OBJECTIVE

To develop a model based on empirical data and human energetics to predict the total energy cost of weight gain and obligatory increase in energy intake and/or decrease in physical activity level associated with weight gain in children and adolescents.

METHODS

One-year changes in weight and body composition and basal metabolic rate (BMR) were measured in 488 Hispanic children and adolescents. Fat-free mass (FFM) and fat mass (FM) were measured by DXA and BMR by calorimetry. Model specifications include the following: body mass (BM) = FFM + FM, each with a specific energy content, cff (1.07 kcal/g FFM) and cf (9.25 kcal/g FM), basal energy expenditure (EE), kff and kf, and energetic conversion efficiency, eff (0.42) for FFM and ef (0.85) for FM. Total energy cost of weight gain is equal to the sum of energy storage, EE associated with increased BM, conversion energy (CE), and diet-induced EE (DIEE).

RESULTS

Sex- and Tanner stage-specific values are indicated for the basal EE of FFM (kff) and the fat fraction in added tissue (fr). Total energy cost of weight gain is partitioned into energy storage (24% to 36%), increase in EE (40% to 57%), CE (8% to 13%), and DIEE (10%). Observed median (10th to 90th percentile) weight gain of 6.1 kg/yr (2.4 to 11.4 kg/yr) corresponds at physical activity level (PAL) = 1.5, 1.75, and 2.0 to a total energy cost of weight gain of 244 (93 to 448 kcal/d), 267 (101 to 485 kcal/d), and 290 kcal/d (110 to 527 kcal/d), respectively, and to a total energy intake of 2695 (1890 to 3730), 3127 (2191 to 4335), and 3551 (2487 to 4930) kcal/d, respectively. If weight gain is caused by a change in PAL alone and PAL(0) = 1.5 at baseline t = 0, the model indicates a drop in PAL of 0.22 (0.08 to 0.34) units, which is equivalent to 60 (18 to 105) min/d of walking at 2.5 mph.

CONCLUSIONS

Halting the development or progression of childhood obesity, as observed in these Hispanic children and adolescents, by counteracting its total energy costs will require a sizable decrease in energy intake and/or reciprocal increase in physical activity.

We performed a comparative investigation of the immunomorphological characteristics of lymphatic and blood microvascular endothelial cells in normal human skin, cutaneous lymphangiomas, and hemangiomas, employing a pre-embedding immunogold electron microscopic technique. We stained for cell membrane proteins that are commonly used for light microscopic characterization of blood endothelial cells. With blood microvascular endothelial cells, we observed uniform labeling of the luminal cell membranes with monoclonal antibodies (MAbs) JC70 (CD31), EN-4 (CD31), BMA120, PAL-E, and QBEND-10 (CD34), and strong staining of the vascular basal lamina for Type IV collagen under normal and pathological conditions. In contrast, lymphatic microvascular endothelial cells in normal human skin and in lymphangiomas displayed, in addition to a luminal labeling, pronounced expression of CD31 and CD34 along the abluminal cell membranes. Moreover, CD31 was preferentially detected within intercellular junctions. The expression of CD34 was mostly confined to abluminal endothelial microprocesses and was upregulated in lymphangiomas and hemangiomas. Type IV collagen partially formed the luminal lining of initial lymphatics and occasionally formed bridges over interendothelial gaps. Our findings suggest a function of transmigration protein CD31 in recruitment of dendritic cells into the lymphatic vasculature. CD34 labeling may indicate early endothelial cell sprouting. The distribution of Type IV collagen also supports its role as a signal for migration and tube formation for lymphatic endothelial cells.

We are developing a high performance double lumen cannula (DLC) for a minimally invasive, ambulatory and percutaneous paracorporeal artificial lung (PAL). The Wang-Zwische (W-Z) DLC was designed for percutaneous insertion into the Internal Jugular (IJ) vein with a drainage lumen open to both the superior vena cava (SVC) and the inferior vena cava (IVC) maximizing venous drainage. A separate collapsible but nondistensible membrane infusion lumen open to the right atrium (RA) achieves minimal recirculation allowing for total gas exchange. The W-Z DLC prototypes are made by a proprietary dip molding process with the "molded in" flat wire spiral stainless steel spring resulting in a flexible yet kink resistant thin wall (0.1 mm) outer cannula with one piece construction. With the ultra thin membrane infusion lumen collapsed, an introducer shaft fits tightly within the drainage lumen to facilitate insertion with placement at the SVC-RA-IVC junction. The W-Z DLC prototypes were tested while connected to a compact pump-gas exchanger circuit in three sheep (2 acute and one 15 day performance study). Insertion was simple, using standard percutaneous insertion techniques. Recirculation was as low as 2%. The 15 day performance study demonstrated our prototype 26 Fr W-Z DLC can achieve 2 L/min blood flow with minimal recirculation. The W-Z DLC design minimizes recirculation rate, maximizes flow lumen cross-sectional area, and maximizes achievable blood flow to enhance gas exchange performance allowing for one site percutaneous venovenous support.

DNA palindromes are rare in humans but are associated with meiosis-specific translocations. The conserved Mre11/Rad50/Nbs1 (MRN) complex is likely directly involved in processing palindromes through the homologous recombination pathway of DNA repair. Using the fission yeast Schizosaccharomyces pombe as a model system, we show that a 160-bp palindrome (M-pal) is a meiotic recombination hotspot and is preferentially eliminated by gene conversion. Importantly, this hotspot depends on the MRN complex for full activity and reveals a new pathway for generating meiotic DNA double-strand breaks (DSBs), separately from the Rec12 (ortholog of Spo11) pathway. We show that MRN-dependent DSBs are formed at or near the M-pal in vivo, and in contrast to the Rec12-dependent breaks, they appear early, during premeiotic replication. Analysis of mrn mutants indicates that the early DSBs are generated by the MRN nuclease activity, demonstrating the previously hypothesized MRN-dependent breakage of hairpins during replication. Our studies provide a genetic and physical basis for frequent translocations between palindromes in human meiosis and identify a conserved meiotic process that constantly selects against palindromes in eukaryotic genomes.

Mitochondria are organelles derived from an intracellular alpha-proteobacterium. The biogenesis of mitochondria relies on the assembly of beta-barrel proteins into the mitochondrial outer membrane, a process inherited from the bacterial ancestor. Caulobacter crescentus is an alpha-proteobacterium, and the BAM (beta-barrel assembly machinery) complex was purified and characterized from this model organism. Like the mitochondrial sorting and assembly machinery complex, we find the BAM complex to be modular in nature. A approximately 150 kDa core BAM complex containing BamA, BamB, BamD, and BamE associates with additional modules in the outer membrane. One of these modules, Pal, is a lipoprotein that provides a means for anchorage to the peptidoglycan layer of the cell wall. We suggest the modular design of the BAM complex facilitates access to substrates from the protein translocase in the inner membrane.

The gene encoding yeast-enhanced green fluorescent protein (GFP) was placed under control of ALS gene promoters in Candida albicans. The <em>PALS</em>-GFP reporter strains were validated using various techniques including a new real-time RT-PCR assay to quantify ALS gene expression. The <em>PALS</em>-GFP reporter strains were grown in media that promoted yeast or germ tube forms, and the resulting fluorescence was measured by flow cytometry. In addition to results that indicate differences in ALS gene expression due to growth medium, growth stage and developmental programme, new data show large differences in transcriptional level among the ALS genes. Expression of ALS1 was associated with transfer of the <em>PALS</em>1-GFP strain to fresh growth medium. ALS3 expression increased markedly when germ tubes were visible microscopically and ALS7 expression exhibited a transient peak between 2 and 3 h following inoculation into fresh YPD medium. Transcription from the ALS1 and ALS3 promoters was strongest among those tested and contrasted markedly with the weaker promoter strength at the ALS5, ALS6, ALS7 and ALS9 loci. These weaker transcriptional responses were also observed using real-time RT-PCR measurements on wild-type C. albicans cells. Assuming a positive correlation between transcriptional level and protein production, these results suggest that some Als proteins are abundant on the C. albicans cell surface while others are produced at a much lower level.

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes both an acidic proteinase and an alkaline proteinase, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Recessive mutations at four unlinked loci, named PALPALPAL mutations was isolated from a wild-type genomic library, which turned out to be a C-terminally truncated form of a 585-residue transcriptional factor of the His2Cys2 zinc finger family, which we propose to call YlRim101p. Another C-terminally truncated version of YlRim101p (419 residues) is encoded by the dominant RPH2 mutation previously isolated as expressing alkaline protease independently of the pH. YlRim101p is homologous to the transcriptional activators Rim101p of Saccharomyces cerevisiae, required for entry into meiosis, and PacC of Aspergillus nidulans and Penicillium chrysogenum, which were recently shown to mediate regulation by ambient pH. YlRim101p appears essential for mating and sporulation and for alkaline proteinase derepression. YlRIM101 expression is autoregulated, maximal at alkaline pH, and strongly impaired by PAL mutations.

The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdc11p and Cdc12p), Candida albicans proteins that accumulate in the hyphal phase (CaCdc3p and CaCdc10p), mouse brain-specific (H5p) and lymphocyte (Diff6p) proteins, Drosophila melanogaster (Dm) protein Pnutp (which is localized to the cleavage furrow of dividing cells), a Diff6p homologue (DmDiff6p), and the Sc septin protein (Sep1hp), a homologue of the 10-nm filament proteins of Sc. One strongly conserved region contains a potential ATP-GTP-binding domain. Primer extension analysis revealed six major transcription start points (tsp) beginning at -142 relative to the ATG start codon. The sequence immediately upstream from the tsp contains consensus binding sites for the HAP2/3/4 and ABFI transcription factors, a T-rich sequence and two putative novel elements for mid to late sporulation, termed SPR3 and PAL. Electrophoretic mobility shift assay (EMSA) and footprint analyses demonstrated that the ABFI protein binds to a region containing the putative ABFI site in vitro, and site-directed mutagenesis showed that the ABFI motif is essential for expression of SPR3 at the appropriate stage in sporulating cells.

Microorganisms are significantly affected when the ambient pH of their environment changes. They must therefore be able to sense and respond to these changes in order to survive. Previous investigators have studied various fungal species to define conserved pH-responsive signaling pathways. One of these pathways, known as the Pal/Rim pathway, is activated in response to alkaline pH signals, ultimately targeting the PacC/Rim101 transcription factor. Although the central signaling components are conserved among divergent filamentous and yeast-like fungi, there is some degree of signaling specificity between fungal species. This specificity exists primarily in the downstream transcriptional targets of this pathway, likely allowing differential adaptation to species-specific environmental niches. In this review, the role of the Pal/Rim pathway in fungal pH response is discussed. Also highlighted are functional differences present in this pathway among human fungal pathogens, differences that allow these specialized microorganisms to survive in the various micro-environments of the infected human host.

OBJECTIVE

To compare the effect of wearing, then ceasing to wear, progressive addition lenses (PALs) versus single vision lenses (SVLs) on myopia progression in children with high accommodative lag to evaluate accommodative lag and mechanical tension as theories of myopia progression.

METHODS

Eighty-five children (age range, 6-11 years) with spherical equivalent (SE) cycloplegic autorefraction between -0.75 D and -4.50 D were randomly assigned to wear SVLs or PALs for 1 year; all children wore SVLs a second year. Children had high accommodative lag and also had near esophoria if their myopia was greater than -2.25 D SE. The primary outcome after each year was the previous year's change in SE.

RESULTS

When the children were randomly assigned to SVLs or PALs, the adjusted 1-year changes in SE were -0.52 D (SVL group) and -0.35 D (PAL group; treatment effect = 0.18 D; P = 0.01). When all children wore SVLs the second year, there was no difference in myopia progression between SVL and former PAL wearers (0.06 D; P = 0.50). Accommodative lag was not associated with myopia progression.

CONCLUSIONS

The statistically significant, but clinically small, PAL effect suggests that treatments aimed at reducing foveal defocus may not be as effective as previously thought in myopic children with high accommodative lag. Finding no evidence of treatment loss after discontinuing PAL wear supports hyperopic defocus-based theories such as accommodative lag; however, not finding an association between accommodative lag and myopia progression is inconsistent with the PAL effect being due to decreased foveal blur during near work. (Clinical Trials.gov number, NCT00335049.).

In order to test the hypothesis that ozone (O3)-induced changes in lung function and respiratory tract injury/inflammation are greater in subjects with asthma than in normal subjects, we exposed 18 asthmatic subjects, on separate days, to O3 (0.2 ppm) and filtered air for 4 h during exercise. Symptom questionnaires were administered before and after exposure, and pulmonary function tests (FEV1, FVC, and specific airway resistance [SRaw]) were performed before, during, and immediately after each exposure. Fiberoptic bronchoscopy, with proximal airway lavage (PAL) of the isolated left main bronchus and bronchoalveolar lavage (BAL; bronchial fraction, the first 10 ml of fluid recovered) of the right middle lobe, was performed 18 h after each exposure. The PAL, bronchial fraction, and BAL fluids were analyzed for the following endpoints: total and differential cell counts; total protein, lactate dehydrogenase (LDH), fibronectin, interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF), myeloperoxidase (MPO), and transforming growth factor-beta (TGF beta 2) concentrations. We found a significant O3 effect on FEV1, FVC, SRaw (p < 0.04) and lower respiratory symptoms (p < 0.001) for the asthmatic subjects. Ozone exposure also significantly increased the percent neutrophils in PAL (p < 0.01); percent neutrophils, total protein, and IL-8 in the bronchial fraction (p < 0.001, p < 0.05, and p < 0.01, respectively); and the percent neutrophils, total protein, LDH, fibronectin, IL-8, GM-CSF, and MPO in BAL (p < 0.001, p < 0.01, p < 0.01, p < 0.001, p < 0.05, p < 0.01, and p < 0.001, respectively) for the asthmatic subjects. There were no significant differences in the lung function responses of the asthmatic subjects in comparison with a group of normal subjects (n = 81) previously studied using an identical protocol, although there was a trend toward a greater O3-induced increase in SRaw in the asthmatic subjects (p < 0.13). In contrast, the asthmatic subjects showed significantly greater (p < 0.05) O3-induced increases in several inflammatory endpoints (percent neutrophils and total protein concentration) in BAL as compared with normal subjects who underwent bronchoscopy (n = 20). Our results indicate that asthmatic persons may be at risk of developing more severe O3-induced respiratory tract injury/inflammation than normal persons, and may help explain the increased asthma morbidity associated with O3 pollution episodes observed in epidemiologic studies.

Chitin, a linear polysaccharide composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc) residues, and chitosan, the fully or partially N-acetylated, water-soluble derivative of chitin composed of (1-->4)-linked GlcNAc and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN), have been proposed as elicitors of defense reactions in higher plants. We tested and compared the ability of purified (1-->4)-linked oligomers of GlcNAc (tetramer to decamer) and of GlcN (pentamer and heptamer) and partially N-acetylated chitosans with degrees of acetylation (DA) of 1%, 15%, 35%, 49%, and 60% and average degrees of polymerization between 540 and 1100 to elicit phenylalanine ammonia-lyase (PAL) and peroxidase (POD) activities, lignin deposition, and microscopically and macroscopically visible necroses when injected into the intercellular spaces of healthy, nonwounded wheat (Triticum aestivum L.) leaves. Purified oligomers of (1-->4)-linked GlcN were not active as elicitors, whereas purified oligomers of (1-->4)-linked GlcNAc with a degree of polymerization>>/= 7 strongly elicited POD activities but not PAL activities. Partially N-acetylated, polymeric chitosans elicited both PAL and POD activities, and maximum elicitation was observed with chitosans of intermediate DAs. All chitosans but not the chitin oligomers induced the deposition of lignin, the appearance of necrotic cells exhibiting yellow autofluorescence under ultraviolet light, and macroscopically visible necroses; those with intermediate DAs were most active. These results suggest that different mechanisms are involved in the elicitation of POD activities by GlcNAc oligomers, and of PAL and POD activities by partially N-acetylated chitosan polymers and that both enzymes have to be activated for lignin biosynthesis and ensuing necrosis to occur.

Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified from differentiating secondary xylem of loblolly pine (Pinus taeda L.). Native molecular weight of the enzyme was estimated to be 280,000, with a subunit molecular weight of 74,000; isoelectric point, 5.8; and Michaelis constant for i-phenylalanine, 27 micromolar. No evidence was obtained for the existence of isoforms of the enzyme, nor for negative cooperativity of substrate binding. Polyclonal antibodies were raised against the phenylalanine ammonia-lyase subunit and used to identify a pal clone in an expression library of xylem complementary DNA (cDNA). Polymerase chain reaction, using oligonucleotide primers made from N-terminal amino acid sequence and from the 5' end of the clone isolated from the expression library, was also used to isolate cDNA clones. These methods yielded cDNA clones covering the protein coding region of the pal messenger RNA. Comparisons of nucleotide sequence of pal cDNAs from pine, bean, sweet potato, and rice showed 60 to 62% identity between the pine clone and the angiosperm clones.

OBJECTIVE

To test the hypothesis that pediatric shock is a common cause of death and functional morbidity and that pediatric advanced life support (PALS)/advanced pediatric life support (APLS) resuscitation in the community hospital setting improves child health outcomes.

METHODS

This study included all children consecutively transported to 5 regional, tertiary care children's hospitals over 4 years, and is a prospective cohort study comparing outcomes in children who did or did not receive PALS/APLS resuscitation in the community hospital.

RESULTS

Shock occurred in 37% of the patients transferred to the tertiary centers. Regardless of trauma status, children with shock had an increased mortality rate compared with those without shock (all patients: 11.4% vs 2.6%), trauma patients (28.3% vs 1.2%), and nontrauma patients (10.5% vs 2.8%). Early shock reversal was associated with reduced mortality (5.06% vs 16.37%) and functional morbidity (1.56% vs 4.11%) rates. Early use of PALS/APLS-recommended interventions was associated with reduced mortality (8.69% vs 15.01%) and functional morbidity (1.24% vs 4.23%) rates. After controlling for center, severity of illness, and trauma status, early reversal of shock and use of PALS/APLS-recommended interventions remained associated with reduced morbidity and mortality rates.

CONCLUSIONS

Shock is common in children who are transferred for tertiary care. Pediatric shock recognition and resuscitation in the community hospital improves survival and functional outcome regardless of diagnostic category. The development of shock/trauma systems for children with and without trauma seems prudent.

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.

BACKGROUND

The benefits of Peer Assisted Learning (PAL) are well established with positive effects on examination scores, student satisfaction and personal and professional development reported. PAL is increasingly utilised as a resource within medical education where the restrictions on resources have forced teachers to look at creating new educational environments which can be delivered at a lower cost. This study sought to evaluate the processes at work as the emphasis of PAL research to date has largely been on the consideration of student outcomes.

METHODS

Fifth-year medical undergraduates, who had completed their communication skills modular training and attended a preparatory workshop, facilitated a role-play session for their second-year colleagues within an Early Patient Contact programme. Semi-structured interviews and focus groups were used to collect data at different time points in order to establish the views of peer learners and tutors towards this new method of teaching. The data was analysed according to the principles framework analysis using N-vivo software. Themes were shared and debated with the multidisciplinary team of authors and a concordance of views on common themes was reached after discussion and debate.

RESULTS

Analysis of the data resulted in the emergence of three thematic categories: Learning Environment, Educational Exchange and Communication and Modelling. The data demonstrated a concordance of the views between peer tutors and learners on barriers and levers of this approach as well as a heightened awareness of the learning environment and the educational exchange occurring therein.

CONCLUSIONS

The data is significant as it not only demonstrates a high level of acceptability among tutors and learners for PAL but also indicates the reciprocity of educational exchange that appears to occur within the PAL setting. This study highlights some of the unique characteristics of PAL and we recommend the development of further qualitative studies around peer learners and tutors views of this process.

BACKGROUND

Binge drinking may lead to brain damage. The aim of the present study was to compare the cognitive abilities of binge and non-binge drinkers in tasks which test functions linked to discrete areas of the prefrontal cortex.

METHODS

Non-binge and binge drinkers were identified according to their binge score derived from the Alcohol Use Questionnaire. Cognitive performance was tested with the Spatial Working Memory task (SWM) linked to the dorsolateral prefrontal cortex, Intra/Extradimensional Shift and reversal task (IED) linked to dorsolateral prefrontal cortex (shift) and to orbitofrontal cortex (reversal), Paired Associates Learning task (PAL) linked to temporal cortex, and Reaction Time Task (RTI) a task measuring motor impulsivity (Inferior frontal gyrus). Personality traits, alcohol outcome expectancies and mood were also evaluated.

RESULTS

Binge drinkers recorded a significantly shorter movement time to target in the RTI, and completed fewer stages on first trial in the PAL, compared with non-bingers. In the IED as well as in the SWM, only female binge drinkers were more impaired than non-binge drinkers.

CONCLUSIONS

Functions linked to dorsolateral prefrontal cortex may be more impaired in female, whereas functions linked with the temporal lobe may be impaired in both male and female binge drinkers compared to non-binge drinkers. Functions linked to orbitofrontal cortex were not impaired. The increased speed of response in the RTI in binge drinkers may indicate an increased motor impulsivity in binge drinkers.

We have advocated the idea of agonist therapy for treating cocaine addiction. This strategy involves administration of stimulant-like medications (eg, monoamine releasers) to alleviate withdrawal symptoms and prevent relapse. A major limitation of this approach is that many candidate medicines possess significant abuse potential because of activation of mesolimbic dopamine (DA) neurons in central nervous system reward circuits. Previous data suggest that serotonin (5-HT) neurons can provide an inhibitory influence over mesolimbic DA neurons. Thus, it might be predicted that the balance between DA and 5-HT transmission is important to consider when developing medications with reduced stimulant side effects. In this article, we discuss several issues related to the development of dual DA/5-HT releasers for the treatment of substance use disorders. First, we discuss evidence supporting the existence of a dual deficit in DA and 5-HT function during withdrawal from chronic cocaine or alcohol abuse. Then we summarize studies that have tested the hypothesis that 5-HT neurons can dampen the effects mediated by mesolimbic DA. For example, it has been shown that pharmacological manipulations that increase extracellular 5-HT attenuate stimulant effects produced by DA release, such as locomotor stimulation and self-administration behavior. Finally, we discuss our recently published data about PAL-287 (naphthylisopropylamine), a novel non-amphetamine DA-/5-HT-releasing agent that suppresses cocaine self-administration but lacks positive reinforcing properties. It is concluded that DA/5-HT releasers might be useful therapeutic adjuncts for the treatment of cocaine and alcohol addiction, obesity, and even attention deficit disorder and depression.

It has been reported that omental fat tissue is a good source of human microvascular endothelial cells. By characterization we demonstrate that the epitheloid cells isolated from omental tissue are not endothelial cells, but mesothelial cells. They contain abundant cytokeratins 8 and 18, which are absent in endothelial cells, and vimentin. No staining with the endothelial-specific antibodies EN-4 and PAL-E is observed. A faint and diffuse staining of von Willebrand factor (vWF) is seen in mesothelial cells, whereas microvascular endothelial cells from subcutaneous fat display vWF in distinct granular structures. Human peritoneal mesothelium produces plasminogen activator-dependent fibrinolytic activity, which is essential in the resolution of fibrous exudates and may therefore be important in preventing the formation of fibrous peritoneal adhesions. This fibrinolytic activity is plasminogen activator-dependent, but has not been fully characterized. We report here that human omental tissue mesothelial cells in vitro produce large amounts of tissue-type plasminogen activator (t-PA), together with type 1 and 2 plasminogen activator inhibitor (PAI-1 and PAI-2). PAI-1 is predominantly secreted into the culture medium, whereas the major part of PAI-2 is found in the cells. No urokinase-type plasminogen activator is detected. On stimulation with the inflammatory mediator tumor necrosis factor (TNF), at least a threefold decrease in t-PA antigen is observed, together with an increase in both PAI-1 and PAI-2. TNF also induces a marked change in cell shape. Whereas TNF and bacterial lipopolysaccharide (LPS) have similar effects on the production of PA inhibitor by human endothelial cells, LPS has no or only a relatively small effect on the fibrinolytic properties of mesothelial cells. The decreased fibrinolytic activity induced by the cytokine TNF may impair the natural dissolution of fibrin deposits at the peritoneum in the presence of an inflammatory reaction.

BACKGROUND

Concerns of a decrease in physical activity levels (PALs) of children and a concurrent increase in childhood obesity exist worldwide. The exact relation between these two parameters however has as yet to be fully defined in children.

OBJECTIVE

This study examined the relation in 47 children, aged 5-10.5 y (mean age 8.4+/-0.9 y) between habitual physical activity, minutes spent in moderate, vigorous and hard intensity activity and body composition parameters.

METHODS

Total energy expenditure (TEE) was calculated using the doubly labelled water technique and basal metabolic rate (BMR) was predicted from Schofield's equations. PAL was determined by PAL=TEE/BMR. Time spent in moderate, vigorous and hard intensity activity was determined by accelerometry, using the Tritrac-R3D. Body fatness and body mass index (BMI) were used as the two measures of body composition.

RESULTS

Body fat and BMI were significantly inversely correlated with PAL (r=-0.43, P=0.002 and r=-0.45, P=0.001). Times spent in vigorous activity and hard activity were significantly correlated to percentage body fat (r=-0.44, P=0.004 and r=-0.39, P=0.014), but not BMI. Children who were in the top tertiles for both vigorous activity and hard activity had significantly lower body fat percentages than those in the middle and lowest tertiles. Moderate intensity activity was not correlated with measures of body composition.

CONCLUSIONS

As well as showing a significant relation between PAL and body composition, these data intimate that there may be a threshold of intensity of physical activity that is influential on body fatness. In light of world trends showing increasing childhood obesity, this study supports the need to further investigate the importance of physical activity for children.

The Aspergillus nidulans pH-responsive transcription factor PacC is modulated by limited, two-step proteolysis. The first, pH-regulated cleavage occurs in the 24-residue highly conserved "signaling protease box" in response to the alkaline pH signal. This is transduced by the Pal signaling pathway, containing the predicted calpain-like cysteine protease and likely signaling protease, PalB. In this work, we carried out classical mutational analysis of the putative signaling protease PalB, and we describe 9 missense and 18 truncating loss-of-function (including null) mutations. Mutations in the region of and affecting directly the predicted catalytic cysteine strongly support the deduction that PalB is a cysteine protease. Truncating and missense mutations affecting the C terminus highlight the importance of this region. Analysis of three-hemagglutinin-tagged PalB in Western blots demonstrates that PalB levels are independent of pH and Pal signal transduction. We have followed the processing of MYC(3)-tagged PacC in Western blots. We show unequivocally that PalB is essential for signaling proteolysis and is definitely not the processing protease. In addition, we have replaced 15 residues of the signaling protease box of MYC(3)-tagged PacC (pacC900) with alanine. The majority of these substitutions are silent. Leu481Ala, Tyr493Ala, and Gln499Ala result in delayed PacC processing in response to shifting from acidic to alkaline medium, as determined by Western blot analysis. Leu498Ala reduces function much more markedly, as determined by plate tests and processing recalcitrance. Excepting Leu498, this demonstrates that PacC signaling proteolysis is largely independent of sequence in the cleavage region.

Previous studies have shown that thyroid hormone receptors can form homo- and heterodimeric complexes when binding to response elements. We report here the binding characteristics of thyroid hormone receptor (TR) homo- and heterodimers binding to synthetic oligonucleotides with directly and palindromically repeated consensus motifs (AGGTCA). Binding assays showed that TR homodimer formation on DNA had a low specificity and cooperativity, and very fast off rates. In contrast, TRs and retinoic acid receptors readily formed heterodimers with higher specificity and affinity on direct repeats of the AGGTCA motif spaced by four or five nucleotides, although these heterodimer/DNA complexes were only moderately stable when compared to DNA-bound TR/retinoid X receptor heterodimers. Also, TR/retinoic acid receptor heteromeric binding to other elements, including the synthetic T3RE-pal element, was of low specificity. These biochemical results suggest that TRs are unlikely to regulate transcription as homodimers in vivo, and that TR heterodimers mediate the effects of thyroid hormone.

Pathologische Anatomie Leiden-endothelium antibody has been used for more than 20 years as a marker for vascular endothelium. Despite its widespread use, the target of this antibody was only recently identified as plasmalemma vesicle-associated protein-1 (PV-1). However, no function has been identified for this molecule. Here we report that activation of human umbilical vein endothelial cells with tumor necrosis factor-alpha resulted in a remarkable redistribution of PV-1 toward the peripheral areas of the cells. Furthermore, in vitro endpoint transmigration experiments showed that transcellularly migrating lymphocytes are surrounded by rings containing PV-1 and caveolin-1. Moreover, PV-1 associates physically with vimentin. In addition, administration of anti-PV-1 antibody during capillary flow assays resulted in a significant inhibition of lymphocyte transmigration through the endothelial cell layer, whereas rolling and adhesion were unaffected. In vivo blockage of PV-1 by an antibody in acute peritonitis and air pouch model resulted in a significant decrease in the number of migrating leukocytes. Here we thus define leukocyte transendothelial migration as the first known function for PV-1.

Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.