The ontogeny of aerobic and diving capacity in the skeletal muscles of Weddell seals.

Our objective was to determine the ontogenetic changes in the skeletal muscles of Weddell seals that transform a non-diving pup into an elite diving adult. Muscle biopsies were collected from pups, juveniles and adults and analyzed for changes in fiber type, mitochondrial density, myoglobin concentrations and aerobic, lipolytic and anaerobic enzyme activities. The fiber type results demonstrated a decrease in slow-twitch oxidative (Type I) fibers and a significant increase in fast-twitch oxidative (Type IIA) fibers as the animals mature. In addition, the volume density of mitochondria and the activity of lipolytic enzymes significantly decreased as the seals matured. To our knowledge, this is the first quantitative account describing a decrease in aerobic fibers shifting towards an increase in fast-twitch oxidative fibers with a significant decrease in mitochondrial density as animals mature. These differences in the muscle physiology of Weddell seals are potentially due to their three very distinct stages of life history: non-diving pup, novice diving juvenile, and elite deep diving adult. During the first few weeks of life, pups are a non-diving terrestrial mammal that must rely on lanugo (natal fur) for thermoregulation in the harsh conditions of Antarctica. The increased aerobic capacity of pups, associated with increased mitochondrial volumes, acts to provide additional thermogenesis. As these future elite divers mature, their skeletal muscles transform to a more sedentary state in order to maintain the low levels of aerobic metabolism associated with long-duration diving.

Failed methotrexate termination of pregnancy: a case report.

We report a case of a fetus with shortened proximal long bones, ambiguous genitalia, intrauterine growth restriction and abnormal umbilical artery Doppler velocities observed on antenatal ultrasound exam. At 34 weeks the patient revealed methotrexate/misoprostol exposure at 6 weeks gestational age in attempted medical termination of pregnancy. On newborn exam, the baby had dysmorphic facial features, a short torso, scoliosis, a micropenis (phallus <1 cm) and shortened proximal long bones both upper and lower extremities. X-ray exam revealed a hemivertebra at T10 level, rib abnormalities, shortened proximal long bones, an absent pubic bone and bilateral knee ossification centers. With methotrexate exposure, improved counseling and surveillance could potentially avoid these significant abnormalities and prevent psychological distress.

A video study of pain relief during newborn male circumcision.

OBJECTIVE: The purpose of this investigation was to compare the effectiveness of dorsal penile nerve block and topical lidocaine-prilocaine anesthesia techniques for pain relief during circumcision. METHODS: In total, 18 healthy term newborn males were divided based on anesthesia. The topical lidocaine-prilocaine group had six males undergoing circumcision and three males undergoing a sham procedure. The dorsal penile nerve block group had six males undergoing circumcision and three males undergoing a sham procedure. The procedures were videotaped and viewed by 90 noninvestigator reviewers who scored the infants' pain using the Neonatal Infant Pain Scale and a numeric pain scale. Statistical analysis utilized a P-value of <0.05 as significant. RESULTS: The median Neonatal Infant Pain Scale and numeric pain scores were significantly lower in the dorsal penile nerve block group than in the topical lidocaine-prilocaine group or the sham group (Neonatal Infant Pain Scale: 1.0 (range 0-6) versus 6.0 (range 2-6) versus 4.0 (range 0-6), P < 0.001, respectively; numeric pain scale: 2.0 (range 0-10) versus 8.0 (range 1-10) versus 4.0 (range 0-10), P < 0.001, respectively). In the sham group, procedures with the dorsal penile nerve block had significantly lower pain scores than those with topical lidocaine-prilocaine (Neonatal Infant Pain Scale: 1.0 (range 0-6) versus 5.0 (range 2- 6), P < 0.001; numeric pain scale: 1.0 (0-8) versus 6.0 (range 0-10), P < 0.001). When reviewers were divided by medical background, gender or parental status, no differences in pain scores were observed. CONCLUSION: The dorsal penile nerve block anesthesia technique for newborn male circumcision was significantly more effective for pain relief than topical lidocaine-prilocaine.

p21 is essential for normal myogenic progenitor cell function in regenerating skeletal muscle.

Despite the ability of myogenic progenitor cells (MPCs) to completely regenerate skeletal muscle following injury, little is known regarding the molecular program that regulates their proliferation and differentiation. Although mice lacking the cyclin-dependent kinase inhibitor p21 (p21-/-), develop normally, we report here that p21-/- MPCs display increased cell number and enhanced cell cycle progression compared with wild-type MPCs. Therefore, we hypothesized that p21-/- mice would demonstrate temporally enhanced regeneration following myotrauma. In response to cardiotoxin-induced injury, p21-/- skeletal muscle regeneration was significantly attenuated vs. regenerating wild-type muscle, contrary to the hypothesis. Regenerating p21-/- skeletal muscle displayed increased proliferative (PCNA positive) nuclei coincident with increased apoptotic nuclei (TUNEL positive) compared with wild-type muscle up to 3 wk after injury. Differentiation of p21-/- MPCs was markedly impaired and associated with increased apoptosis compared with wild-type MPCs, confirming that the impaired differentiation of the p21-/- MPCs was a cell autonomous event. No dysregulation of p27, p53, or p57 protein expression in differentiating p21-/- MPCs compared with wild-type MPCs was observed, suggesting that other compensatory mechanisms are responsible for the regeneration that ultimately occurs. On the basis of these findings, we propose that p21 is essential for the coordination of cell cycle exit and differentiation in the adult MPC population and that in the absence of p21, skeletal muscle regeneration is markedly impaired.

Sepsis after Bartholin's duct abscess marsupialization in a gravida.

BACKGROUND: Little information exists regarding sepsis following marsupialization of a Bartholin's duct abscess. We report a gravida who became septic after marsupialization. CASE: A 30-year-old primigravida at 32 weeks' gestation underwent marsupialization of a Bartholin's gland abscess. Postoperatively, she developed fever with maternal and fetal tachycardia. She was admitted to the hospital and started on broad-spectrum antibiotics. Her temperature increased to 39 degrees C, and she became hypotensive. Blood work demonstrated evidence of disseminated intravascular coagulopathy. The patient was stabilized with aggressive fluid resuscitation, antibiotics, transfusion of blood products and oxygen therapy. Within 24 hours, the fever and coagulopathy resolved. She was discharged on postoperative day 5 and gave birth without complications at 38 weeks' gestation. CONCLUSION: Pregnant women undergoing marsupialization of a Bartholin's gland abscess should be considered at high risk and managed accordingly.

Functional and molecular adaptations in skeletal muscle of myoglobin-mutant mice.

Myoglobin is a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers and facilitates oxygen delivery during periods of high metabolic demand. Myoglobin content in skeletal muscle increases in response to hypoxic conditions. However, we previously reported that myoglobin-null mice are viable and fertile. In the present study, we define important functional, cellular, and molecular compensatory adaptations in the absence of myoglobin. Mice without myoglobin manifest adaptations in skeletal muscle that include a fiber type transition (type I to type II in the soleus muscle), increased expression of the hypoxia-inducible transcription factors hypoxia-inducible factor (HIF)-1alpha and HIF-2 (endothelial PAS domain protein), stress proteins such as heat shock protein 27, and the angiogenic growth factor vascular endothelial growth factor (soleus muscle), as well as increased nitric oxide metabolism (extensor digitorum longus). The resulting changes in angiogenesis, nitric oxide metabolism, and vasomotor regulation are likely to account for preserved exercise capacity of animals lacking myoglobin. These results demonstrate that mammalian organisms are capable of a broad spectrum of adaptive responses that can compensate for a potentially serious defect in cellular oxygen transport.

Myogenic satellite cells: physiology to molecular biology.

Adult skeletal muscle has a remarkable ability to regenerate following myotrauma. Because adult myofibers are terminally differentiated, the regeneration of skeletal muscle is largely dependent on a small population of resident cells termed satellite cells. Although this population of cells was identified 40 years ago, little is known regarding the molecular phenotype or regulation of the satellite cell. The use of cell culture techniques and transgenic animal models has improved our understanding of this unique cell population; however, the capacity and potential of these cells remain ill-defined. This review will highlight the origin and unique markers of the satellite cell population, the regulation by growth factors, and the response to physiological and pathological stimuli. We conclude by highlighting the potential therapeutic uses of satellite cells and identifying future research goals for the study of satellite cell biology.

Adaptive mechanisms that preserve cardiac function in mice without myoglobin.

Mice lacking myoglobin survive to adulthood and meet the circulatory demands of exercise and pregnancy without cardiac decompensation. In the present study, we show that many myoglobin-deficient embryos die in utero at midgestation with signs of cardiac failure. Fetal mice that survive to gestational day 12.5, however, suffer no subsequent excess mortality. Survival in the absence of myoglobin is associated with increased vascularity and the induction of genes encoding the hypoxia-inducible transcription factors 1alpha and 2, stress proteins such as heat shock protein 27, and vascular endothelial growth factor. These adaptations are evident in late fetal life, persist into adulthood, and are sufficient to maintain normal myocardial oxygen consumption during stressed conditions. These data reveal that myoglobin is necessary to support cardiac function during development, but adaptive responses evoked in some animals can fully compensate for the defect in cellular oxygen transport resulting from the loss of myoglobin.

Life without myoglobin.

Hemoproteins are widely distributed among prokaryotes, unicellular eukaryotes, plants and animals [1]. Myoglobin, a cytoplasmic hemoprotein that is restricted to cardiomyocytes and oxidative skeletal myofibers in vertebrates, has been proposed to facilitate oxygen transport to the mitochondria [1-3]. This cytoplasmic hemoprotein was the first protein to be subjected to definitive structural analysis and has been a subject of long-standing and ongoing interest to biologists [1-3]. Recently, we utilized gene disruption technology to generate mice that are viable and fertile despite a complete absence of myoglobin [4]. This unexpected result led us to reexamine existing paradigms regarding the function of myoglobin in striated muscle.

Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF.

Myocyte nuclear factor (MNF) is a winged helix transcription factor that is expressed selectively in myogenic stem cells (satellite cells) of adult animals. Using a gene knockout strategy to generate a functional null allele at the Mnf locus, we observed that mice lacking MNF are viable, but severely runted. Skeletal muscles of Mnf-/- animals are atrophic, and satellite cell function is impaired. Muscle regeneration after injury is delayed and incomplete, and the normal timing of expression of cell cycle regulators and myogenic determination genes is dysregulated. Mnf mutant mice were intercrossed with mdx mice that lack dystrophin and exhibit only a subtle myopathic phenotype. In contrast, mdx mice that also lack MNF die in the first few weeks of life with a severe myopathy. Haploinsufficiency at the Mnf locus (Mnf+/-) also exacerbates the mdx phenotype to more closely resemble Duchenne's muscular dystrophy in humans. We conclude that MNF acts to regulate genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury. Animals deficient in MNF may prove useful for evaluation of potential therapeutic interventions to promote muscle regeneration for patients having Duchenne's muscular dystrophy.

Obstetric conditions and erythropoietin levels.

OBJECTIVE: Our purpose was to evaluate and compare erythropoietin levels as related to obstetric conditions, including acute and chronic bleeding, preeclampsia, and multiple gestations. STUDY DESIGN: During April 1999 all women in the labor and delivery unit with delivery expected to occur within 24 to 72 hours of admission had erythropoietin and hematocrit values obtained. First-trimester hematocrit values, obstetric problems, medications, and history of vaginal bleeding were obtained from patient interview, examination, and the prenatal record. Statistics were analyzed by the Student t test and chi(2). RESULTS: During a 1-month period, 302 consecutive women were divided into 5 groups on the basis of obstetric events. Group 1 consisted of women with normal, uncomplicated term singleton gestations (n = 230); group 2, women with acute vaginal bleeding (n = 10); group 3, women with chronic vaginal bleeding (n = 29); group 4, women with multiple gestations (n = 13); and group 5, women with preeclampsia (n = 16). The mean erythropoietin level in group 1 (20. 2 +/- 10.3 mU/mL) was significantly different from values in the other 4 groups (group 2, 74.2 +/- 29.2 mU/mL; group 3, 65.0 +/- 33.0 mU/mL; group 4, 34.8 +/- 16.8 mU/mL; group 5, 43.4 +/- 11.4 mU/mL; P <.001). The admission hematocrit for group 1 (0.369 +/- 0.029) was significantly greater than for groups 2 and 3 (group 2, 0.323 +/- 0. 024; group 3, 0.321 +/- 0.023; P <.001) and significantly lower than for group 5 (0.384 +/- 0.022; P <.05). CONCLUSION: The maternal serum erythropoietin level varies depending on the events occurring during gestation. Acute and chronic bleeding, multiple gestations, and preeclampsia are all associated with various serum erythropoietin levels.

The winged-helix/forkhead protein myocyte nuclear factor beta (MNF-beta) forms a co-repressor complex with mammalian sin3B.

Winged-helix/forkhead proteins regulate developmental events in both invertebrate and vertebrate organisms, but biochemical functions that establish a mechanism of action have been defined for only a few members of this extensive gene family. Here we demonstrate that MNF (myocyte nuclear factor)-beta, a winged-helix protein expressed selectively and transiently in myogenic precursor cells of the heart and skeletal muscles, collaborates with proteins of the mammalian Sin3 (mSin3) family to repress transcription. Mutated forms of MNF-beta that fail to bind mSin3 are defective in transcriptional repression and in negative growth regulation, an overexpression phenotype revealed in oncogenic transformation assays. These data extend the known repertoire of transcription factors with which mSin3 proteins can function as co-repressors to include members of the winged-helix gene family. Transcriptional repression by MNF-beta-mSin3 complexes may contribute to the co-ordination of cellular proliferation and terminal differentiation of myogenic precursor cells.

Mice without myoglobin.

Myoglobin, an intracellular haemoprotein expressed in the heart and oxidative skeletal myofibres of vertebrates, binds molecular oxygen and may facilitate oxygen transport from erythrocytes to mitochondria, thereby maintaining cellular respiration during periods of high physiological demand. Here we show, however, that mice without myoglobin, generated by gene-knockout technology, are fertile and exhibit normal exercise capacity and a normal ventilatory response to low oxygen levels (hypoxia). Heart and soleus muscles from these animals are depigmented, but function normally in standard assays of muscle performance in vitro across a range of work conditions and oxygen availability. These data show that myoglobin is not required to meet the metabolic requirements of pregnancy or exercise in a terrestrial mammal, and raise new questions about oxygen transport and metabolic regulation in working muscles.

Differential expression of mitochondrial DNA replication factors in mammalian tissues.

Mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication are regulated during development and in response to physiological stresses, but the regulatory events that control the abundance of mtDNA in cells of higher eukaryotes have not been defined at a molecular level. In this study, we observed that expression of the catalytic subunit of DNA polymerase gamma (POLgammaCAT) mRNA varies little among different tissues and is not increased by continuous neural activation of skeletal muscle, a potent stimulus to mitochondrial biogenesis. Increased copy number for the POLgamma locus in a human cell line bearing a partial duplication of chromosome 15 increased the abundance of POLgammaCAT mRNA without up-regulation of mtDNA. In contrast, expression of mitochondrial single-stranded DNA-binding (mtSSB) mRNA is regulated coordinately with variations in the abundance of mtDNA among tissues of mammalian organisms and is up-regulated in association with the enhanced mitochondrial biogenesis that characterizes early postnatal development of the heart and the adaptive response of skeletal myofibers to motor nerve stimulation. In addition, we noted that expression of mtSSB is concentrated within perinuclear mitochondria that constitute active sites of mtDNA replication. We conclude that constitutive expression of the gene encoding the catalytic subunit of mitochondrial DNA polymerase is sufficient to support physiological variations in mtDNA replication among specialized cell types, whereas expression of the mtSSB gene is controlled by molecular mechanisms acting to regulate mtDNA replication or stability in mammalian cells.

Persistent expression of MNF identifies myogenic stem cells in postnatal muscles.

Skeletal muscles contain an undifferentiated myogenic stem cell pool (satellite cells) that can be mobilized to regenerate myofibers in response to injury. We have determined that the winged helix transcription factor MNF is expressed selectively in quiescent satellite cells, which do not express known regulators of the myogenic program. Following muscle injury, MNF is present transiently in proliferating satellite cells and in centralized nuclei of regenerating myofibers, but expression declines as these fibers mature, until only the residual stem cell pool continues to express detectable levels of MNF. MNF also is expressed selectively but transiently at embryonic stages of myogenesis in the developing myotome, limb bud precursors, and heart tube, but by late fetal stages of development, MNF is down-regulated within differentiated cardiac and skeletal myocytes, and persistently high expression is observed only in satellite cells. These data identify MNF as a marker of quiescent satellite cells and suggest that downstream genes controlled by MNF serve to modulate proliferative growth or differentiation in this unique cell population.

Temporospatial expression of the small HSP/alpha B-crystallin in cardiac and skeletal muscle during mouse development.

Although the small (22 Kd) heat shock protein/alpha B-crystallin functions as a major structural protein and molecular chaperone in the vertebrate lens, little is known about the protein's role in nonlenticular tissues such as the heart and skeletal muscle. Recent studies have demonstrated that alpha B-crystallin expression is uniquely regulated during myogenesis in vitro. We report here for the first time that the temporal and spatial expression of alpha B-crystallin is similarly regulated in vivo during mouse embryogenesis. Expression of alpha B-crystallin mRNA was detected by in situ hybridization in the primitive heart at 8.5 days postconception (p.c.) and in the myotome of the somites at 10.5 days p.c. This tissue-restricted pattern was corroborated by immunohistochemical studies. alpha B-crystallin mRNA and protein expression were uniform in the developing atria and ventricles without regional differences or gradients. alpha B-crystallin expression was absent in the endocardial cushion, pulmonary trunk, aorta, and endothelium. Examination of muscle precursors revealed expression throughout the dorsoventral aspect of the myotomes and in developing skeletal muscle. Our findings suggest that alpha B-crystallin may serve pivotal roles as a structural protein and a molecular chaperone in myofiber stabilization of metabolically active tissues during early embryogenesis. Thus, early alpha B-crystallin expression in myogenic lineages supports the hypothesis that the putative functions of alpha B-crystallin are coupled to the activation of genetic programs responsible for myogenic differentiation and cardiac morphogenesis.

Postnatal development and plasticity of specialized muscle fiber characteristics in the hindlimb.

Recent progress in defining molecular components of pathways controlling early stages of myogenesis has been substantial, but regulatory factors that govern the striking functional specialization of adult skeletal muscle fibers in vertebrate organisms have not yet been identified. A more detailed understanding of the temporal and spatial patterns by which specialized fiber characteristics arise may provide clues to the identity of the relevant regulatory factors. In this study, we used immunohistochemical, in situ hybridization, and Northern blot analyses to examine the time course and spatial characteristics of expression of myoglobin protein and mRNA during development of the distal hindlimb in the mouse. In adult animals, myoglobin is expressed selectively in oxidative, mitochondria-rich, fatigue-resistant myofibers, and it provides a convenient marker for this particular subset of specialized fibers. We observed only minimal expression of myoglobin in the hindlimb prior to the second day after birth, but a rapid and large (50-fold) induction of this gene in the ensuing neonatal period. Myoglobin expression was limited, however, to fibers located centrally within the limb which coexpress myosin isoforms characteristic of type I, IIA, and IIX fibers. This induction of myoglobin expression within the early postnatal period was accompanied by increased expression of nuclear genes encoding mitochondrial proteins, and exhibited a time course similar to the upregulation of myoglobin and mitochondrial proteins, and exhibited a time course similar to the upregulation of myoglobin and mitochondrial protein expression that can be induced in adult muscle fibers by continuous motor nerve stimulation. This comparison suggests that progressive locomotor activity of neonatal animals may provide signals which trigger the development of the specialized features of oxidative, fatigue-resistant skeletal muscle fibers.

Are there really alternatives to the use of fetal tissue from elective abortions in transplantation research?

PIP: Researchers believe fetal tissue can be easily transplanted into and cure people with incurable debilitating diseases such as Parkinson's disease. In 1988, the Reagan Administration stopped funding transplantation research of fetal tissue from induced abortions. An advisory panel later decided that it is an acceptable public policy as long as certain conditions a re met. Yet the Bush Administration continued the ban. In 1992, it erroneously claimed that transplantation research could use alternative sources of fetal tissue. 1 alternative is fetal tissue obtained from ectopic pregnancies. Yet spontaneously aborted ectopic pregnancies tend not to produce recognizable or viable in culture fetal tissue and if they do the tissue has been ischemic for days. Ectopic pregnancies requiring surgical sterilization tend to be morphologically abnormal. The only likelihood of viable fetal tissue form ectopic pregnancies is a fetus with myocardial contractility before surgery. The administration also recommended use of fetal tissue from spontaneous abortions but these fetuses often have a major chromosomal or other fatal defect. Researchers cannot use chromosomally abnormal fetal tissue since it growth, development, and function are unreliable. Expulsion of the necrotic fetus tends to occur a couple of weeks after death. The Bush Administration also proposed use of tissue from stillbirths but their tissue tends to be nonviable and the tissue, even if it were viable, is generally not at the developmental stage needed for transplantation. The placenta and yolk sac were other suggested alternatives, but the placenta is likely to be less immunogenic than embryonic tissue. It can help develop certain cell lines which produce insulin or neurotransmitters like dopamine, however. The yolk sac could replace fetal liver cells in transplantation. Nevertheless the only advantage of using the suggested alternatives is the perception of them raising less ethical concern than fetal tissue from an induced abortion.^ieng

Epidermolysis bullosa acquisita induced by GM-CSF: a role for eosinophils in treatment-related toxicity.

A patient treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) developed eosinophilia and epidermolysis bullosa acquisita. The bullae were subepidermal, and filled with an inflammatory infiltrate composed predominantly of eosinophils. Immunofluorescence studies disclosed linear deposition of IgG, IgA and C3 at the basement membrane zone and immunoelectron microscopy demonstrated antibody deposition in the lamina densa and sublamina densa region; however, the patient's serum did not contain circulating antibody to basement membrane zone antigens. Staining with monoclonal antibodies revealed dense deposits of both eosinophil peroxidase and eosinophil major basic protein at the dermal-epidermal junction. The eosinophilia and skin lesions resolved upon discontinuation of GM-CSF. This case provides evidence for two hypotheses: (1) GM-CSF induced proliferation and activation of eosinophils may contribute to some of the toxicities of GM-CSF treatment, and (2) activated granulocytes, including eosinophils, may mediate blister formation in epidermolysis bullosa acquisita.

Effects of islet hormones on amylase secretion and localization of somatostatin binding sites.

The interaction of insulin and somatostatin on amylase secretion was examined in the isolated perfused rat pancreas. Exogenous insulin (10 mU/ml) significantly potentiated cholecystokinin- (CCK; 0.5 mU/ml) stimulated amylase secretion (12.47 +/- 2.9 micrograms/ml, n = 7). Glucose (16.7 mM) stimulated endogenous insulin secretion (523 +/- 66 microU/ml) and also significantly enhanced CCK-stimulated amylase secretion (13.41 +/- 2.8 micrograms/ml, n = 11). When somatostatin was included in the perfusion media, containing insulin and CCK, amylase secretion was reduced to 3.17 +/- 0.83 micrograms/ml (n = 7), a level comparable to that of CCK-stimulated amylase secretion alone. Similarly, addition of exogenous somatostatin to perfusion media, containing 16.7 mM glucose and CCK, reduced amylase secretion to 4.29 +/- 1.09 micrograms/ml (n = 9). The effect of somatostatin and insulin on carbamylcholine-stimulated amylase secretion was also examined. Exogenous insulin (50 mU/ml) potentiated carbamylcholine- (10(-8) M) stimulated amylase secretion, and addition of exogenous somatostatin to the media containing both insulin and carbamylcholine suppressed the insulin potentiation. Uptake of 125I-[Tyr11]somatostatin in the perfused pancreas was saturable as it decreased significantly with the addition of excess unlabeled somatostatin. Autoradiograms revealed uptake of the ligand by both the endocrine islets and the exocrine pancreas with the highest density of grains observed over the acini. These results support the hypothesis that islet peptides modulate the exocrine pancreas, that somatostatin inhibits amylase secretion by inhibiting the action of insulin, and that somatostatin may act directly on the exocrine pancreas via specific receptors on acinar cells.