Lentivirus-mediated carboxyl-terminal modulator protein gene transfection via aerosol in lungs of K-ras null mice.

The low efficiency of conventional therapies in achieving long-term survival of lung cancer patients calls for development of novel options. Aerosol gene delivery may provide the alternative for safe and effective treatment for lung cancer. Therefore, current study was performed to elucidate the potential effects of C-terminal modulator protein (CTMP) via aerosol on lung tumorigenesis. Lentiviral vector-CTMP was delivered into K-ras null lung cancer mice through the nose-only inhalation system for 30 min. After 48 h, the potential effects of CTMP on Akt1-related signals and cell cycle regulation in the lungs were evaluated by western blot, immunohistochemistry and zymography. Lentivirus-based CTMP delivery inhibited the Akt1 activity through selective suppression of Akt1 phosphorylation at Ser473. Aerosol delivery of CTMP inhibited proteins important for Akt1 signals, cell cycle and tumor metastasis in lungs of K-ras null mice. Together, our results suggest that lentivirus-mediated aerosol delivery of CTMP may be compatible with noninvasive in vivo gene therapy. Our results emphasize the importance of noninvasive-targeted delivery of CTMP for lung cancer therapy in the future. While the studies are conducted in mice, it is envisioned that noninvasive targeting the specific genes responsible for cancer progression is an attractive strategy for effective anticancer therapeutics.

Aerosol-delivered programmed cell death 4 enhanced apoptosis, controlled cell cycle and suppressed AP-1 activity in the lungs of AP-1 luciferase reporter mice.

The long-term survival of lung cancer patients treated with conventional therapies remains poor and therefore the need for novel approaches remains high. This has led to the re-emergence of aerosol delivery as a therapeutic intervention. In this study, glucosylated polyethylenimine (GPEI) was used as carrier to investigate programmed cell death 4 (PDCD4) and PDCD4 mutant (D418A), an eIF4A-binding mutant, on PDCD4-related signaling and activator protein-1 (AP-1) activity in the lungs of AP-1 luciferase reporter mice. After confirming the efficiency of GPEI as a carrier in lungs, the effects of aerosol-delivered PDCD4 were investigated in AP-1 luciferase reporter mice. Aerosol delivery of GPEI/PDCD4 through a nose-only inhalation facilitated the apoptosis of lungs whereas aerosol PDCD4 mutant did not. Also, such aerosol delivery regulated proteins relevant to cell-cycle control and suppressed AP-1 activity. Results obtained by western blot analysis, immunohistochemistry, luciferase assay and deoxynucleotidyl-transferase-mediated nick end labeling study suggest that combined actions such as facilitating apoptosis, controlling cell cycle and suppression of AP-1 activity by PDCD4 may provide useful tool for designing lung tumor prevention and treatment by which PDCD4 functions as a transformation suppressor in the future.

Aerosol delivery of Akt controls protein translation in the lungs of dual luciferase reporter mice.

Lung cancer has emerged as a leading cause of cancer death in the world; however, most of the current conventional therapies are not sufficiently effective in altering the progression of disease. Therefore, development of novel treatment approaches is needed. Although several genes and methods have been used for cancer gene therapy, a number of problems such as specificity, efficacy and toxicity reduce their application. This has led to re-emergence of aerosol gene delivery as a noninvasive method for lung cancer treatment. In this study, nano-sized glucosylated polyethyleneimine (GPEI) was used as a gene delivery carrier to investigate the effects of Akt wild type (WT) and kinase deficient (KD) on Akt-related signaling pathways and protein translation in the lungs of CMV- LucR-cMyc-IRES-LucF dual reporter mice. These mice are a powerful tool for the discrimination between cap-dependent/-independent protein translation. Aerosols containing self-assembled nano-sized GPEI/Akt WT or GPEI/Akt KD were delivered into the lungs of reporter mice through nose-only-inhalation-chamber with the aid of nebulizer. Aerosol delivery of Akt WT caused the increase of protein expression levels of Akt-related signals, whereas aerosol delivery of Akt KD did not. Furthermore, dual luciferase activity assay showed that aerosol delivery of Akt WT enhanced cap-dependent protein translation, whereas a reduction in cap-dependent protein translation by Akt KD was observed. Our results clearly showed that targeting Akt may be a good strategy for prevention as well as treatment of lung cancer. These studies suggest that our aerosol delivery is compatible for in vivo gene delivery which could be used as a noninvasive gene therapy in the future.

Gene array analysis of osteoblast differentiation.

We have used gene array technology to chart changes in gene expression during differentiation of the mouse calvarial-derived MC3T3-E1 cell line to an osteoblast-like phenotype. Expression was analyzed on a mouse gene array panel of 588 cDNAs representing tightly regulated genes with key roles in various biological processes. When compared with NIH3T3 fibroblasts, MC3T3-E1 cells showed generally higher expression of cyclins and Bcl-2 family members, as well as specific expression of products such as the CD44 antigen, which is consistent with their calvarial origin. MC3T3-E1 cells also showed a surprisingly high level of p53. Differentiation in MC3T3-E1 cells involves withdrawal from the cell cycle by day 7, accompanied by matrix accumulation and, ultimately, mineralization. Gene expression patterns in induced MC3T3-E1 cells generally reflected these stages. Cyclins were sharply down-regulated, and expression of certain antiproliferative factors and tissue-restricted genes was induced. Many of the observed changes, such as the induction of follistatin, bone morphogenetic protein receptor 1A, transforming growth factor beta, and matrix remodeling factors, reflect expected patterns and support the physiological relevance of the results. Other observed changes were not anticipated and offer new insight into the osteoblast differentiation process. An example is the sharp induction of the Tob antiproliferative factor, which has previously been associated specifically with terminal differentiation in muscles. Another example is the induction of the DNA damage-associated proteins EI24 and Gadd45, apparently as a normal aspect of osteoblast differentiation. The oxidative stress-induced protein A170 and the transcription factor Nrf2, which regulates metabolic responses to oxidative stress, were also induced. This response may reflect the in vivo requirement for vascularization during bone growth and fracture repair. Other induced factors include tumor necrosis factor receptor-associated factor-1 (1-TRAF), which is a nuclear factor kappaB activator, cellular retinoic acid-binding protein II (CRABP-II), and the transcription factors S-II, SP2, and SEF2 (ITF2/E2:2). SEF2 is the first basic helix-loop-helix protein found to be up-regulated during osteoblast differentiation. Northern blots confirm the induction of SEF2.

Phosphate is a specific signal for induction of osteopontin gene expression.

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue.

E1A blocks hyperphosphorylation of p130 and p107 without affecting the phosphorylation status of the retinoblastoma protein.

The phosphorylation status of the pRB family of growth suppressor proteins is regulated in a cell cycle entry-, progression-, and exit-dependent manner in normal cells. We have shown previously that p130, a member of this family, exhibits patterns of phosphorylated forms associated with various cell growth and differentiation stages. However, human 293 cells, which are transformed cells that express the adenoviral oncoproteins E1A and E1B, exhibit an abnormal pattern of p130 phosphorylated forms. Here we report that, unlike pRB, the phosphorylation status of both p130 and p107 is not modulated during the cell cycle in 293 cells as it is in other cells. Conditional overexpression of individual G(1)/S cyclins in 293 cells does not alter the phosphorylation status of p130, suggesting that the expression of E1A and/or E1B blocks hyperphosphorylation of p130. In agreement with these observations, transient cotransfection of vectors expressing E1A 12S, but not E1B, in combination with pocket proteins into U-2 OS cells blocks hyperphosphorylation of both p130 and p107. However, the phosphorylation status of pRB is not altered by cotransfection of E1A 12S vectors. Moreover, MC3T3-E1 preosteoblasts stably expressing E1A 12S also exhibit a block in hyperphosphorylation of endogenous p130 and p107. Direct binding of E1A to p130 and p107 is not required for the phosphorylation block since E1A 12S mutants defective in binding to the pRB family also block hyperphosphorylation of p130 and p107. Our data reported here identify a novel function of E1A, which affects p130 and p107 but does not affect pRB. Since E1A does not bind the hyperphosphorylated forms of p130, this function of E1A might prevent the existence of "free" hyperphosphorylated p130, which could act as a CDK inhibitor.

Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts.

We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targets p300/CBP, but not the pRB family. Using these constructs to dissect osteoblast differentiation, we found that targeting of p300/CBP appears to be sufficient to repress alkaline phosphatase expression, although a low but functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-stage markers such as osteocalcin and osteopontin. Surprisingly, cell lines exhibiting severe repression of alkaline phosphatase activity differentiate to a mineral-secreting phenotype much like normal MC3T3-E1 cells. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induction of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in conditions in which endogenous alkaline phosphatase is severely repressed.

Nonpeptide endothelin receptor antagonists. VIII: attentuation of acute hypoxia-induced pulmonary hypertension in the dog.

It has been proposed that endothelin-1 (ET-1), a potent endogenous vasoactive peptide, may play an important role in the regulation of pulmonary blood flow. The purpose of the present study was to characterize the effects of ET-1 and a nonpeptide mixed ET(A) and ET(B) receptor antagonist, SB 209670, in isolated segments of the canine pulmonary artery and to examine the effects of SB 209670 in a canine model of acute hypoxia-induced pulmonary hypertension. In isolated segments of the pulmonary artery, SB 209670 (3-300 nM) produced a concentration-dependent antagonism of contraction elicited by ET-1 (pA2 = 8.9; slope = 0.9) and had no effect on phenylephrine responses. In addition, SB 209670 antagonized the small, endothelium-dependent relaxation induced by sarafotoxin 6c in phenylephrine (10 microM)-precontracted vessels (pKB = 8.6). In anesthetized dogs, the driving pressure across the pulmonary circulation increased approximately 100% during the hypoxic period (area under the curve [AUC] = 267.1 +/- 25.3 mm Hg x min). SB 209670 treatment (3 and 30 microg/kg/min i.v.) reduced pulmonary vascular resistance and produced a profound dose-related inhibition of hypoxia-induced pulmonary hypertension (AUC = 158.3 +/- 22.7 mm Hg x min and 50.1 +/- 4.9 mm Hg x min, respectively). None of the other hemodynamic or arterial blood gas parameters differed significantly in the vehicle and treatment groups. In addition, SB 209670 produced a significant reversal of hypoxia-induced pulmonary hypertension (AUC = 267.1 +/- 25.3 mm Hg x min vs. 167.8 +/- 23.4 mm Hg x min) when administered at the plateau of the hypoxic response. It was found that SB 209670 administration significantly elevated plasma levels of ET-1-LI (> or = 25-fold). These results suggest that ET-1 is an important mediator of hypoxia-induced pulmonary hypertension in the dog and that SB 209670, a potent and selective mixed ET(A) and ET(B)receptor antagonist in the pulmonary circulation, may represent an important therapeutic approach to the treatment of pulmonary hypertension.

An analytical comparison of the three most commonly used prostate-specific antigen assays: Tandem-R, Tandem-E, and IMx.

OBJECTIVES: To compare the similarity of individual prostate-specific antigen (PSA) results when using the Tandem-R PSA assay, the Tandem-E PSA assay, or the IMx PSA assay; to assess the lot-to-lot variation within (intra-assay interlot) and between (interassay interlot) the Tandem-R, Tandem-E, and IMx PSA assays; and to evaluate the individual and overall potential lot-to-lot bias of the Tandem-R, Tandem-E, and IMx PSA assays. METHODS: Forty-nine serum samples (PSA values from 0 to 85 ng/mL) were each tested by three separate lots (manufacturer's reagent materials) of Tandem-R, Tandem-E, and IMx PSA assays. Therefore, a total of nine different lots were utilized per patient sample in this investigation. Analyses primarily focused on three ranges: 0 to 10 ng/mL (low), 10 to 85 ng/mL (high), and 0 to 85 ng/mL (overall). RESULTS: In the 0 to 10 ng/mL range, 93% of the assay comparisons yielded an actual difference of less than 1 ng/mL. All three assays demonstrated excellent correlation within and between their three respective lots, within all three ranges. The 95% confidence intervals around the percent coefficient of variation (CV) demonstrated similar results for each assay (CV range, 3.2% to 6.0%). All lots demonstrated an average actual PSA bias of less than +/- 1 ng/mL. The average percent PSA bias was also similar between all three assay systems. All lots demonstrated a less than +/- 4% bias. CONCLUSIONS: Overall, the Tandem-R and IMx PSA assays yielded slightly lower results than the Tandem-E PSA assay. However, these differences were not statistically significant. In addition, the overall lot-to-lot variation (intra-assay and interassay) was not statistically significant, and the actual or percent PSA bias was minimal with these three assays. Therefore, a clinician can feel confident that a patient's serum sample should yield a similar and interchangeable result, whether it is determined by the Tandem-R, Tandem-E, or IMx PSA assay system.

Pharmacological evidence for the presence of three distinct functional endothelin receptor subtypes in the rabbit lateral saphenous vein.

1. Contraction of the rabbit isolated saphenous vein is mediated by a heterogeneous endothelin (ET) receptor population. This study has characterized these receptor subtypes by use of several pharmacologically distinct ET receptor agonists and antagonists. 2. ET-1, ET-3, sarafotoxin S6c (STXc) and [Ala3,11]ET-1 produced biphasic, concentration-dependent contractions of the saphenous vein, responses which were best fitted by a two-site model comprised of a high (pM) and a low (nM) affinity site. In contrast, IRL 1620 only recognized one of these sites. ET(16-21) was devoid of contractile activity. ET-1, ET-3 and STXc were equipotent at the high affinity site (pD2s of 12.0 +/- 0.2, 12.2 +/- 0.2 and 12.3 +/- 0.3) indicating that this site had the characteristics of an ETB receptor. In contrast, the low affinity site had the functional characteristics of an ETc receptor since the pD2s for ET-3 (10.2 +/- 0.3) and STXc (10.6 +/- 0.3) were significantly greater than that for ET-1 (9.1 +/- 0.1). These contractile responses were insensitive to BQ-123, confirming that ETA receptors were not involved in mediating this effect. 3. SB 209670 differentially antagonized the high affinity phases of the isopeptide concentration-response curves in a fashion dependent on the competing agonist: relative to the KB obtained against STXc (0.15 nM). SB 209670 was 10 fold less potent when ET-1 was used as the competing agonist. This differential effect was not evident at the low affinity site (KB = 38 nM). SB 209670 produced parallel, concentration-dependent rightward shifts in the concentration-response curve to STXc Ro 47-0203 was approximately 1 to 2 orders of magnitude less potent than SB 209670 at inhibiting the high affinity component of the concentration-response curve to STXc, whereas BQ-788 and Ro 46-2005 were approximately 3 orders of magnitude less potent than SB 209670. In addition to RES-701 and BQ-123, the high affinity site was insensitive to PD 142893 suggesting that it may represent an ETB2 receptor. Ro 47-0203 and SB 209670 were equipotent at inhibiting the low affinity component of the STXc concentration-response curve. Although Ro 46-2005, BQ-788, PD 142893 and RES-701 produced significant antagonism at the low affinity site, they were at least ten fold less potent than SB 209670. 4. ET-1, ET-3 and STXc produced endothelium-dependent vasorelaxation in the precontracted saphenous vein. Antagonist IC50s were approximated as being: SB 209670, 3 nM; BQ-788 and RES 701,300 nM; Ro 46-2005 and PD 142893, 3 microM; BQ-123, > 10 .M, consistent with vasorelaxation being mediated by an ETB1 receptor.5. In summary, three pharmacologically distinct ET receptor subtypes have been identified in the rabbits aphenous vein. Two contractile receptors are present on the vascular smooth muscle, a high affinity site with the characteristics of an ETB2 receptor and a distinct lower affinity site with the characteristics of an ETc receptor. In addition, an ETBI receptor is present on the endothelium which mediates the vasodilator actions of this peptide family.

Pharmacologic evidence for the presence of three functional endothelin receptor subtypes in rabbit saphenous vein.

The contractile responses to endothelin-1 (ET-1), endothelin-3 (ET-3), and sarafotoxin S6c (STXc) were best-fitted by a two-site model in the rabbit isolated saphenous vein. Agonists were equipotent at the high-affinity site (pD2s 12.0 +/- 0.2, 12.2 +/- 0.2, and 12.3 +/- 0.3, respectively), characteristic of an ETB receptor, whereas ET-3 and STXc were significantly more potent than ET-1 as vasoconstrictors at the low-affinity site (pD2s 10.2 +/- 0.3, 10.6 +/- 0.3, and 9.1 +/- 0.1, respectively), characteristic of an ETC-like receptor. The high affinity, ETB-mediated response was inhibited by SB 209670, Ro 47-0203, BQ-788, and Ro 46-2005 (Kbs of 0.15, 14, 110, and 280 nM against STXc, respectively) but was insensitive to PD 142893, RES-701 and BQ-123 (Kbs > or = 10 microM), consistent with this response being mediated by the ETB2-like receptor subtype. The low-affinity ETC-like-mediated component was inhibited by SB 209670, Ro 47-0203, BQ-788, Ro 46-2005, PD 142893, and RES-701 (Kbs 38 nM, 62 nM, 670 nM, 580 nM, 1.7 microM, and 2.0 microM, respectively), but was insensitive to BQ-123 (Kb > or = 10 microM). ET-3 produced endothelium-dependent vasorelaxation in the presence of active tone. Antagonist IC50s were approximated as being: SB 209670 3 nM; BQ-788 and RES 701,300 nM; Ro 46-2005 and PD 142893 3 microM; BQ-123 > or = 10 microM. These values were consistent with vasorelaxation being mediated by an ETB1-like receptor. Therefore, three pharmacologically distinct ET receptor subtypes have been identified in rabbit saphenous vein. Two contractile receptors, ETB2-like and ETC-like, are present on the vascular smooth-muscle receptor, whereas an ETB1-like receptor, which mediates the vasodilator actions of this peptide family, is present on the endothelium.

Agonist-dependent inhibition by peptide and nonpeptide endothelin receptor antagonists in the rabbit isolated pulmonary artery.

This study examined the effect of peptide and nonpeptide endothelin (ET) receptor antagonists on the contractile actions of ET-1 and sarafotoxin S6c (STXc) in the rabbit isolated pulmonary artery (RbPA). The peptide antagonists BQ-123, BQ-788, and RES-701 (10 microM) did not inhibit ET-1-induced RbPA contractions. The nonpeptide antagonists PD 156123, Ro 47-0203, and SB 217242 (10 microM) also had no effect on ET-1--induced contractions. In contrast, the nonpeptide antagonists SB 209670 and L-749,239 (10 microM) both caused a shift to the right of the ET-1 concentration-response curve (Kbs of 231 nM and 1.42 microM, respectively) and a two- to three-fold increase in nH (Hill slope), without altering the Rmax (maximal agonist-induced contraction). Contractile responses to STXc were unaffected by BQ-123, RES-701, and PD 156123 (10 microM). SB 209670, SB 217242, L-749,239, Ro 47-0203, and BQ-788 (10 microM) caused a shift to the right of the STXc response curve (Kbs of 16, 353, 202, 1,303 and 1,499 nM, respectively) and an increase in nH and Rmax. SB 209670 and L-749,239 were both approximately 10-fold more potent in antagonizing STXc than ET-1. Assuming, at best, Kbs of 10 microM for SB 217242, Ro 47-0203, and BQ-788 against ET-1, these antagonists were also at least 10-fold more potent in blocking STXc. The effects of various incubation and washout times on the antagonistic properties of SB 209670 (1 microM) against STXc were studied. Washing the antagonist from the tissue bath caused a 100-fold decrease in affinity (Kbs of 23-2,373 nM at 0 and 30 min, respectively) and a return to near control levels of nH (within 30 min). In contrast, the augmented Rmax response did not return to control levels until 6 hr later. As the incubation time was increased, the augmented Rmax was sustained (6 h), but affinity decreased (Kbs of 21 and 793 nM after 5 min and 6 h of incubation), and nH returned to near control values. In conclusion, agonist-dependent inhibition by ETB receptor antagonists is observed in the RbPA. Heterogenous populations of ET receptors, differential binding domains, and/or functionally uncoupled receptors may account for this phenomenon.

Nonpeptide endothelin receptor antagonists. II. Pharmacological characterization of SB 209670.

The pharmacological characterization of SB 209670, a highly potent nonpeptide endothelin ETA/ETB receptor antagonist was performed. SB 209670 produced concentration-dependent, parallel rightward shifts in the ET-1 concentration-response curves in the isolated rat aorta (ETA receptor-mediated vascular contraction). The Kb for inhibition of ETA receptor-mediated contraction by SB 209670 was 0.4 +/- 0.04 nM. Inhibition by SB 209670 was stereoselective as the (+)-antipode of SB 209670 was approximately 575-fold more potent than the (-)-antipode. Relative to other ET receptor antagonists, the potency of SB 209670 for inhibiting ETA receptor-mediated vascular contraction was 45-, 180- and 775-fold more potent than the ETA selective antagonist BQ-123, or the mixed ETA/ETB receptor antagonists bosentan or PD142893, respectively. The pharmacological antagonism produced by SB 209670 was specific for ET-1. SB 209670 inhibited ETB receptors in the isolated rabbit pulmonary artery as demonstrated by concentration-dependent, parallel rightward shifts in either the ET-1 or sarafotoxin S6c (S6c) concentration-response curves. The Kb values for inhibition were 200 +/- 9 and 52 +/- 14 nM for ET-1 and S6c, respectively. In contrast, neither the ETB selective antagonist RES-701 (10 microM) nor BQ-123 (10 microM) inhibited S6c-mediated vasoconstriction. However, PD 142893 (10 microM) and bosentan (10 microM) produced a small rightward shift in the S6c concentration-response curve, each with Kb values of 1.5 to 3.7 microM. In isolated human circumflex coronary arteries, (+/-)-SB 209670 inhibited ET-1 mediated contraction with a Kb value of 7 +/- 3 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Acceleration forces at eye level experienced with rotation on the horizontal bar.

Negative acceleration forces (-Gz) experienced at eye level have been associated with preretinal hemorrhage and headache. These signs and symptoms were found in individuals who experienced negative (toward the head) force while rotating on a horizontal bar or hanging from a trapeze. Lightweight accelerometers were used to measure -Gz experienced at eye level in children and adult gymnasts performing a single-knee backswing on a horizontal bar. Rate of onset of -Gz, peak -Gz, time experiencing -Gz, area of curve (G.second), and mean force (area/time) were calculated. There was no significant difference between the children and the adult gymnasts in any of the above parameters. The best gymnast had a maximum rate of onset of 38.15 G/s and the maximum negative force experienced was 5.52 G. The maximum rate of onset for a child was 41.56 G/s and the maximum negative force experienced was 5.73 G. Compared with -Gz tolerance curves generated on a centrifuge the best gymnast would have become symptomatic while performing this maneuver in 6 s. The best child would have become symptomatic in 25 s. These tolerance limits can be easily exceeded by gymnasts and by the monkey-bar enthusiast.