental days each. Prior to experimental day, subjects began passive dehydration after noon until the next morning. On experimental day subjects submaximally exercised for two hours in the environmental chamber (35°C, 32% r.h.) to additionally dehydrate to a final value of −4–5% body mass. Subjects rested and rehydrated for 60 minutes and then completed an exercise challenge (25 minutes run at 70% VO2max, 800 m maximal sprint, 5 minutes maximal self-paced box-lifting) in the heat. This study found, according to predefined hypotheses, that intracellular hsp72, extracellular hsp70, and NFAT5 were elevated acutely with stress with acute decrease during rehydration. Values stayed elevated through 24 hours following experimental day. In response to one hour heat shock, PBMCs exhibited less annexin V expression with timepoints where hsp72 levels were concurrently higher when compared among PBMC samples collected at various timepoints. CD40L decreased acutely with stress and increased during recovery, as seen with sCD40L in other studies. ^ Conclusive inferences from this studies are that hsp72 protects cells in response to acute stress and recovery periods, NFAT5 does so in response to hydration status, and surface CD40L appears to decrease with acute stress and increase during recovery to baseline levels.

Effect of acclimation to mild hypertonic stress on protein degradation activity.

<p><i>A:</i> Effect of treatment of control and acclimated worms with vehicle only (1% DMSO) or 20 mM chloroquine (CQ) and 100 µM MG-132 on spontaneous aggregation of Q35::YFP. (<i>n</i> = 9–15). *P<0.003 compared to vehicle-treated control worms. **P<0.0001 compared to drug-treated control worms. <i>B:</i> Effect of RNAi silencing of Hos genes on spontaneous Q35::YFP aggregation in control and acclimated worms. Animals were fed bacteria expressing nonspecific (control) dsRNA or dsRNA targeting proteasome (<i>pas-6</i> and <i>rpn-3</i>) and lysosome (<i>vha-13</i>) components, or a putative lysosomal serine carboxypeptidase (F13D12.6). (n = 16–51). *P<0.001 compared to control or acclimated worms fed a nonspecific dsRNA. **P<0.03 compared to unacclimated <i>vha-13(RNAi)</i> worms. <i>C:</i> Percent of red mutant ubiquitin (UbG76V) tagged Dendra2 remaining in body wall muscle cells 24 h after photoconversion in control and 200 mM NaCl acclimated worms exposed to control or hypertonic growth media. Control and acclimated animals were exposed to 400 mM and 600 mM NaCl, respectively. (<i>n</i> = 3–8). *P<0.01 compared to unstressed worms. <i>D:</i> Percent change in ³⁵S-methionine labeled total protein levels in control and acclimated worms treated with 500 µg/ml of cycloheximide for 6 h to inhibit protein synthesis. (<i>n</i> = 3).</p

Effect of elevated glycerol levels on hypertonic stress-induced aggregation of endogenous proteins.

<p><i>A:</i> Effect of increasing NaCl concentrations on motility in control, acclimated, <i>osm-11</i> and acclimated <i>gpdh-1; gpdh-2</i> worms. <i>gpdh-1; gpdh-2</i> mutants lack functional GPDH-1 and GPDH-2 enzymes resulting in greatly reduced glycerol accumulation under hypertonic stress conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034153#pone.0034153-Lamitina2" target="_blank">[12]</a>. (<i>n</i> = 5–18 experiments with 15–60 worms/experiment). <i>B:</i> Left panel, relative insoluble protein in acclimated <i>gpdh-1; gpdh-2</i> worms maintained in either 200 mM NaCl or exposed to 500 mM NaCl for 4 h. Insoluble protein was quantified as a fraction of total protein and is plotted relative to that observed in worms maintained on 200 mM NaCl. (<i>n</i> = 3 experiments with 4000–5000 worms/experiment). Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from acclimated <i>gpdh-1; gpdh-2</i> worms maintained in 200 mM NaCl or exposed to 500 mM NaCl. <i>C:</i> Left panel, relative insoluble protein in <i>osm-11</i> worms grown under control conditions (51 mM NaCl) or exposed to 700 mM NaCl for 4 h. Insoluble protein was quantified and plotted in the same manner as described in <i>B</i>. (<i>n</i> = 3 samples of 4000–5000 worms/sample). *P<0.03 compared to animals maintained on 51 mM NaCl. Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from <i>osm-11</i> worms exposed to 51 or 700 mM NaCl.</p

Effect of acute hypertonic stress on ³⁵S-methionine incorporation into total protein.

<p>Worms were transferred to 200 mM NaCl agar plates at time 0 and ³⁵S-methionine incorporation into total protein was quantified 20 min and 1, 12 and 48 h after transfer. Values are expressed relative to unstressed control worms (i.e., time 0). (<i>n</i> = 3). *P<0.05 compared to control worms.</p

Effect of elevated glycerol levels on aging-induced aggregation of Q35::YFP.

<p><i>A:</i> Whole worm glycerol levels in controls worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. (<i>n</i> = 4 samples of ∼4000 worms/sample). <i>B:</i> Time course of aging-induced Q35::YFP aggregation in control, acclimated and <i>osm-11</i> mutant animals. (<i>n</i> = 7 experiments with 10–15 worms/experiment).</p

Effect of elevated glycerol levels on the properties of age-induced Q35::YFP aggregates.

<p><i>A:</i> Fluorescence micrographs of aggregate morphology in body wall muscle cells of control worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. Images were taken from 7-day old adult worms. Scale bar is 10 µm. <i>B:</i> Time course of bleaching and fluorescence recovery in aggregates of young (4-day old) and old (10-day old) adult control, acclimated and <i>osm-11</i> worms (<i>n</i> = 3). <i>C:</i> Aggregate toxicity in control, acclimated and <i>osm-11</i> worms. Toxicity is measured as reductions in motility, which is mediated by body wall muscle cells. (<i>n</i> = 5–12).</p