Fetal surgery for myelomeningocele: history, research, clinical trials, and future directions.

Myelomeningocele, more commonly known as spina bifida, is the most common neural tube defect worldwide. In the United States, it is the primary cause of lifelong childhood paralysis with approximately four children born daily with this devastating disease. To minimize damage to the exposed spinal cord and prevent ascending central nervous system infections, postnatal closure of the spinal defect has been the standard of care for decades. Research into the mechanism of spinal cord injury in those with spina bifida revealed that damage continues to accrue during the gestational period. Prenatal defect closure via in utero surgery was proposed to prevent this early deterioration of the spinal cord, and early animal research demonstrated that prenatal repair was feasible and promising. This paved the way for the first human prenatal repairs in the mid-to-late 1990s. Following the promising outcomes observed during the first human cases, a randomized controlled trial, the Management of Myelomeningocele Study (MOMS), was conducted comparing postnatal repair of spina bifida to prenatal repair. The MOMS trial demonstrated that to those undergoing prenatal repair of spina bifida had a decreased need for ventriculoperitoneal shunting and improved lower extremity motor function. With the success of the MOMS trial, in utero repair is now considered the standard of care in those who meet the criteria for prenatal repair. This review will provide an overview of spina bifida and its impact, highlight the historical changes in care, describe the early research and theory that made prenatal repair an option, discuss the clinical experiences with human fetal repair and briefly touch on future research directions for those with myelomeningocele.

Expression and cellular localization of the classical progesterone receptor in healthy and amyotrophic lateral sclerosis affected spinal cord.

BACKGROUND AND PURPOSE: Previous studies have suggested that elevated progesterone levels are associated with a slower disease course in amyotrophic lateral sclerosis (ALS). Given that the effects of progesterone are mediated in part by the classical progesterone receptor (PR), the expression and cellular localization of the A and B isoforms (PR-A and PR-B, respectively) of the PR in control (neuropathologically normal) and ALS-affected spinal cord (SC) were examined. METHODS: Semi-quantitative RT-PCR, immunohistochemistry and immunofluorescence analyses of the cervical and lumbar SC of post-mortem ALS patients (n = 19) and control subjects (n = 10) were performed. Primers and antibodies used allowed the detection of both PR-A and PR-B isoforms together (PR-A+B) or PR-B isoform alone. RESULTS: Lumbar PR-A+B and cervical PR-B mRNA expression were significantly higher in ALS than controls. In both ALS and controls, PR-A+B immunoreactivity (IR) was occasionally detected in motor neurons. In contrast, PR-A+B IR was prominent in axonal processes and vessels. This was more evident in nerve roots and large arteries in ALS compared with controls. Colocalization of PR-A+B with markers of neurons, axonal processes and vascular endothelium was also observed. CONCLUSIONS: Evidence that both PR-A and PR-B isoforms are expressed in the human SC is provided, with some regional variation in isoform expression between ALS and controls. The IR was more prominent in nerve roots and large arteries in ALS, suggesting a potential role in the degenerative process.

Mutations at arg486 and glu571 in human topoisomerase IIalpha confer resistance to amsacrine: relevance for antitumor drug resistance in human cells.

Human topoisomerase II, a nuclear protein involved in chromosome segregation, is the target of amsacrine and other clinically important anticancer drugs. The enzyme is expressed as alpha and beta isoforms whose mutation/down-regulation has been implicated in drug resistance. To understand the role of target mutations in cellular drug resistance, we have used yeast to select and characterize plasmid-borne human topoisomerase IIalpha mutants resistant to amsacrine. Single point changes of Glu571 to Lys (E571K) or Arg486 to Lys (R486K) in the conserved PLRGK motif, both of which reside in the GyrB homology domain of human topoisomerase IIalpha, were frequently selected and could be shown in vivo to confer >25-fold and >100-fold resistance, respectively, to amsacrine and approximately 3-fold cross-resistance to etoposide. Highly purified E571K and R486K human topoisomerase IIalpha proteins required 100-fold higher levels of amsacrine to induce DNA cleavage similar to that of wild-type protein, consistent with a resistance mechanism involving reduced cleavable complex formation. Our functional studies of the R486K mutation, previously identified in two amsacrine-resistant human cell lines and in human biopsy material, establish unequivocally that it confers resistance, and suggest mechanisms for its phenotypic expression in vivo. These results differ significantly from previous work using yeast topoisomerase II as a model system: introduction of the equivalent mutation to R486K (R476K) into the yeast enzyme did not give amsacrine resistance. We conclude that species-specific differences in topoisomerase II enzymes can affect the drug resistance phenotype of particular mutations and highlight the need to study the relevant human homolog.

Effects of meal form and composition on plasma testosterone, cortisol, and insulin following resistance exercise.

The purpose of this study was to examine the effects of postexercise feeding on plasma levels of insulin, testosterone, cortisol, and testosterone:cortisol (T:C). Ten experienced, resistance trained males (20.7 +/- 0.95 years) were given whole food (WF: protein 38 g; carbohydrate 70 g; fat 7 g), a supplemental drink (SD; isocaloric and isonitrogenous to WF), an isocaloric carbohydrate beverage (C), or a placebo beverage (P) immediately, 2 and 4 hours after a standardized weight training protocol on 4 days, each separated by 1 week, in a repeated measures design. Subjects also received a standardized meal at 7 and 12 hours postexercise. Insulin, testosterone, and cortisol were measured pre-exercise and during 24 hours of recovery (at 0.5, 2.5, 4.5, 8, and 24 hours) using venous blood samples. Significant (condition 3 time) interactions were found for insulin, testosterone, and T:C, but not for cortisol (p <. 05). The SD yielded a greater response for insulin than all other conditions. Conversely, P demonstrated the greatest values for testosterone and T:C at 2.5 and 4.5 hours postexercise. Cortisol did not vary between conditions and there were no condition effects for insulin, testosterone, cortisol, and T:C at 8 or 24 hours. In conclusion, the efficacy of postexercise feeding for optimizing T:C and muscle growth is unclear; however, consumption of SD appears to maximize circulating insulin for several hours following resistance exercise.

Noninvasive estimation of the maximal lactate steady state in trained cyclists.

PURPOSE: The purposes of this study were to estimate noninvasively the maximal lactate steady state (MLSS) in trained cyclists on a windload simulator with a velocity based technique and to determine whether the HR at MLSS (HR(MLSS)) elicited a similar blood lactate concentration (BLC) during field testing. METHODS: To determine and verify MLSS, 10 male cyclists performed five to seven laboratory trials on separate days, including a VO2max test; a 5-km time trial (TT); and two or more 30-min trials at specific percentages of each subject's average 5-km TT speed (AVS5km). Mean+/-SD for the following variables were obtained at MLSS: velocity was 90.3+/-2.7% of the AVS5km, BLC was 5.4+/-1.6 mM, RPE was 15+/-2.1, VO2 was 80+/-6.3% of VO2max, and HR was 167+/-9.5 beats x min(-1), which was 88+/-3.8% of the mean maximum HR. Field tests included three laps of an 8-km road circuit at HR(MLSS) +/-3 beats x min(-1) and one lap at maximum sustainable velocity (a road TT). RESULTS: There were no significant differences in BLC, HR, and RPE between the three steady-state road laps and the lab MLSS trial. There was also good agreement between the road and lab MLSS velocity/TT velocity ratios. CONCLUSIONS: Our data suggest that 5-km TT cycling velocity, as measured on a windload simulator, may be used to estimate MLSS and the HR at MLSS for training purposes.

Impact of rapid weight loss on cognitive function in collegiate wrestlers.

PURPOSE: The purpose of this study was to examine the effects of rapid weight loss (RWL) on cognitive function in collegiate wrestlers. METHODS: Wrestlers (N = 14) and controls (N = 15) were college-aged males who were tested at three different times: baseline, RWL, and rehydration. Wrestlers practiced RWL in preparation for competition while controls maintained normal body weight and dietary practices throughout the study. At each test session, blood glucose, hemoglobin (Hgb), hematocrit (Hct), plasma volume (PV), and body weight were measured. Subjects also completed mood and hypoglycemic profiles as well as five short cognitive tests. RESULTS: Two-way ANOVA and post-hoc tests revealed poorer performance for wrestlers than controls in two recall tests as well as greater mood negativity for the wrestlers after RWL. With RWL, between group differences were also evident in hypoglycemic profile, blood glucose, PV, and body weight. All measures returned to near baseline values after rehydration, suggesting that all physiological and cognitive effects associated with RWL were reversible. It is possible that precompetition anxiety may partially explain the results. CONCLUSIONS: RWL in collegiate wrestlers before a competition appears to cause physiological effects that are accompanied by transient mood reduction and impairment of short-term memory. The potential negative impact of this practice on the student-athlete should be considered.

Transcutaneous electrical muscle stimulation does not alter regional arm adiposity and muscularity.

The present study evaluated the effects of an electrical muscle stimulation (EMS) training protocol claimed to elicit site-specific fat reduction and muscle hypertrophy. The protocol includes 6 weeks (3 sessions/week, 10 contractions/session) of high frequency (2500 Hz) transcutaneous EMS training on upper arm muscle and fat tissue. Two groups of 10 subjects were stimulated either on the dominant biceps brachii (B) or triceps brachii (T). The non-dominant arm served as a control. Post-training measures of upper arm fat and muscle tissue evaluated with girths, skinfolds, and ultrasound measures of fat did not change following EMS training of the treatment and control arms (p > 0.05). Upper arm x-ray measurements to estimate total upper arm volume, muscle plus bone volume, and fat volume also did not change significantly for groups B and T following training (p > 0.05). The results thus indicate no selective upper arm muscle hypertrophy or site-specific fat reduction with high frequency EMS training of healthy muscle. Am. J Hum. Biol. 10:317-326, 1998. © 1998 Wiley-Liss, Inc.

Water running with and without a flotation vest in competitive and recreational runners.

The purpose of this study was to determine whether competitive and recreational runners would replicate land training intensity during water immersion (WI) running with (V) and without (NV) a flotation vest and during treadmill running (Tm). Seven female competitive runners (CR) and seven female noncompetitive runners (NR) were asked to replicate preferred land training intensity characteristic of a 45-min run under three conditions (Tm, V, and NV). When 20-min submaximal runs at the preferred land training intensity were performed for Tm, V, and NV conditions, CR were able to elicit a similar submaximal VO2 for all three conditions. In contrast, the NR group had a significantly (P < 0.05) lower VO2 (27%), HR (23%), VE (26%) and %VO2max (27%) during V versus Tm condition. During the NV condition, NR had a significantly lower VO2 (13%), %VO2max (13%), and a higher RPE compared with Tm running, and a significantly higher VO2 (16%), HR (15%), VE (24%), %VO2max (15%) and RPE compared with the V condition. Competitive runners were able to achieve training intensities similar to land training for WI running with or without a flotation vest. However, recreational runners failed to replicate land training pace, where intensity was significantly lower during WI running without a vest and lowest with a vest, despite efforts to maintain a similar level of exertion.

Identification of yeast DNA topoisomerase II mutants resistant to the antitumor drug doxorubicin: implications for the mechanisms of doxorubicin action and cytotoxicity.

Doxorubicin is a therapeutically useful anticancer drug that exerts multiple biological effects. Its antitumor and cardiotoxic properties have been ascribed to anthracycline-mediated free radical damage to DNA and membranes. Evidence for this idea comes in part from the selection by doxorubicin from stationary phase yeast cells of mutants (petites) deficient in mitochondrial respiration and therefore defective in free radical generation. However, doxorubicin also binds to DNA topoisomerase II, converting the enzyme into a DNA damaging agent through the trapping of a covalent enzyme-DNA complex termed the 'cleavable complex.' We have used yeast to determine whether stabilization of cleavable complexes plays a role in doxorubicin action and cytotoxicity. A plasmid-borne yeast TOP2 gene was mutagenized with hydroxylamine and used to transform drug-permeable yeast strain JN394t2-4, which carries a temperature-sensitive top2-4 mutation in its chromosomal TOP2 gene. Selection in growth medium at the nonpermissive temperature of 35 degrees in the presence of doxorubicin resulted in the isolation of plasmid-borne top2 mutants specifying functional doxorubicin-resistant DNA topoisomerase II. Single-point changes of Gly748 to Glu or Ala642 to Ser in yeast topoisomerase II, which lie in and adjacent to the CAP-like DNA binding domain, respectively, were identified as responsible for resistance to doxorubicin, implicating these regions in drug action. None of the mutants selected in JN394t2-4, which has a rad52 defect in double-strand DNA break repair, was respiration-deficient. We conclude that topoisomerase II is an intracellular target for doxorubicin and that the genetic background and/or cell proliferation status can determine the relative importance of topoisomerase II- versus free radical-killing.

Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast.

Candida albicans topoisomerase II, encoded by the TOP2 gene, mediates chromosome segregation by a double-strand DNA break mechanism and is a potential target for anti-fungal therapy. In this paper, we report the characterization of the C. albicans TOP2 gene and its use to develop a yeast system that allows the identification and study of anti-fungal topoisomerase II inhibitors in vivo. The gene, specifying a 1461-residue polypeptide with only 40% identity with human topoisomerase IIalpha and beta isoforms, was isolated from C. albicans on a 6.3 kb EcoRI fragment that mapped to chromosome 4. It was used to construct a plasmid in which TOP2 expresses a recombinant enzyme (residues 57-1461 of C. albicans topoisomerase II fused to the first five residues of Saccharomyces cerevisiae topoisomerase II) under the control of a galactose-inducible promoter. The plasmid rescued the lethal phenotype of a temperature-sensitive S. cerevisiae DNA topoisomerase II mutant allowing growth at 35 degrees C. Yeast cells, bearing ISE2 permeability and rad52 double-strand-break-repair mutations the growth of which at 35 degrees C was dependent on C. albicans topoisomerase II, were killed by the known topoisomerase II inhibitors amsacrine and doxorubicin. Parallel experiments in yeast expressing human topoisomerase IIalpha allowed the relative sensitivities of the fungal and host topoisomerases to be examined in the same genetic background. To compare the killing in vivo with drug inhibition in vitro, the recombinant C. albicans topoisomerase II protein was expressed and purified to near-homogeneity from S. cerevisiae yielding a 160 kDa polypeptide that displayed the expected ATP-dependent DNA-relaxation and DNA-decatenation activities. The enzyme, whether examined in vitro or complementing in S. cerevisiae, was comparably sensitive to amsacrine and doxorubicin. Our results suggest that potential topoisomerase II-targeting anti-fungal inhibitors can be identified and studied in S. cerevisiae.

Modification in movement accuracy in the triphasic pattern during a rapid forearm-flexion task.

The present study was designed to investigate modifications in the triphasic EMG pattern during a forearm-flexion task at maximum speed which required three levels of movement accuracy. 36 subjects participated in 4 training sessions, performing a total of 200 repetitions of each movement. The fastest movement time was associated with the least accurate movement task. Likewise, the slowest movement time was found for the movement requiring the greatest accuracy. Differences in the duration and amplitude of agonist 1 activity, the start of agonist 2 activity, and the start and amplitude of antagonist activity were observed for the three movements. The results indicate that agonist 1 provides a propulsive force to initiate limb movement. The antagonist EMG activity was thought responsible for braking and correcting limb movement. Modifications in agonist 2 activity suggest this burst is related to movement velocity.