Analysis of hippocampal local field potentials by diffusion mapped delay coordinates.

Spatial navigation through novel spaces and to known goal locations recruits multiple integrated structures in the mammalian brain. Within this extended network, the hippocampus enables formation and retrieval of cognitive spatial maps and contributes to decision making at choice points. Exploration and navigation to known goal locations produce synchronous activity of hippocampal neurons resulting in rhythmic oscillation events in local networks. Power of specific oscillatory frequencies and numbers of these events recorded in local field potentials correlate with distinct cognitive aspects of spatial navigation. Typically, oscillatory power in brain circuits is analyzed with Fourier transforms or short-time Fourier methods, which involve assumptions about the signal that are likely not true and fail to succinctly capture potentially informative features. To avoid such assumptions, we applied a method that combines manifold discovery techniques with dynamical systems theory, namely diffusion maps and Takens' time-delay embedding theory, that avoids limitations seen in traditional methods. This method, called diffusion mapped delay coordinates (DMDC), when applied to hippocampal signals recorded from juvenile rats freely navigating a Y-maze, replicates some outcomes seen with standard approaches and identifies age differences in dynamic states that traditional analyses are unable to detect. Thus, DMDC may serve as a suitable complement to more traditional analyses of LFPs recorded from behaving subjects that may enhance information yield.

The expression of ceruloplasmin in astrocytes is essential for postnatal myelination and myelin maintenance in the adult brain.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux. The absence of this protein in humans and rodents produces progressive neurodegeneration with brain iron accumulation. Astrocytes express high levels of Cp and iron efflux from these cells has been shown to be central for oligodendrocyte maturation and myelination. To explore the role of astrocytic Cp in brain development and aging we generated a specific conditional KO mouse for Cp in astrocytes (Cp cKO). Deletion of Cp in astrocytes during the first postnatal week induced hypomyelination and a significant delay in oligodendrocyte maturation. This abnormal myelin synthesis was exacerbated throughout the first two postnatal months and accompanied by a reduction in oligodendrocyte iron content, as well as an increase in brain oxidative stress. In contrast to young animals, deletion of astrocytic Cp at 8 months of age engendered iron accumulation in several brain areas and neurodegeneration in cortical regions. Aged Cp cKO mice also showed myelin loss and oxidative stress in oligodendrocytes and neurons, and at 18 months of age, developed abnormal behavioral profiles, including deficits in locomotion and short-term memory. In summary, our results demonstrate that iron efflux-mediated by astrocytic Cp-is essential for both early oligodendrocyte maturation and myelin integrity in the mature brain. Additionally, our data suggest that astrocytic Cp activity is central to prevent iron accumulation and iron-induced oxidative stress in the aging CNS.

The Arf6 activator Efa6/PSD3 confers regional specificity and modulates ethanol consumption in Drosophila and humans.

Ubiquitously expressed genes have been implicated in a variety of specific behaviors, including responses to ethanol. However, the mechanisms that confer this behavioral specificity have remained elusive. Previously, we showed that the ubiquitously expressed small GTPase Arf6 is required for normal ethanol-induced sedation in adult Drosophila. Here, we show that this behavioral response also requires Efa6, one of (at least) three Drosophila Arf6 guanine exchange factors. Ethanol-naive Arf6 and Efa6 mutants were sensitive to ethanol-induced sedation and lacked rapid tolerance upon re-exposure to ethanol, when compared with wild-type flies. In contrast to wild-type flies, both Arf6 and Efa6 mutants preferred alcohol-containing food without prior ethanol experience. An analysis of the human ortholog of Arf6 and orthologs of Efa6 (PSD1-4) revealed that the minor G allele of single nucleotide polymorphism (SNP) rs13265422 in PSD3, as well as a haplotype containing rs13265422, was associated with an increased frequency of drinking and binge drinking episodes in adolescents. The same haplotype was also associated with increased alcohol dependence in an independent European cohort. Unlike the ubiquitously expressed human Arf6 GTPase, PSD3 localization is restricted to the brain, particularly the prefrontal cortex (PFC). Functional magnetic resonance imaging revealed that the same PSD3 haplotype was also associated with a differential functional magnetic resonance imaging signal in the PFC during a Go/No-Go task, which engages PFC-mediated executive control. Our translational analysis, therefore, suggests that PSD3 confers regional specificity to ubiquitous Arf6 in the PFC to modulate human alcohol-drinking behaviors.

Life-course evidence of birth weight effects on bone mass: systematic review and meta-analysis.

A systematic review of the literature was performed in July 2011. Original papers based on longitudinal studies measuring spine, femoral neck, or total body bone mass by DXA were included (n = 17). Birth weight was positively associated with bone mass among children. The association was unclear among adolescents and weak among adults. This study aims to evaluate the association between birth weight and bone mass in future ages through a systematic review of literature and meta-analysis. A systematic review of the literature was performed in July 2011 in Medline, Web of Science and LILACS bases using key terms: ("birth size" OR "birth weight" OR birthweight OR prematurity OR premature OR "gestational age") AND (osteoporosis OR "bone mass" OR "bone density" OR "bone mineral density" OR "bone mineral content" OR "bone area") AND (longitudinal OR cohort). Original papers based on longitudinal studies measuring lumbar spine, femoral neck or total body bone mass by dual-emission X-ray absorptiometry (DXA) were included. A meta-analysis was performed using birth weight and bone mass density and/or content as continuous variables and adjusted for current height and/or weight. A total of 218 articles were retrieved from which 17 were selected and grouped into three categories according to age: studies with children; with adolescents and young adults, and studies with adults (older than 25). Five papers were included in the meta-analysis. Positive association between birth weight and bone mass was clear among children, unclear among adolescents, and weak among adults. The effect on bone mass content was stronger than those on body mass density regardless of age. Birth weight influences positively bone health in later life. Preventive health policies dealing with early-life modifiable risk factors, as birth weight, should be encouraged to attain an optimal peak bone mass as an strategy to decrease osteoporosis in the elderly.

Telehealth Care in a Pediatric and Adolescent Gynecology Clinic During the COVID-19 Pandemic.

BACKGROUND: Since the onset of the COVID-19 pandemic, health care systems have increased their telehealth services to meet the changing public health needs. Before the pandemic, telehealth was used primarily in surgical specialties for postoperative visits and rural medicine. However, out of necessity, nearly all medical and surgical subspecialties incorporated this virtual technology to improve patient health care access in a short time. Few studies have addressed telehealth in pediatric and adolescent gynecology (PAG) to date. STUDY OBJECTIVE: To describe the large-scale utilization of telehealth visits, assess patient experience, and improve access to care in a large academic ambulatory gynecology PAG clinic METHODS: This retrospective, cross-sectional quality improvement study was performed by administering patient surveys and compiling aggregate data from the EPIC electronic health record in the Division of Pediatric and Adolescent Gynecology clinics at a single children's hospital between March 2020 and March 2021. Patient demographic characteristics, payer characteristics, visit type and purpose, and patient experience were reviewed. INTERVENTIONS: Wider expansion of telehealth in PAG clinics at a single institution RESULTS: A total of 6159 telehealth appointments were performed, involving 6 clinic sites and 9 providers. Telehealth visits constituted 50% of the total ambulatory volume (12,527). Most patients were located within the institution's state (99.5%), and the remaining called into their telehealth visits from a neighboring state. Most patients were 18 years of age or younger (73%). Video visits lasted 15-30 minutes and included routine follow-up (66.3%), new/consult visits (28.4%), postoperative visits (1.6%), and urgent follow-up (0.2%). The patient population was ethnically diverse by self-identification: 61.4% White, 38.4% Hispanic, 16% Black, 4.4% Asian, and 0.4% Native Hawaiian/American Indian/Alaska Native. Payer mix included self-pay (45.5%), private payer (32.2%), and Medicaid/CHIP (22.3%). Conditions seen ranged from menstrual management (71%) and routine preventive or acute gynecologic concerns (21%) to surgical evaluation for congenital anomalies, endometriosis, fertility preservation, and genital concerns or pelvic masses (8%). Telehealth visits met patient expectations for 87.3% of respondents. Patient-reported opportunities for improvement included improving set-up instructions and more consistent audio/video connections. Challenges identified by providers included difficulty utilizing interpreters, technology limitations, and privacy constraints during HEADSS examination. CONCLUSIONS: This study demonstrates how a large, diverse volume of patients with PAG needs received appropriate care through a telehealth format during the COVID-19 pandemic. Patients were satisfied with the services, but opportunities for improvement were elicited to allow for continued refining of this health care delivery tool in the future.

Ceruloplasmin deletion in myelinating glial cells induces myelin disruption and oxidative stress in the central and peripheral nervous systems.

Ceruloplasmin (Cp) is a ferroxidase enzyme that is essential for cell iron efflux and has been postulated to have a neuroprotective role. During the myelination process, oligodendrocytes (OLs) and Schwann cells (SCs) express high levels of Cp, but the role of this enzyme in glial cell development and function is completely unknown. To define the function of Cp in the myelination of the central and peripheral nervous systems, we have conditionally knocked-out Cp specifically in OLs and SCs during early postnatal development as well as in aged mice. Cp ablation in early OLs (postnatal day 2, P2) significantly affects the differentiation of these cells and the synthesis of myelin through the first four postnatal weeks. The total number of mature myelinating OLs was reduced, and the density of apoptotic OLs was increased. These changes were accompanied with reductions in the percentage of myelinated axons and increases in the g-ratio of myelinated fibers. Cp ablation in young myelinating OLs (P30 or P60) did not affect myelin synthesis and/or OL numbers, however, Cp loss in aged OLs (8 months) induced cell iron overload, apoptotic cell death, brain oxidative stress, neurodegeneration and myelin disruption. Furthermore, Cp deletion in SCs affected postnatal SC development and myelination and produced motor coordination deficits as well as oxidative stress in young and aged peripheral nerves. Together, our data indicate that Cp ferroxidase activity is essential for OLs and SCs maturation during early postnatal development and iron homeostasis in matured myelinating cells. Additionally, our results suggest that Cp expression in myelinating glial cells is crucial to prevent oxidative stress and neurodegeneration in the central and peripheral nervous systems.

Growth from birth to adulthood and abdominal obesity in a Brazilian birth cohort.

BACKGROUND: Rapid weight gain in childhood may increase the risk of chronic adult diseases. Few studies have examined the effects of lifecourse weight gain on waist circumference (WC), hip circumference (HC), or waist-to-hip ratio (WHR). OBJECTIVE: To evaluate the effects of birthweight and weight gain from birth to age 23 years on WC, HC, and WHR in young adults. DESIGN: Population-based birth cohort study started in 1982. A sample of 856 individuals was examined in 2006. Conditional growth analyses were carried out with adjustment for confounders. WC and HC were also mutually adjusted. RESULTS: Weight gains during all age ranges studied (birthweight, 0-2, 2-4, 4-15, 15-18/19, and 18/19-23 years) were positively associated with WC and HC in both sexes. These effects were strongest from 4 to 15 years range (beta = 5.0 cm for both circumferences). Proxies for visceral adipose tissue (WHR and WC adjusted for HC) were associated with weight gain after 2 years in females and after 4 years in males. Subcutaneous adipose and muscular tissues, assessed by HC adjusted for WC, were associated with birthweight and weight gain from 0 to 2 years in both sexes, and again with weight gains from 4 to 18 years in males and 4 to 15 years in females. CONCLUSIONS: Weight gains in utero and in the first 2 years had long-term effects on HC, but weight gain after age 4 years was strongly associated with WC. Weight gains up to age 2 years may reduce cardiovascular risk associated with adult fat patterns in a middle-income setting.

Two known therapies could be useful as adjuvant therapy in critical patients infected by COVID-19. / Dos terapias conocidas podrían ser efectivas como adyuvantes en el paciente crítico infectado por COVID-19.

Pneumonia caused by coronavirus, which originated in Wuhan, China, in late 2019, has been spread around the world already becoming a pandemic. Unfortunately, there is not yet a specific vaccine or effective antiviral drug for treating COVID-19. Many of these patients deteriorate rapidly and require intubation and are mechanically ventilated, which is causing the collapse of the health system in many countries due to lack of ventilators and intensive care beds. In this document we review two simple adjuvant therapies to administer, without side effects, and low cost that could be useful for the treatment of acute severe coronavirus infection associated with acute respiratory syndrome (SARS-CoV-2). VitaminC, a potent antioxidant, has emerged as a relevant therapy due to its potential benefits when administered intravenous. The potential effect of vitaminC in reducing inflammation in the lungs could play a key role in lung injury caused by coronavirus infection. Another potential effective therapy is ozone: it has been extensively studied and used for many years and its effectiveness has been demonstrated so far in multiples studies. Nevertheless, our goal is not to make an exhaustive review of these therapies but spread the beneficial effects themselves. Obviously clinical trials are necessaries, but due to the potential benefit of these two therapies we highly recommended to add to the therapeutic arsenal.

Characterization of a functional NTPDase in the endoplasmic reticulum of rat submandibular salivary gland.

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent with a NTPDase identity. The presence of a NTPDase was demonstrated by immunohistochemistry in rat SMG. Immunoreactivity was stronger in ductal cells than in mucous and serous acini. Although this enzyme was observed in the plasma membrane, colocalization with the ER marker calnexin revealed a specific subcellular localization in this organelle of all three types of cell. The putative function of this NTPDase activity in salivary glands is discussed.

A protocol of human animal interaction to habituate young sheep and goats for behavioural studies.

Animal habituation is key to obtain reliable data on behavioural studies but detailed procedures to achieve it are scarce. This study designed a set of actions to habituate sheep and goats to human observers. Pelibuey sheep (n = 15) and Criollo goats (n = 10) were classified as (a) avoider, flight from human interaction, or (b) follower, seek human interaction. Habituation was measured by the reduction of flight distance by avoiders, or number of followers in the presence of observers. The habituation protocol consisted of a gradually increased series of five manoeuvres, either challenge (for avoiders) or evasion (for seekers), performed first inside a pen and subsequently in a grass paddock. Habituation was considered successful when animals could be observed from a 1-m distance without flight or following the observer. In the pen, habituation took 12 and 13 days for sheep and goats, respectively. Meanwhile, in the grass paddock habituation took 10 days, for both species. The number of challenge and evasion series was negatively correlated with the flight distance in sheep and with the number of followers in goats. This protocol is simple and practical to implement and enables animal habituation for behavioural studies.

Plasma spectroscopy analysis technique based on optimization algorithms and spectral synthesis for arc-welding quality assurance.

A new plasma spectroscopy analysis technique based on the generation of synthetic spectra by means of optimization processes is presented in this paper. The technique has been developed for its application in arc-welding quality assurance. The new approach has been checked through several experimental tests, yielding results in reasonably good agreement with the ones offered by the traditional spectroscopic analysis technique.

The influence of action execution on end-state comfort and underlying movement kinematics: An examination of right and left handed participants.

People typically move in an anticipatory manner, planning the intended action in advance to minimize the energy costs associated with producing the action (e.g., Rosenbaum et al., 2009). This is exemplified behaviorally in the end-state comfort effect, which is characterized by the selection of an uncomfortable initial posture to enable a comfortable posture upon completion of the movement (Rosenbaum et al., 1990). The main objective of this study was to further investigate the end-state comfort effect in left- and right-handers (N=20). More specifically, to: (a) understand the influence of mode of action execution; and (b) delineate the role of handedness. The overturned glass task (Fischman, 1997) was used as means of assessment, where participants were asked to demonstrate picking up a glass to pour water in four modes of execution: (1) pantomime without a stimulus; (2) pantomime with image of the glass as a guide; (3) pantomime with glass as a guide; and (4) grasping the glass. End-state comfort was displayed regardless of mode of execution, hand used to complete the task or handedness group. However, kinematic analysis revealed distinct differences, highlighting how movement parameters are altered as a result the mode of action execution.

Golli Myelin Basic Proteins Modulate Voltage-Operated Ca(++) Influx and Development in Cortical and Hippocampal Neurons.

The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.

PU.1 (Spi-1) autoregulates its expression in myeloid cells.

PU.1 (Spi-1), a member of the Ets transcription factor family, is predominantly expressed in myeloid (granulocytes, monocytes and macrophages) and B cells. PU.1 is upregulated early during commitment of multipotential progenitors to the myeloid lineages and inhibition of PU.1 function in human CD34+ progenitors prior to this upregulation blocks myeloid colony formation. Since PU.1 expression appears to play a role in hematopoietic development, we characterized the PU.1 promoter. Here we report that the murine PU.1 promoter, as well as the human promoter, demonstrate tissue-specific reporter gene expression in myeloid cell lines but not in T cells and HeLa (non-hematopoietic cells) cells. Deletion analysis of the PU.1 promoter indicates that tissue-specific functional elements are encoded in the -61 to -39 bp and -7 to +34 bp regions. The first region contains a functional octamer (Oct) site at -54 bp and an Sp1 site at -39 bp. The second contains a binding site at +20 bp for both PU.1 itself and the related ets family member Spi-B. In vivo footprinting assays demonstrate that a hypersensitive band was detected at the PU.1 site in myeloid cells but not in HeLa. A mutation of the PU.1 site which abolished PU.1 binding caused a significant decrease in promoter activity. Mutation of the Oct and/or Sp1 site results in a lesser decrease of promoter activity in myeloid cells. Co-transfection of PU.1 or Spi-B in cells lacking PU.1 and Spi-B specifically transactivated a minimal promoter containing the PU.1 binding site, indicating that PU.1 can activate its own promoter elements in an autoregulatory loop. Positive autoregulation of the PU.1 promoter may play an important role in the function of PU.1 in myeloid cells.

Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2(+)-dependent adenosinetriphosphatase.

The Ca2(+)-dependent adenosinetriphosphatase (Ca2(+)-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is phosphorylated by inorganic phosphate (Pi) in the absence of Ca2+. The reaction can be described by the following simplified scheme: [formula: see text] where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E.Pi is a noncovalent and acid-labile complex. The reaction is Mg2(+)-dependent. Membrane fragments deposited on Millipore filters were successively perfused with two solutions, at constant flow. The effluent samples were analyzed. The perfused solutions were Ca2+ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [32P]Pi, 32P binding is only detected in the presence of Mg2+, indicating the formation of the phosphoenzymes (E.Pi and E-P). Following perfusions of the phosphoenzymes with 5% trichloroacetic acid, 32P release indicates the amount of the acid-labile moiety (E.Pi). After phosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 +/- 0.12, and 1.48 +/- 0.10 nmol of Pi/mg of protein ( +/- S.E., n = 5), after phosphorylations with 20 microM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2(+)-ATPase cycle. Keq = k2/k-2), in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg.Pi complex, since Mg2+ is necessary for step 1 in the above scheme.

Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes.

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca2+ to provide the simultaneous activation of the (Ca2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 degrees C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.

Manganese as a cosubstrate for the phosphorylation of the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase with orthophosphate.

The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P(i) was characterized using Mn as a Mg analogue. Steady state and transient fluorescence and radioisotopic techniques were used; the affinities of Mn and P(i) for the enzyme and the rate constants of the phosphorylation and dephosphorylation reactions were determined, under several conditions. The reactions were carried out at pH 5.5 to minimize the binding of contaminant Ca to the transport sites, thus avoiding the use of Ca chelators. The apparent affinity of Mn binding at low [Mn] is larger in the absence of P(i) (35 microM) than in the presence of saturating P(i) (70 microM). On the contrary, the apparent affinity of Mn for the formation of the phosphoenzyme increases, from 1.5 mM to 0.15 mM, upon increasing [P(i)] in the millimolar range. The apparent affinty of P(i) for the formation of the phosphoenzyme also increases, from 2.2 mM to 0.2 mM, upon increasing [Mn] in the millimolar range. The equilibrium of the phosphoenzyme with the noncovalent Mn.P(i). Enzyme complex favors the covalent species. The simulation of a reaction model including the random binding of 2 Mn and I P(i) per mol of ATPase and a noncovalent complex in equilibrium with the phosphoenzyme, using a set of equilibrium constants deduced from the results, agree with the experimental data.

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle.

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.

Voltage-gated Ca2+ entry promotes oligodendrocyte progenitor cell maturation and myelination in vitro.

We have previously shown that the expression of voltage-operated Ca(++) channels (VOCCs) is highly regulated in the oligodendroglial lineage and is essential for proper oligodendrocyte progenitor cell (OPC) migration. Here we assessed the role of VOCCs, in particular the L-type, in oligodendrocyte maturation. We used pharmacological treatments to activate or block voltage-gated Ca(++) uptake and siRNAs to specifically knock down the L-type VOCC in primary cultures of mouse OPCs. Activation of VOCCs by plasma membrane depolarization increased OPC morphological differentiation as well as the expression of mature oligodendrocyte markers. On the contrary, inhibition of L-type Ca(++) channels significantly delayed OPC development. OPCs transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents showed reduce Ca(++) influx by ~75% after plasma membrane depolarization, indicating that Cav1.2 is heavily involved in mediating voltage-operated Ca(++) entry in OPCs. Cav1.2 knockdown induced a decrease in the proportion of oligodendrocytes that expressed myelin proteins, and an increase in cells that retained immature oligodendrocyte markers. Moreover, OPC proliferation, but not cell viability, was negatively affected after L-type Ca(++) channel knockdown. Additionally, we have tested the ability of L-type VOCCs to facilitate axon-glial interaction during the first steps of myelin formation using an in vitro co-culture system of OPCs with cortical neurons. Unlike control OPCs, Cav1.2 deficient oligodendrocytes displayed a simple morphology, low levels of myelin proteins expression and appeared to be less capable of establishing contacts with neurites and axons. Together, this set of in vitro experiments characterizes the involvement of L-type VOCCs on OPC maturation as well as the role played by these Ca(++) channels during the early phases of myelination.

Jaw dysfunction is associated with neck disability and muscle tenderness in subjects with and without chronic temporomandibular disorders.

PURPOSE: Tender points in the neck are common in patients with temporomandibular disorders (TMD). However, the correlation among neck disability, jaw dysfunction, and muscle tenderness in subjects with TMD still needs further investigation. This study investigated the correlation among neck disability, jaw dysfunction, and muscle tenderness in subjects with and without chronic TMD. Participants. Forty females between 19 and 49 years old were included in this study. There were 20 healthy controls and 20 subjects who had chronic TMD and neck disability. METHODS: Subjects completed the neck disability index and the limitations of daily functions in TMD questionnaires. Tenderness of the masticatory and cervical muscles was measured using an algometer. RESULTS: The correlation between jaw disability and neck disability was significantly high (r = 0.915, P < 0.05). The correlation between level of muscle tenderness in the masticatory and cervical muscles with jaw dysfunction and neck disability showed fair to moderate correlations (r = 0.32-0.65). CONCLUSION: High levels of muscle tenderness in upper trapezius and temporalis muscles correlated with high levels of jaw and neck dysfunction. Moreover, high levels of neck disability correlated with high levels of jaw disability. These findings emphasize the importance of considering the neck and its structures when evaluating and treating patients with TMD.