Receptor Tyrosine Kinases ror1/2 and ryk Are Co-expressed with Multiple Wnt Signaling Components During Early Development of Sea Urchin Embryos.

AbstractA combination of receptors, co-receptors, and secreted Wnt modulators form protein complexes at the cell surface that activate one or more of the three different Wnt signaling pathways (Wnt/ß-catenin, Wnt/JNK, and Wnt/Ca2+). Two or more of these pathways are often active in the same cellular territories, forming Wnt signaling networks; however, the molecular mechanisms necessary to integrate information from these pathways in these situations are unclear in any in vivo model system. Recent studies have implicated two Wnt binding receptor tyrosine kinases, receptor tyrosine kinase-like orphan receptor (Ror) and related-to-receptor tyrosine kinase (Ryk), in the regulation of canonical and non-canonical Wnt signaling pathways, depending on the context; however, the spatiotemporal expression of these genes in relation to Wnt signaling components has not been well characterized in most deuterostome model systems. Here we use a combination of phylogenetic and spatiotemporal gene expression analyses to characterize Ror and Ryk orthologs in sea urchin embryos. Our phylogenetic analysis indicates that both ror1/2 and ryk originated as single genes from the metazoan ancestor. Expression analyses indicate that ror1/2 and ryk are expressed in the same domains of many Wnt ligands and Frizzled receptors essential for the specification and patterning of germ layers along the early anterior-posterior axis. In addition, both genes are co-expressed with Wnt signaling components in the gut, ventral ectoderm, and anterior neuroectoderm territories later in development. Together, our results indicate that Ror and Ryk have a complex evolutionary history and that their spatiotemporal expression suggests that they could contribute to the complexity of Wnt signaling in early sea urchin embryogenesis.

Cytochemistry of phosphatases of the sarcoplasmic reticulum. II. In situ localization of the Mg-dependent enzyme.

The distribution of the Mg-dependent ATPase associated with a microsomal fraction of rabbit psoas muscle was studied histochemically and its localization in relation to the vesicles of the fraction and to the structure of intact fixed muscle was determined. Although enzyme activity was retained after fixation in hydroxyadipaldehyde and in glyoxal, it was lost after fixation in glutaraldehyde or after 4 hr fixation in formaldehyde. Activity was optimally demonstrated when incubations were conducted at 17 degrees C, in media containing 125 mM Trismaleate buffer, pH 7.5, 5 mM ATP, 4 mM MgCl(2), and 1 mM Pb(NO(3))(2). After such incubations, activity was present throughout the sarcoplasmic reticulum, but was absent from the T system. Activation by Na or K could not be demonstrated histochemically. However, the other biochemical properties of the enzyme in the isolated vesicles and in intact muscle were similar with respect to Mg dependence, substrate specificity, inhibition by Ca, N-ethyl maleimide, p-hydroxymercuribenzoate, and lack of inhibition by ouabain.

Cytochemistry of phosphatases of the sarcoplasmic reticulum. I. Biochemical studies.

A microsomal fraction was isolated from rabbit psoas muscle by a modification of Muscatello's method. The fraction contained a Mg-dependent ATPase which had a pH optimum of 7.5. Activity was further stimulated by addition of Na or K or other monovalent cations to the reaction mixture, but synergistic activation by Na and K, and ouabain inhibition, could not be demonstrated. The enzyme hydrolyzed only ATP (adenosine triphosphate) and ITP (inosine triphosphate) at appreciable rates, but Na or K stimulated activity only when ATP was used as substrate. Activity was inhibited by Ca and by low concentrations of Na deoxycholate, and was sensitive to inhibition by thiol group reagents. The enzyme could be distinguished from another enzyme, also present in the fraction, which was Ca-activated, and which exhibited a wider substrate specificity, different pH activation characteristics, lower specific activity, lack of stimulation by Na or K, and less sensitivity to inhibition by deoxycholate and by thiol group reagents. These findings formed the basis for demonstration of the Mg-dependent ATPase in situ.

Alteration of tight junctional permeability in the rat parotid gland after isoproterenol stimulation.

The permeability of junctional complexes to ultrastructural tracers of different molecular weight and the freeze-fracture appearance of junctional structure were investigated in the resting and stimulated rat parotid gland. Tracers were administered retrogradely via the main excretory duct, and allowed to flow by gravity (16 mmHg) into the gland for 15-60 min. Secretion was induced in some animals by intraperitoneal injection of isoproterenol. In resting glands, the tracers microperoxidase , cytochrome c, myoglobin, tyrosinase (subunits), and hemoglobin were restricted to the luminal space of the acini and ducts. In glands stimulated 1-4 h before tracer administration, reaction product for microperoxidase , cytochrome c, myoglobin, and tyrosinase was found in the intercellular and interstitial spaces, whereas hemoglobin was usually retained in the lumina. In contrast, horseradish peroxidase and lactoperoxidase appeared to penetrate the tight junctions and reaction product was localized in the extracellular spaces in both resting and stimulated glands. Diffuse cytoplasmic staining for horseradish peroxidase and lactoperoxidase was frequently observed in acinar and duct cells. The distribution of horseradish peroxidase was similar in both Sprague-Dawley and Wistar-Furth rats, and at concentrations of 0.1-10 mg/ml in the tracer solution. Freeze-fracture replicas of stimulated acinar cells revealed an increased irregularity of the tight junction meshwork, but no obvious gaps or discontinuities were observed. These findings indicate that (a) tight junctions in the resting rat parotid gland are impermeable to tracers of molecular weight greater than or equal to 1,900; (b) stimulation with isoproterenol results in a transient increase in junctional permeability allowing passage of tracers of molecular weight less than or equal to 34,500; (c) junctional permeability cannot be directly correlated with junctional structure; and (d) the behavior of horseradish peroxidase and lactoperoxidase in the rat parotid gland is inconsistent with their molecular weights. Cell membrane damage due to the enzymatic activity or binding of these two tracers may account for the observed distribution.

Changes in tight junctions of thyroid epithelium with changes in thyroid activity.

The morphology of the tight junction of rat thyroid epithelium was examined in freeze-fractured material fixed in glutaraldehyde and briefly glycerinated. In normal thyroids the overall appearance of this junctional specialization resembled that of other cell types in many respects. Short-term changes in thyroid activity and hypophysectomy for 3 wk did not obviously affect the appearance of tight junctions. Feeding of the goitrogen, thiouracil, which stimulates secretion of thyroid-stimulating hormone, resulted in the appearance of some very narrow and some very wide, tight junctions or sometimes junctions with both wide and narrow regions within the same cell.

Metabolic studies of isolated human eccrine sweat glands.

This paper describes a method for isolating and studying the metabolism of human eccrine sweat glands. (a) Electron microscopy of glands which had been isolated and then incubated for an hour revealed no apparent alteration in morphology. (b) Known variation in gland size (male > female > children) was reflected in the relative rates of lactate production. (c) Lactate production was approximately 1.5 nmoles/gland per hr in the absence of glucose and rose to 2.7 at physiological concentrations of glucose (5.6 mmoles/liter). This amount of lactate production agrees well with the amounts found in sweat. (d) Both adrenergic (epinephrine) and cholinergic (methacholine) stimuli increased lactate production. (e) Glycogen depletion was demonstrated during incubation. (f) O(2) consumption was measured and aerobic metabolism was found to account for less than 1% of the energy derived from anaerobic pathways. These studies demonstrate that the large amounts of lactate appearing in human eccrine sweat can be accounted for by glandular metabolism and that both glycogen and glucose can be used as substrates.

Electron microscopic identification of adrenergic nerve endings on thyroid epithelial cells.

Electron microscopic observations were made of the microinnervation of the thyroids of normal mice and of mice given false adrenergic neurotransmitters, and the effects of the drugs on thyroidal norepinephrine uptake were measured. In addition to adrenergic nerve terminals on thyroid blood vessels, structures resembling nerve endings were seen in close apposition to the bases of thyroid epithelial cells. Because they take up false adrenergic neurotransmitters, and undergo degenerative change after administration of cytotoxic false neurotransmitters (6-hydroxydopamine and 5,7-dihydroxytryptamine), which also reduce thyroid norepinephrine uptake, they probably represent adrenergic nerve terminals on thyroid follicles. If so, they afford a morphological basis for the direct effects of catecholamines on thyroid function, which have been reported.

Immunocytochemical localization of catechol-O-methyltransferase in the pregnant rat uterus.

Light microscopic immunocytochemical observations of catechol-O-methyl-transferase (COMT) localization in the pregnant rat uterus were made with a specific antibody to soluble rat liver COMT and the peroxidase-antiperoxidase technique. The changes in cellular localization of COMT were followed from the onset of pregnancy to 12 h after delivery of the last fetus. COMT first appeared on day 5 of pregnancy in endometrial epithelial cells of the uterus. On day 7, COMT appeared in the decidual cells on the antimesometrial side of the uterus close to the implanting blastocyst. Two morphologically distinct, COMT-positive, decidual cells were observed: polygonal cells and stellate cells. After parturition, COMT-positive decidual cells remained in the decidua basalis. The possible physiological roles of COMT in the pregnant uterus are discussed.

Gap junctions in thyroid epithelium of the rat.

Gap junctions were observed on the lateral plasma membrane of the typical thyroid epithelial cell in freeze-fracture preparations of rat thyroid gland. The gap junctions were 0.1 to 0.4 mum in diameter and were composed of closely packed, approximately 80 nm particles. They were located from 1 to 6 mum from the apical surface of the cell.

Anesthetic regimen effects on a pediatric porcine model of asphyxial arrest.

The effects of three anesthetic regimens on an established model of pediatric porcine hypoxic-hypercarbic arrest were examined. Twenty-four preadolescent miniature piglets were paralyzed, mechanically ventilated and anesthetized with one of three regimens: IM + IV pentobarbital (n = 8); IM + IV ketamine (n = 8); or IM ketamine+inhaled isoflurane (n = 8). Asphyxial cardiopulmonary arrest was induced and, after and 8 min cardiac arrest nonintervention interval, a standardized protocol of manual CPR with mechanical ventilation was performed. Outcome variables included incidence of ventricular fibrillation, time to cardiac arrest, endogenous plasma epinephrine levels and arteriovenous epinephrine gradients. IV Ketamine anesthesia produced the highest incidence of ventricular fibrillation (P < 0.01 vs. pentobarbital and isoflurane). Time to asphyxia induced cardiac arrest was greatest for the pentobarbital group (P < 0.05 vs. ketamine and isoflurane). During induction of asphyxial cardiac arrest (low cardiac flow), endogenous venous epinephrine accumulation was highest in the pentobarbital anesthetized group (P < 0.05). After 8 min of untreated cardiac arrest and 1 min of CPR (low flow), arterial epinephrine levels were highest in the ketamine group (P < 0.05). Endogenous epinephrine gradients were venous > arterial in all groups at the end of the 8 min cardiac arrest non-intervention interval (no flow). After 1 min of CPR, the gradients had either equalized or reversed to arterial > venous in all groups except for pentobarbital. As designed and expected, return of spontaneous circulation did not occur in any animal. We conclude that, in developing models of porcine asphyxial cardiopulmonary arrest and resuscitation to simulate pediatric human arrest, variations in anesthetic regimen produce significant differences in parameters that are important to consider: time to asphyxia induced cardiac arrest, fibrillation threshold, plasma epinephrine level and arteriovenous epinephrine gradient. Anesthetic effects need to be carefully considered and clearly explained to facilitate the interpretation of studies of interventions in cardiopulmonary arrest and resuscitation.

Heliox improves pulmonary mechanics in a pediatric porcine model of induced severe bronchospasm and independent lung mechanical ventilation.

BACKGROUND: A helium-oxygen gas mixture (heliox) has low gas density and low turbulence and resistance through narrowed airways. The effects of heliox on pulmonary mechanics following severe methacholine-induced bronchospasm were investigated and compared to those of a nitrogen-oxygen gas mixture (nitrox) in an innovative pediatric porcine, independent lung, mechanical ventilation model. RESULTS: All of the lungs showed evidence of severe bronchospasm after methacholine challenge. Prospective definition of 'heliox response' was a 15% or greater improvement in lung function in the lung receiving heliox compared with the matched lung receiving nitrox. Seven out of 10 pigs responded to heliox therapy with respect to resistance and eight out of 10 pigs responded to heliox therapy with respect to compliance and tidal volume (P < 0.03). After crossover from nitrox to heliox, eight out of eight lungs significantly improved with respect to tidal volume, resistance and compliance (P < 0.001). After crossover from heliox to nitrox all eight lungs showed a significant increase in resistance and a significant decrease in tidal volume (P < 0.001). CONCLUSIONS: In a pediatric porcine model of acute, severe methacholine-induced bronchospasm and independent lung mechanical ventilation, administration of heliox improves pulmonary mechanics, gas flow, and ventilation. Administration of heliox should be considered for support of pediatric patients with acute, severe bronchospasm requiring mechanical ventilation through small artificial airways.

A comparison of the distribution of enzymatically and non-enzymatically produced lead phosphate in insect flight muscle.

Lead phosphate precipitates were produced in indirect flight muscles of Phormia regina by sequential incubation in solutions containing lead and inorganic phosphate and their distribution was compared with those produced by ATP hydrolysis in the presence of lead. Enzymatically produced precipitates were associated almost exclusively with thick filaments. Non-enzymatically produced precipitates were associated with thick filaments but were also found associated with thin filaments in significant numbers.

Changes in tight junctions of rat intestinal crypt cells associated with changes in their mitotic activity.

The freeze-fracture appearance of tight junctions between rat duodenal crypt cells was studied in normal, mitotically suppressed, and mitotically enhanced animals. In normal animals crypt cell tight junctions present a pleomorphic appearance. The population includes junctions resembling postmitotic junctions of the intestinal villus, junctions composed largely or completely of particle chains, and regions at the cell apex in which junctions are absent for 3-4 micron distances laterally. Mitotic suppression by inhibition of DNA synthesis with cytosine arabinoside results in the disappearance of pleomorphism and crypt tight junctions progressively come to resemble those of the intestinal villus. With recovery from the drug and further synchronization with Colcemid, the crypt cells undergo a mitotic burst, and all varieties of unusual junctional configurations are observed with increased frequency.

Morphology of the granular secretory glands in skin of poison-dart frogs (Dendrobatidae).

The granular glands of nine species of dendrobatid frogs were examined using light and electron microscopy. The glands are surrounded by a discontinuous layer of smooth muscle cells. Within the glands proper the secretory cells form a true syncytium. Multiple flattened nuclei lie at the periphery of the gland. The peripheral cytoplasm also contains mitochondria, rough surfaced endoplasmic reticulum, the Golgi apparatus, and an abundance of smooth endoplasmic reticulum. Centrally, most of the gland is filled with membrane-bound granules surrounded by amorphous cytoplasm. Few other organelles are found in this region. Early in the secretory cycle, the central part of the gland is filled with flocculent material which appears to be progressively partitioned off by membranes to form the droplet anlage. As granules form, the structure of the contents becomes progressively more vesicular. Dense vesicles, which bud off from the Golgi apparatus, fuse with the granular membrane during the development of granules, and might contain enzymes involved in toxin synthesis. The granules at this point resemble multivesicular bodies. Their structure is similar in all species of dendrobatid frogs even though the different frogs secrete substances of different chemical structure and toxicity.

Tracer and freeze fracture observations on developing tight junctions in fetal rat thyroid.

The development of tight junctions in fetal rat thyroid from the sixteenth to the twentieth days of gestation was examined with conventional ultrastructural methods and freeze-fracture preparations. These results were compared with those obtained using lanthanum hydroxide and horseradish peroxidase (HRP) tracers. Tight junctions appear to arise on the plasma membranes of fetal thyroid cells by the aggregation and fusion of linear particle chains which appear at several discrete sites on the plasma membrane of developing follicular cells. Tracer studies show that they are effective barriers to the passage of HRP from the outset, are freely penetrated by La3+ at the sixteenth and seventeenth days of gestation, but progressively lose permeability to La3+ from the seventeenth to twentieth days of gestation. However, freeze-fracture observations suggest that La3+ must penetrate into the follicular lumen through the tight junction elements, for the follicular lumen, when it appears, is always completely surrounded by a continuous though sometimes rudimentary meshwork of tight junction elements. The results suggest that the tight junction forms an effective barrier to the passage of large macromolecules, e.g. thyroglobulin, from very early stages in its development. The La3+ results suggest that decreased resistance of the intercellular pathway, possibly related to the development of transepithelial potentials, may occur during this period in development.

Behavioral covariation in the treatment of delusional verbalization with contingency management.

A 23-year-old female with the dual diagnosis of mild mental retardation and emotional disturbance was referred for treatment because of frequent disruptive outbursts. A brief inpatient behavior analysis suggested that delusional verbalization reliably preceded all other aberrant behaviors. Consequently treatment focused mainly on delusional verbalization; the additional aberrant behaviors were observed for behavioral covariation. The treatment package consisted of verbal instructions, modeling of appropriate eating behaviors, contingent verbal reprimand and a 2-min removal of food, and differential reinforcement of other behaviors. The design was a multiple baseline across settings. Results indicate a marked decrease in delusional verbalization in all settings, and, a correlated decrease for disruption, screaming, and out-of-seat behaviors.

The reduction of seizure-like behaviors through contingency management.

A 22-year-old mildly retarded male, diagnosed as epileptic and paranoid schizophrenic, was admitted to an inpatient behavior unit for a medication assessment and possible behavioral treatment of uncontrolled seizure-like behavior. Since the patient had rarely been allowed out of his living unit in the past year, treatment consisted of a half-hour walk with a preferred staff member made contingent upon a 24-hr period of time free of any occurrence of seizure-like behaviors. An ABAB reversal design was used to assess experimental control. Data indicated that seizures decreased from a mean of 0.70 per day during the initial baseline phase to 0.36 per day during the final treatment phase. Additionally, the duration of seizures decreased from a mean of 50 min per seizure during the initial baseline phase to 17 min per seizure during the final treatment phase, although the contingency was applied irrespective of duration of seizure-like activity. The patient was discharged to a transitional setting until a community placement could be located.

Activities of an ethics consultation service in a Tertiary Military Medical Center.

The Joint Commission on Accreditation of Healthcare Organizations requires hospitals to have a mechanism to address issues of medical ethics. Most hospitals, especially those in the military, have an ethics committee composed solely of members who serve as an additional duty. To enhance the ethics consultation service, the 59th Medical Wing created a position under the chief of the medical staff for a full-time, fellowship-trained, medical ethicist. After establishment of this position, the number of consultations increased, a systematic program for caregiver education was developed and delivered, and an organizational presence was achieved by instituting positions on the institutional review board, the executive committee of the medical staff, and the credentials committee. Issues in medical care are becoming increasingly complicated, due in large part to financial stresses and technological advancements. Ethics consultation can help prevent and resolve many of these problems. This report discusses the activities of the first year of a full-time ethicist in a tertiary military medical center.

Increasing distance and independence of ambulation in elderly nursing home residents.

We tested the effectiveness of a prompt-and-praise procedure for increasing walking distance and independence in eight nursing home residents. Walking distance and method of ambulation were observed just prior to mealtimes; in addition, mobility and social interaction were time-sampled in the living areas throughout the day. Treatment was lagged in a multiple baseline design across lunch and dinner meals within subjects, and across subjects within each of three units. In the mealtime setting, two subjects began walking the maximum scored distance during baseline; the other six subjects showed a marked increase in walking beginning with the first meal in which the intervention was applied. Six of the eight subjects also progressed to more independent means of ambulation. Generalization of walking to the second meal was observed in all four of the subjects in whom this could be assessed. Generalization across subjects was not observed nor was generalization to the living areas. Staff successfully implemented the procedures in the mealtime settings and the effects were maintained at the 4-month follow-up.