Mirror neuron system activation in children with developmental coordination disorder: A replication functional MRI study.

BACKGROUND: It has been hypothesised that abnormal functioning of the mirror neuron system (MNS) may lead to deficits in imitation and the internal representation of movement, potentially contributing to the motor impairments associated with developmental coordination disorder (DCD). AIMS: Using fMRI, this study examined brain activation patterns in children with and without DCD on a finger adduction/abduction task during four MNS activation states: observation; motor imagery; execution; and imitation. METHODS AND PROCEDURES: Nineteen boys (8.25-12.75 years) participated, including 10 children with DCD (≤16th percentile on MABC-2; no ADHD/ASD), and nine typically developing controls (≥25th percentile on MABC-2). OUTCOMES AND RESULTS: Even though children with DCD displayed deficits behaviourally on imitation (Sensory Integration & Praxis Test Subtests) and motor imagery assessments prior to scanning, no differences in MNS activation were seen between the DCD and control groups at a neurological level, with both groups activating mirror regions effectively across conditions. Small clusters of decreased activation during imitation were identified in non-mirror regions in the DCD group, including the thalamus, caudate, and posterior cingulate - regions involved in motor planning and attentional processes. CONCLUSIONS AND IMPLICATIONS: The results of this study do not provide support for the MNS dysfunction theory as a possible causal mechanism for DCD. Further research to explore attentional and motor planning processes and how they may interact at a network level may enhance our understanding of this complex disorder.

Tests of general relativity from timing the double pulsar.

The double pulsar system PSR J0737-3039A/B is unique in that both neutron stars are detectable as radio pulsars. They are also known to have much higher mean orbital velocities and accelerations than those of other binary pulsars. The system is therefore a good candidate for testing Einstein's theory of general relativity and alternative theories of gravity in the strong-field regime. We report on precision timing observations taken over the 2.5 years since its discovery and present four independent strong-field tests of general relativity. These tests use the theory-independent mass ratio of the two stars. By measuring relativistic corrections to the Keplerian description of the orbital motion, we find that the "post-Keplerian" parameter s agrees with the value predicted by general relativity within an uncertainty of 0.05%, the most precise test yet obtained. We also show that the transverse velocity of the system's center of mass is extremely small. Combined with the system's location near the Sun, this result suggests that future tests of gravitational theories with the double pulsar will supersede the best current solar system tests. It also implies that the second-born pulsar may not have formed through the core collapse of a helium star, as is usually assumed.

Diaphragm structure and function in the Florida manatee (Trichechus manatus latirostris).

Relative to many other mammals, little is known about the functional morphology of the four extant species of the order Sirenia. In this study, 166 Florida manatee (Trichechus manatus latirostris) carcasses fresh enough to collect detailed anatomical information were examined to describe the gross anatomy of the diaphragm. Our results show that the Florida manatee's diaphragm differs from those of other mammals in that it: lies in a dorsal plane, rather than in the more typical transverse plane; is located dorsal to the heart and does not attach to the sternum; and attaches medially at the "I"-shaped central tendon to bony projections extending ventrally from the vertebral bodies, forming two distinct hemidiaphragms. The manatee's transverse septum is a separate structure that lies at a right angle to the diaphragm and separates the heart from the liver and other viscera. The extreme muscularity of the diaphragm and the ability of manatees to adjust their position in the water column with minimal external movement suggest that diaphragmatic contractions may change the volume of each pleural cavity to affect buoyancy, roll, and pitch. We also hypothesize that such contractions, in concert with contractions of powerful abdominal muscles, may compress gas in the massive large intestine, and thereby also contribute to buoyancy control.

Determination of the analgesic dose-response relationship for epidural fentanyl and sufentanil with bupivacaine 0.125% in laboring patients.

STUDY OBJECTIVE: To establish the analgesic effective doses as defined as a visual analog pain scale (VAS) of at least 10 for 95% of parturients (ED95) receiving either epidural fentanyl or sufentanil with bupivacaine 0.125% for labor analgesia. DESIGN: Double-blind, randomized controlled study. SETTING: Two tertiary-care teaching hospitals. PATIENTS: 100 female patients, at full-term pregnancy, in active early labor (< 5 cm cervical dilation) and requesting obstetric anesthesia services for labor analgesia. INTERVENTIONS: Patients were randomized and equally distributed to receive one of ten epidural dosing regimens of bupivacaine 0.125% alone or with either fentanyl 25, 50, 75, or 100 micrograms or sufentanil 5, 10, 15, 20, or 25 micrograms in a 10-ml bolus after a 3-ml test dose of bupivacaine 0.25%. MEASUREMENTS AND MAIN RESULTS: VAS scores were obtained from each parturient using a 10-cm plastic VAS slide rule at times 0, 1, 5, 10, 15, 20, 25, and 30 minutes, and then again when the patient requested additional analgesia. Analgesic duration and demographic and obstetric data also were obtained. Using a log-probit dose-response analysis, analgesic success as defined as a VAS of at least 10 with each opioid dose was plotted and an ED95 value of 8 micrograms and 50 micrograms was established for sufentanil and fentanyl, respectively, in bupivacaine 0.125%. No statistical difference was detected for analgesic duration or incidence of side effects between analgesic groups. CONCLUSIONS: Epidural analgesia with fentanyl and sufentanil in bupivacaine 0.125% behaves in a dose-response fashion allowing for the determination of equipotent dose of each.

Analysis of aerial survey data on Florida manatee using Markov chain Monte Carlo.

We assess population trends of the Atlantic coast population of Florida manatee, Trichechus manatus latirostris, by reanalyzing aerial survey data collected between 1982 and 1992. To do so, we develop an explicit biological model that accounts for the method by which the manatees are counted, the mammals' movement between surveys, and the behavior of the population total over time. Bayesian inference, enabled by Markov chain Monte Carlo, is used to combine the survey data with the biological model. We compute marginal posterior distributions for all model parameters and predictive distributions for future counts. Several conclusions, such as a decreasing population growth rate and low sighting probabilities, are consistent across different prior specifications.

The temporal relationship between protein phosphatase, ICE/CED-3 proteases, intracellular acidification, and DNA fragmentation in apoptosis.

Apoptosis occurs during development and tissue homeostasis, and under conditions of physical and chemical stress. During apoptosis, cells digest their DNA, decrease intracellular pH, shrink, exhibit protein phosphatase activity, and activate members of the ICE/CED-3 family of proteases. This protease activity is identified by cleavage of poly(ADP-ribose) polymerase (PARP). Phosphatase activity during apoptosis is observed as dephosphorylation of the retinoblastoma susceptibility protein (Rb). Serine/threonine phosphatase inhibitors can prevent dephosphorylation of Rb and apoptosis, suggesting that Rb dephosphorylation is an indication of a critical regulator of apoptosis. The experiments described here were designed to establish the temporal relationship between these events. Apoptosis was induced in human ML-1 cells by the topoisomerase inhibitor etoposide. An inhibitor of the ICE/CED-3 protease family, z-VAD-fluoromethylketone (FMK), showed concentration-dependent protection from PARP cleavage, intracellular acidification, DNA digestion, early changes in membrane permeability, and cell shrinkage, thereby placing all of these events downstream of the ICE/CED-3 protease action. However, z-VAD-FMK did not prevent the dephosphorylation of Rb, placing this change upstream of the protease. These results suggest that the imbalance between protein phosphatase and kinase that is responsible for the dephosphorylation of Rb is also responsible for the activation of ICE/CED-3 proteases, which in turn is responsible for all the other events associated with apoptosis.

Detection of apoptosis by flow cytometry of cells simultaneously stained for intracellular pH (carboxy SNARF-1) and membrane permeability (Hoechst 33342).

Intracellular acidification appears to be a frequent and possibly universal occurrence during apoptosis. We have used carboxy SNARF-1 to measure intracellular pH by flow cytometry. We report here that the addition of Hoechst 33342 concurrently with carboxy SNARF-1 provides a clearer discrimination of the apoptotic population. Apoptotic cells accumulate Hoechst 33342 more rapidly than do viable nonapoptotic cells. The cells with greater Hoechst 33342 fluorescence are the same cells as those with decreased intracellular pH; these cells also have decreased volume. The different parameters in this analysis are presented for three models of apoptosis: human myeloblastoid ML-1 cells incubated with etoposide, murine cytotoxic T cells following withdrawal of interleukin-2, and Chinese hamster ovary cells incubated with staurosporine. In most circumstances, carboxy SNARF-1 provides excellent resolution of viable and apoptotic cells. However, the addition of Hoechst 33342 is particularly valuable when carboxy SNARF-1 alone can not fully discriminate the two cell populations. This situation occurs in Chinese hamster ovary cells, which undergo a smaller degree of acidification than do the other cell models. This situation also occurs when the extracellular pH is experimentally reduced to investigate the mechanism of pH dysregulation; the apoptotic cells appear to retain the ability to regulate intracellular pH at low pH, although they are defective in proton export at neutral pH.

Intracellular calcium stores are not required for Bcl-2-mediated protection from apoptosis.

The ability of Bcl-2 to inhibit cell death is well documented but its mechanism of action remains elusive. Recent reports have suggested that Bcl-2 prevents apoptosis by inhibiting the release of Ca2+ from the thapsigargin-sensitive Ca2+ store. The mobilization of Ca2+ from this store has been implicated as a signal regulating apoptotic cell death induced by glucocorticoids and by interleukin-3 withdrawal. The present study was designed to determine if Bcl-2 would still inhibit apoptosis after depletion of intracellular Ca2+ stores. We compared the response of two Chinese hamster ovary cell lines (5AHSmyc and 5A300bcl-2.2) following incubation with the calcium ionophore ionomycin to deplete intracellular Ca2+ stores. Continued incubation of 5AHSmyc cells in calcium-free media induced substantial apoptotic DNA fragmentation within 4 h and >95% loss of viability within 48 h. However, 5A300bcl-2.2 cells showed no evidence of DNA fragmentation or loss of viability over the same time period. Intracellular Ca2+ was analyzed with the Ca2+-sensitive fluorescent dye INDO-1 and confirmed that ionomycin was capable of releasing Ca2+ from intracellular stores in both cell lines. These results show that depletion of intracellular Ca2+ stores induces apoptosis and that these Ca2+ stores are not required for the protection afforded by Bcl-2.

Internal initiation of translation of hepatitis C virus RNA: the ribosome entry site is at the authentic initiation codon.

Hepatitis C viral (HCV) RNA includes an internal ribosome entry segment (IRES) that extends some 30 nt into the coding region and promotes internal initiation of translation at the authentic initiation codon at nt 342. The 5'-boundary of this IRES was mapped by in vitro translation and transfection assays and was found to lie between nt 42 and 71. Within these IRES boundaries there are, in most HCV strains, three AUG triplets upstream of the authentic initiation site. Although the first, 5'-proximal, of these is absolutely conserved, a mutational analysis showed that it is not a functional initiation codon. In particular, the G residue could be substituted provided compensatory mutations were made to maintain base pairing. The other two upstream AUGs are not absolutely conserved, and mutation of the third (5'-distal) had little effect on IRES activity. When an additional AUG codon was introduced by single-site mutation just upstream of the authentic initiation codon, it was found to be used when most of the IRES had been deleted to generate an RNA translated by the scanning ribosome mechanism, but was not used in the background of the full-length IRES when internal initiation is operative. These results argue that the IRES promotes direct ribosome entry immediately at, or indeed very close to, the authentic initiation codon, and that the upstream AUGs do not serve as ribosome entry sites.

The involvement of protein phosphatases in the activation of ICE/CED-3 protease, intracellular acidification, DNA digestion, and apoptosis.

Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.

Structure and function of the gastrointestinal tract of the Florida manatee, Trichechus manatus latirostris.

BACKGROUND: The Florida manatee, Trichechus manatus latirostris (Sirenia: Trichechidae), is the largest herbivorous marine mammal. Previously, components of the gastrointestinal (GI) tract of the species have been described, but no comprehensive descriptions of the gross and microscopic anatomy existed. This study integrates function and structure of the entire Florida manatee GI tract. METHODS: The GI tracts of several recently dead Florida manatees were examined from the following viewpoints: gross anatomical studies of preserved and unpreserved specimens, histology and histochemistry, and ultrastructure. RESULTS: The manatee GI tract has an enlarged hindgut, as do other nonruminant herbivores (i.e., hindgut digesters such as horses), but it also has important adaptations not seen in most other mammals. These structural adaptations include a discrete accessory digestive gland (the cardiac gland), submucosal mucous glands along the greater curvature of the stomach, and unkeratinized, stratified squamous epithelial cells overlying the glandular mucosae of the pyloric antrum, midgut cecum, colon, and rectum. CONCLUSIONS: The adaptations described above may relate to osmoregulation as well as to herbivory. The Florida manatee GI tract is most similar to those of other members of the Order Sirenia and to that of the herbivorous green sea turtle (Chelonia mydas), but it also shows superficial similarities to those of phylogenetically close Orders, the Proboscidea and Hyracoidea. The immense size of both the manatee and its large intestine suggests that, relative to smaller hindgut digesters, manatees have a slow rate of passage of digesta and efficient breakdown of fibrous plant material.

BCL-2 and MCL-1 expression in Chinese hamster ovary cells inhibits intracellular acidification and apoptosis induced by staurosporine.

Multiple physiological and pharmacological stimuli induce cells to die by apoptosis. In many cases, this apoptosis is inhibited by BCL-2, suggesting the involvement of a common regulatory pathway. One frequent characteristic of apoptosis is the digestion of DNA into oligonucleosome-length fragments. Intracellular acidification and increased intracellular calcium have been variously implicated in activating the endonuclease responsible for this DNA digestion. To explore the involvement of these potential signals in endonuclease activation, we have analyzed three Chinese hamster ovary cell lines: a parental line, one expressing a cDNA encoding BCL-2, and the third expressing the BCL-2 family member MCL-1. Apoptosis was induced with the protein kinase inhibitor staurosporine and intracellular pH and calcium were measured by flow cytometry. We found that both MCL-1 and BCL-2 inhibited DNA digestion compared to the parent cell line, although BCL-2 was more potent in this regard. Concurrent with DNA digestion, we observed intracellular acidification; MCL-1 and BCL-2 inhibited intracellular acidification to an extent commensurate with their ability to inhibit DNA digestion. In contrast, staurosporine caused a dose-dependent increase in intracellular calcium in all three cell lines, demonstrating that intracellular free calcium levels did not correlate with the induction of apoptosis. These results suggest that BCL-2 and MCL-1 may regulate a pathway for intracellular pH homeostasis during apoptotic cell death.

Major-locus contributions to variability of the craniofacial feature dystopia canthorum in Waardenburg syndrome.

We used segregation analysis to investigate the genetic basis of variation in dystopia canthorum, one of the key diagnostic features of Waardenburg syndrome type 1 (WS1). We sought to determine whether the W-index, a quantitative measure of this craniofacial feature, is influenced primarily either by allelic variation in the PAX3 disease gene or other major loci, by polygenic background effects, or by all of these potential sources of genetic variation. We studied both WS1-affected individuals and their WS1-unaffected relatives. After adjustment of the W-index for WS1 disease status, segregation analyses by the regression approach indicated major-locus control of this variation, although residual parent-offspring and sib-sib correlations are consistent with additional (possibly polygenic) effects. Separate analyses of WS1-affected and WS1-unaffected individuals suggest that epistatic interactions between disease alleles at the PAX3 WS1 locus and a second major locus influence variation in dystopia canthorum. Our approach should be applicable for assessing the genetic architecture of variation associated with other genetic diseases.

Unique features of internal initiation of hepatitis C virus RNA translation.

The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.

Subparsec-scale structure and evolution of Centaurus A (NGC5128).

We present a series of 8.4-GHz very-long-baseline radio interferometry images of the nucleus of Centaurus A (NGC5128) made with a Southern Hemisphere array, representing a 3.3-year monitoring effort. The nuclear radio jet is approximately 50 milliarcseconds in extent, or at the 3.5-megaparsec distance of NGC5128, approximately 1 parsec in length. Subluminal motion is seen and structural changes are observed on time scales shorter than 4 months. High-resolution observations at 4.8 and 8.4 GHz made in November 1992 reveal a complex morphology and allow us to unambiguously identify the self-absorbed core located at the southwestern end of the jet.

Analysis of locus heterogeneity in Waardenburg syndrome types 1 and 2 using highly informative microsatellite markers.

We performed linkage and locus heterogeneity analyses of Waardenburg syndrome (WS) types 1 and 2 using 9 DNA markers from 2q35-q37, including two highly polymorphic microsatellites very closely linked to the PAX3 candidate gene. None of 5 WS type 2 (WS2) families showed linkage to the PAX3 candidate region. We localized the marker D2S102 to less than 1 cM from PAX3 (lod = 33.7, theta = 0), but a complete absence of crossovers prevented determining whether it maps distal or proximal to PAX3. Study of 14 WS type 1 (WS1) families yielded a maximum lod score of 27.81 at PAX3, theta f = 0.010, theta = 0.007 assuming homogeneity. However, we found significant evidence of locus heterogeneity, with one family initially classified as WS1 unlinked to the PAX3 region. Reevaluation of the clinical features of this family revealed atypical morphology of inner canthi. This produced the appearance of dystopia canthorum and high W-index scores. While our one unlinked WS1 family exhibits atypical canthal morphology, our type 1 families with classic dystopia appear to be homogeneously linked to PAX3. These and other findings identify precautions that need to be addressed before using PAX3-linked markers for diagnostic purposes.

Analysis of variability of clinical manifestations in Waardenburg syndrome.

Expression of clinical findings of Waardenburg syndrome type 1 (WS1) and type 2 (WS2) is extremely variable. Using our collection of 26 WS1 and 8 WS2 families, we analyzed the occurrence, severity, and symmetry of clinical manifestations associated with WS. We found significant differences between WS1 and WS2 in deafness, and in pigmentary and craniofacial anomalies. Factor analysis was used to identify manifestations which covaried, resulting in 2 orthogonal factors. Since mean factor scores were found to differ when compared between WS1 and WS2, we suggest that these factors could be useful in distinguishing WS types. We found that the WS gene was transmitted from mothers more often than from fathers. We also extensively examined the W-Index, a continuous measure of dystopia canthorum. Our data suggest that use of the W-Index to discriminate between affected WS1 and WS2 individuals may be problematic since 1) ranges of W-Index scores of affected and unaffected individuals overlapped considerably within both WS1 and WS2, and 2) a considerable number of both affected and unaffected WS2 individuals exhibited W-index scores consistent with dystopia canthorum. Misclassification of families may have implications for risk assessment of deafness, since WS2 families have been reported to have greater incidence of deafness, as confirmed in our study.