Search

Background: Malnutrition is a pro-inflammatory state, yet data on nutritional risk factors and development of acute graft-versus-host disease (aGVHD) are extremely limited., Procedure: We conducted a retrospective cohort analysis of pediatric patients up to age 21 years who underwent allogeneic hematopoietic stem cell transplantation (HSCT) at the Children's Hospital of Philadelphia from January 2011 to September 2014 to determine whether malnutrition was associated with development of aGVHD and early mortality. We identified body mass index (BMI) percentile and serum albumin levels as potential markers of malnutrition and defined two composite nutritional risk variables as any of the following: albumin < 2.8 g/dl, weight loss ≥10% from baseline, and low BMI [<25th (NUT25) or <5th percentile (NUT5)]. Nutritional markers and GVHD grade were assessed at baseline, 30, 60, and 90 days post-HSCT, and patients were censored upon development of GVHD., Results: BMI <25th or <5th percentile, NUT25, and NUT5 at the beginning of any 30-day period predicted a three- to fourfold risk of developing of severe (grade III-IV) aGVHD in the subsequent 30 days in models adjusted for age, sex, donor source, and degree of human leukocyte antigen matching. Mortality at day 100 was low, but NUT25 risk at baseline conferred an increased risk of death (7.9% vs. 1%, P = 0.035)., Conclusions: Malnutrition is a targetable risk factor in pediatric HSCT; prospective trials are needed to investigate this relationship further and identify effective nutritional interventions., (© 2017 Wiley Periodicals, Inc.)

Objective: To assess the long-term efficacy of antireflux surgery on Barrett's esophagus (BE) using BRAVO wireless pH monitoring., Background: BE is associated with chronic gastroesophageal reflux and esophageal cancer. Till date, studies have failed to demonstrate that preventing gastroesophageal reflux with antireflux surgery halts the progression of BE, often because of difficulties in objectively proving an effective antireflux barrier., Methods: Since 1991, all patients undergoing antireflux surgery across 2 hospital sites have been followed in a prospective database. Patients with BE and at least 5 years follow up after antireflux surgery were identified. All patients completed a clinical outcome questionnaire and underwent endoscopic assessment and histological evaluation of their BE. Fourty-eight hours pH monitoring was then performed with the wireless BRAVO system., Results: A total of 50 patients (40 males:10 females) were included in the study, with an average follow up of 11.9 years. Approximately, 92% (46/50) reported their outcome of surgery as "excellent" or "good" and 86% (43/50) reported "none" or "mild" symptoms. Histological regression of BE was seen in 41% (20/49). Lower esophageal acid exposure (percentage time pH < 4) was significantly greater in those with no pathological regression (P = 0.008). Moreover, 64% (32/50) showed endoscopic reduction in the length of BE. Acid exposure was also significantly less in the group showing endoscopic reduction of BE (%time pH < 4, 0.2 vs 3.6, P = 0.007)., Conclusions: Antireflux surgery is safe and effective in patients with Barrett's esophagus. An intact fundoplication, as assessed with BRAVO wireless pH monitoring, suggests that antireflux surgery may halt the progression of Barrett's esophagus, and this might reduce the risk of cancer development.

Recent years have seen increasing study of stimulus-responsive hydrogels constructed from aptamer-connected DNA building blocks. Presumably due to a lack of simple, quantitative tools with which to measure gel responsiveness, however, the literature describing these materials is largely qualitative. In response, we demonstrate here simple, time-resolved, multiscale methods for measuring the response kinetics of these materials. Specifically, by employing trace amounts of fluorophore-quencher labeled cross-linkers and the rheology of entrapped fluorescent particles, we simultaneously measure dissolution at molecular, hundred-nanometer, and hundred-micron length-scales. For our test-bed system, an adenine-responsive hydrogel, we find biphasic response kinetics dependent on both effector concentration and depth within the gel and a dissolution pattern uniform at scales longer than a few times the monomer-monomer distance. Likewise, we find that, in agreement with theoretical predictions, dissolution kinetics over the hundred nanometer length scale exhibit a power-law-like dependence on the fraction of disrupted cross-links before a distinct crossover from solid-like to liquid-like behavior.

Optimal therapy for relapsed APL in pediatric patients is controversial. Allogeneic HSCT is an alternative, with event-free survival of 70-75%. We report a pediatric patient with APL who relapsed 28 months after CBT from her sibling and then was treated with BMT from the same donor. Bone marrow was selected for higher cell dose, donor availability, and partial donor chimerism. Persistent molecular remission was achieved, currently at 65 months after BMT. This case suggests the potential role of GVL activity in APL and illustrates the use of different cell sources from the same donor in allogeneic transplantation for pediatric patients., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: We present a case of confirmed clozapine-induced myocarditis in a patient who was not naïve to the drug., Method: This patient, who had been stable on clozapine for 10 years, relapsed following self-cessation. Asymptomatic throughout inpatient re-titration, serum cardiac enzymes were nonetheless routinely taken., Results: Occult myocarditis was only discovered due to an elevated Troponin I, and was confirmed by cardiac imaging., Conclusions: Once thought to be the preserve of initial exposure to the medication, clozapine-induced myocarditis can occur at any re-titration point if the immunological milieu permits. We therefore recommend routine monitoring of serum cardiac enzymes with all patients undergoing titration of clozapine, regardless of whether they have previously been stable on the drug., (© The Royal Australian and New Zealand College of Psychiatrists 2014.)

Evidence suggests that patients with irritable bowel syndrome (IBS) are more vigilant to pain-associated stimuli. The aims of this study were to compare women with IBS (n = 20) to healthy control (HC, n = 20) women on pain sensitivity, conditioned pain modulation (CPM) efficiency, and salivary cortisol levels before and after the CPM test and to examine the relationship of CPM efficiency with gastrointestinal pain, somatic pain, psychological distress symptoms, and salivary cortisol levels in each group. Women, aged 20-42 years, gave consent, completed questionnaires, and kept a symptom diary for 2 weeks. CPM efficiency was tested with a heat test stimulus and cold water condition stimulus in a laboratory between 8 and 10 a.m. on a follicular phase day. Salivary cortisol samples were collected just before and after the experimental testing. Compared to the HC group, women with IBS reported more days with gastrointestinal and somatic pain/discomfort, psychological distress, fatigue, and feeling stressed. During the CPM baseline testing, women with IBS reported greater pain sensitivity compared to the HC group. There was no significant group difference in salivary cortisol levels nor in CPM efficiency, though a post-hoc analysis showed a higher prevalence of impaired CPM efficiency among IBS subjects with more severe lower-GI symptoms. In the IBS group, reduced CPM efficiency was associated with daily abdominal pain/discomfort and psychological distress. Overall, women with IBS exhibited an increased sensitivity to thermal stimuli. Impaired CPM was present in a subset of women with IBS., (© The Author(s) 2014.)

Drought conditions in Amazonia are associated with increased fire incidence, enhancing aerosol emissions with degradation in air quality. Quantifying the synergic influence of climate and human-driven environmental changes on human health is, therefore, critical for identifying climate change adaptation pathways for this vulnerable region. Here we show a significant increase (1.2%-267%) in hospitalisations for respiratory diseases in children under-five in municipalities highly exposed to drought. Aerosol was the primary driver of hospitalisations in drought affected municipalities during 2005, while human development conditions mitigated the impacts in 2010. Our results demonstrated that drought events deteriorated children's respiratory health particularly during 2005 when the drought was more geographically concentrated. This indicates that if governments act on curbing fire usage and effectively plan public health provision, as a climate change adaptation procedure, health quality would improve and public expenditure for treatment would decrease in the region during future drought events.

Background: To explore whether poor initial insight during a first episode of mania with psychotic features was predictive of poor psychosocial and clinical outcomes at 18 months., Methods: Secondary analysis was performed on data collected during an 8-week RCT comparing the efficacy of olanzapine versus chlorpromazine as an adjunct to lithium, and at 18-month follow-up. 74 participants were divided into three groups (no insight, partial insight, and full insight) according to the insight item from the Young Mania Rating Scale (YMRS). Differences between these three groups were examined at baseline and at 18 months on measures of symptoms (YMRS, HAMD-21, and CGI-S), and social and occupational functioning (SOFAS). Baseline differences between the three groups were determined using general linear models and chi-squared analyses. Group differences from baseline to 18-month follow-up were determined using repeated measures general linear models., Results: At baseline there were significant differences between the three insight groups in terms of mania and functioning, but at 18 months all groups had improved significantly in terms of psychopathology, mania, depression and social and occupational functioning. There were no significant differences between the three groups at study completion with respect to these domains., Limitations: The study was limited by the lack of availability of a more detailed rating scale for insight, and it did not account for the duration of untreated psychosis (DUI)., Conclusions: Poor initial insight during a first episode of mania with psychotic features does not predict poor clinical and psychosocial outcome at 18 months., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Background: There is a learning curve associated with laparoscopic antireflux surgery which has an impact on patient outcomes. It is unclear, however, whether this can be eliminated by supervision of early cases by experienced surgeons. The aim of this study was to evaluate the impact of training under supervision on outcomes for laparoscopic fundoplication., Method: Patients undergoing primary laparoscopic antireflux surgery from 1995 to 2009 were identified from a prospective database. Patients were classified according to whether they were operated on by an experienced consultant or supervised trainee, and sub-categorised according to the presence of a very large hiatus hernia. A standardised questionnaire was used to assess outcomes for heartburn, dysphagia and satisfaction at 1 and 5 years follow-up. Outcomes for the study groups were compared., Results: One thousand seven hundred and ten patients underwent surgery; 1,112 were operated on by consultants and 598 by trainees. The peri-operative complication rate was not different between the groups, although in patients operated on by trainees, there were increased rates of endoscopic dilatation (9 vs. 5 % p = 0.014) and re-operation (9 vs. 6 %, p = 0.031), and a lower satisfaction rate (76 vs. 82 %, p = 0.044) within 5 years of surgery. All other outcomes were similar for trainees vs. consultants., Conclusion: The learning curve for laparoscopic fundoplication had a small, but statistically significant, impact on patient outcomes, with slightly lesser outcomes when surgery was undertaken by trainees, even when supervised by experienced surgeons. Although the differences were not large, they raise questions about equipoise and highlight ethical dilemmas with teaching new generations of surgeons.

DNA protection during starvation (Dps) proteins, dodecameric assemblies of four-helix bundle subunits, contribute to protection against reactive oxygen species. Deinococcus radiodurans, which is characterized by resistance to DNA damaging agents, encodes two Dps homologs, of which Dps-1 binds DNA with high affinity. DNA binding requires N-terminal extensions preceding the four-helix bundle core. Composed of six Dps-1 dimers, each capable of DNA binding by N-terminal extensions interacting in consecutive DNA major grooves, dodecameric Dps-1 would be predicted to feature six DNA binding sites. Using electrophoretic mobility shift assays and intrinsic tryptophan fluorescence, we show that dodecameric Dps-1 binds 22-bp DNA with a stoichiometry of 1:6, consistent with the existence of six DNA binding sites. The stoichiometry of Dps-1 binding to 26-bp DNA is 1:4, suggesting that two Dps-1 dodecamers can simultaneously occupy opposite faces of this DNA. Mutagenesis of an arginine (Arg132) on the surface of Dps-1 leads to a reduction in DNA binding. Altogether, our data suggest that duplex DNA lies along the dimer interface, interacting with Arg132 and the N-terminal α-helices, and they extend the hexagonal packing model for Dps-DNA assemblies by specifying the basis for occupancy of available DNA binding sites., (Copyright © 2011 Wiley Periodicals, Inc.)

Metastatic melanoma is a fatal disease due to the lack of successful therapies and biomarkers for early detection and its incidence has been increasing. Genetic studies have defined recurrent chromosomal aberrations, suggesting the location of either tumor suppressor genes or oncogenes. Transcription factor 21 (TCF21) belongs to the class A of the basic helix-loop-helix family with reported functions in early lung and kidney development as well as tumor suppressor function in the malignancies of the lung and head and neck. In this study, we combined quantitative DNA methylation analysis in patient biopsies and in their derived cell lines to demonstrate that TCF21 expression is downregulated in metastatic melanoma by promoter hypermethylation and TCF21 promoter DNA methylation is correlated with decreased survival in metastatic skin melanoma patients. In addition, the chromosomal location of TCF21 on 6q23-q24 coincides with the location of a postulated metastasis suppressor in melanoma. Functionally, TCF21 binds the promoter of the melanoma metastasis-suppressing gene, KiSS1, and enhances its gene expression through interaction with E12, a TCF3 isoform and with TCF12. Loss of TCF21 expression results in loss of KISS1 expression through loss of direct interaction of TCF21 at the KISS1 promoter. Finally, overexpression of TCF21 inhibits motility of C8161 melanoma cells. These data suggest that epigenetic downregulation of TCF21 is functionally involved in melanoma progression and that it may serve as a biomarker for aggressive tumor behavior.

In industrial countries, a number of factors put indigenous peoples at increased risk of HIV infection. National surveillance data between 1999 and 2008 provided diagnoses for Aboriginal and Torres Strait Islanders (Australia), First Nations, Inuit and Métis (Canada excluding Ontario and Quebec) and Māori (New Zealand). Each country provided similar data for a non-indigenous comparison population. Direct standardisation used the 2001 Canadian Aboriginal male population for comparison of five-year diagnosis rates in 1999-2003 and 2004-2008. Using the general population as denominators, we report diagnosis ratios for presumed heterosexual transmission, men who have sex with men (MSM) and intravenous drug users (IDU). Age standardised HIV diagnosis rates in indigenous peoples in Canada in 2004-2008 (178.1 and 178.4/100 000 for men and women respectively) were higher than in Australia (48.5 and 12.9/100 000) and New Zealand (41.9 and 4.3/100 000). Higher HIV diagnosis rates related to heterosexual contact among Aboriginal peoples, especially women, in Canada confirm a widening epidemic beyond the conventional risk groups. This potential of a generalised epidemic requires urgent attention in Aboriginal communities; available evidence can inform policy and action by all stakeholders. Although less striking in Australia and New Zealand, these findings may be relevant to indigenous peoples in other countries.

Laser-induced breakdown spectroscopy (LIBS) has been employed for the analysis of slurry samples. Quantitative analysis of slurry samples is crucial and challenging. The problems associated with slurry samples include splashing, surface turbulence, and the difficulties of obtaining reproducible samples due to sedimentation. The LIBS analysis has achieved limited success due to inherent disadvantages when applied to slurry samples. In order to achieve improved measurement precision and accuracy, a spin-on-glass sampling method was evaluated. Five elements (Al, Ca, Fe, Ni, and Si) were examined in five slurry simulants containing varying amounts of each ion. Three calibration models were developed by using univariate calibration, multiple linear regression, and partial least square regression. LIBS analysis results obtained from the partial least square regression model were determined to be the best fit to results obtained from inductively coupled plasma optical emission spectroscopy analysis.

We showed previously that murine naive CD4(+) T cells and T(H)1 cell clones express the beta2-adrenergic receptor (β(2)AR), while T(H)2 cell clones do not. We report here that naive CD4(+) T cells that differentiated for 1-5 days under T(H)1 driving conditions increased β(2)AR gene expression, while cells cultured under T(H)2 driving conditions decrease β(2)AR gene expression. Chromatin immunoprecipitation revealed that the increase in β(2)AR gene expression in T(H)1 cells is mediated by an increase in histone 3 (H3) and H4 acetylation, as well as an increase in histone 3 lysine 4 (H3K4) methylation. Conversely, the decrease in β(2)AR gene expression in T(H)2 cells is mediated by a decrease in H3 and H4 acetylation and a decrease in H3K4 methylation, as well as an increase H3K9 and H3K27 methylation. The histone changes could be detected as early as 3 days of differentiating conditions. Genomic bisulfite sequencing showed that the level of methylated CpG dinucleotides within the promoter of the β(2)AR gene was increased in T(H)2 cells as compared to naive and T(H)1 cells. Collectively, these results suggest that epigenetic mechanisms mediate maintenance and repression, respectively, of the β(2)AR gene expression in T(H)1- and T(H)2-driven cells, providing a potential mechanism by which the level of β(2)AR expression might be modulated pharmacologically within immune cells and other cell types in which the expression profile may change during a disease process., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Activated extracellular signal-regulated kinase (ERK) signaling mediated plasticity-related gene transcription has been proposed for one possible mechanism by which 17β-estradiol (E2) enhances synaptic plasticity and memory. Because activated ERK also enhances plasticity-related mRNA translation in the dendrites of neurons, we sought to determine the effects of E2 on activation of ERK, phosphorylation of translation initiation factors, and dendritic mRNA translation in hippocampal neurons. Acute E2 application resulted in a rapid, transient increase in phosphorylation of translation initiation factors, ribosomal protein (S6) and eIF4E binding protein1 (4EBP1), in an activated ERK-dependent manner. Since phosphorylation of these translation factors enhance mRNA translation, we tested E2's effect on dendritic mRNA translation. Using a green fluorescent protein (GFP)-based dendritic mRNA translation reporter (reporter plasmid construct consisted of a GFP gene fused to the 3' untranslated region (UTR) from CAMKIIα, which contains dendritic resident mRNA targeting and mRNA translational regulatory elements) we showed that E2 treatment resulted in increased somatic and dendritic GFP mRNA translation in GFP-reporter transfected hippocampal neurons. Translation inhibitor anisomycin and ERK inhibitor U0126 blocked E2 effects. Taken together, our results provide a novel mechanism by which E2 may trigger local protein synthesis of α-CaMKII in the dendrites, which is necessary for modulation of synaptic plasticity., (Published by Elsevier Ltd.)

Difficulties identifying appetite signals and emotions have been implicated in the development and maintenance of disordered eating. The current study evaluated the mediating roles of appetite awareness and emotional awareness in a brief eating disorders prevention program designed to help participants identify and respond to internal appetite signals. A series of regression analyses was carried out to test the mediator effects of appetite and emotional awareness. Appetite awareness, but not emotional awareness, mediated improvements in binge eating symptoms as well as eating- and weight-control self-efficacy. Appetite awareness appears to be an effective target for eating disorders prevention programs.

Background: Restriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning.", Results: We report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation., Conclusion: The new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation.

Alterations in the genome and the epigenome are common in most cancers. Changes in epigenetic signatures, including aberrant DNA methylation and histone deacetylation, are among the most prevalent modifications in cancer and lead to dramatic changes in gene expression patterns. Because DNA methylation and histone deacetylation are reversible processes, they have become attractive as targets for cancer epigenetic therapy, both as single agents and as 'enhancing' agents for other treatment strategies. In this review we discuss our current view of the mammalian epigenome, this view has changed over the years because of the availability of novel technologies. We further demonstrate how the profound understanding of epigenetic alterations in cancer will help develop novel strategies for epigenetic therapies.

Background: Previous studies of individual genes have shown that in a self-enforcing way, dimethylation at histone 3 lysine 9 (dimethyl-H3K9) and DNA methylation cooperate to maintain a repressive mode of inactive genes. Less clear is whether this cooperation is generalized in mammalian genomes, such as mouse genome. Here we use epigenomic tools to simultaneously interrogate chromatin modifications and DNA methylation in a mouse leukemia cell line, L1210., Results: Histone modifications on H3K9 and DNA methylation in L1210 were profiled by both global CpG island array and custom mouse promoter array analysis. We used chromatin immunoprecipitation microarray (ChIP-chip) to examine acetyl-H3K9 and dimethyl-H3K9. We found that the relative level of acetyl-H3K9 at different chromatin positions has a wider range of distribution than that of dimethyl-H3K9. We then used differential methylation hybridization (DMH) and the restriction landmark genome scanning (RLGS) to analyze the DNA methylation status of the same targets investigated by ChIP-chip. The results of epigenomic profiling, which have been independently confirmed for individual loci, show an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing. In contrast to the previous notion, dimethyl-H3K9 seems to be less distinct in specifying silencing for the genes tested., Conclusion: This study demonstrates in L1210 leukemia cells a diverse relationship between histone modifications and DNA methylation in the maintenance of gene silencing. Acetyl-H3K9 shows an inverse relationship between DNA methylation and histone acetylation in regulating gene silencing as expected. However, dimethyl-H3K9 seems to be less distinct in relation to promoter methylation. Meanwhile, a combination of epigenomic tools is of help in understanding the heterogeneity of epigenetic regulation, which may further our vision accumulated from single-gene studies.

Tumor suppressor CCAAT enhancer binding protein alpha (C/EBPalpha) is a transcription factor involved in cell cycle control and cellular differentiation. In a recent study, microarray expression profiling on head and neck squamous cell carcinoma (HNSCC) samples identified significant C/EBPalpha down-regulation, correlating with poor prognosis. However, the mechanisms of C/EBPalpha down-regulation remained elusive. C/EBPalpha has been previously found to provide an antiproliferative role in lung cancer, and our laboratory showed that its down-regulation involves epigenetic mechanisms. This prompted us to investigate the involvement of epigenetics in down-regulating C/EBPalpha in HNSCC. Here, we show that C/EBPalpha is down-regulated in HNSCC by loss of heterozygosity and DNA methylation, but not by gene mutation. We found a consistently methylated upstream regulatory region (-1,399 bp to -1,253 bp in relation to the transcription start site) in 68% of the HNSCC tumor samples, and DNA demethylation using 5-aza-2'-deoxycytidine treatment was able to significantly restore C/EBPalpha mRNA expression in the HNSCC cell lines we tested. In addition, C/EBPalpha overexpression in a HNSCC cell line (SCC22B) revealed its ability to provide tumor suppressor activity in HNSCC in vitro and in vivo. In conclusion, we showed for the first time not only that C/EBPalpha has tumor suppressor activity in HNSCC, but also that it is down-regulated by DNA promoter methylation.

Providing small grants to community organizations can be an effective way to encourage changes in the environment that support better nutrition. This is effective because these organizations can provide insights into their communities, ready-made relationships with community members, and the trust of the community. Small-grants programs are more likely to be successful when they are tailored to the needs of individual communities, led by organizations that have established reputations with the community, fully supported by the lead community organization, and engage local partners that complement the skills and resources of the lead organization. An evaluation of a small-grants program, Grants for Healthy Youth, found that grantees developed unique approaches to improving their community nutrition environments, gained experience and skills in program development, built partnerships, and received recognition for their project work. Grantees faced some common barriers, especially with program evaluation. Small-grants programs can be an effective way to improve community nutrition environments, but granting agencies need to provide effective technical assistance to communities throughout the process.

Background: It has been suggested that current UK thresholds for treating paracetamol overdose should be reduced, following case reports of patients developing fatal liver failure after presenting with paracetamol concentrations below these thresholds., Aim: To determine the frequency of severe liver dysfunction following paracetamol overdose when paracetamol concentrations are below current UK antidote thresholds., Design: Retrospective case note review., Methods: Details were collected from all patients admitted to liver transplant units in Newcastle and Edinburgh with paracetamol-induced hepatotoxicity., Results: Of 696 patients admitted to the two liver units following paracetamol overdose, 14 presented between 4 and 15 h after overdose with paracetamol concentrations below current UK treatment thresholds (estimated annual population rate 0.15/million person-years). Over the period of study, >100 000 presentations with paracetamol overdose would be expected in the catchment populations for these liver units., Discussion: In view of the rarity of this event, this research does not suggest a need to lower the current thresholds for antidotal treatment.

The goal of this study was to identify recurrent regions of genomic gain or loss in giant-cell tumor of bone (GCTb). Array comparative genomic hybridization (aCGH) was performed for 20 frozen tumor samples of GCTb. A separate subset of 59 GCTb with outcome data was used for validation. The most frequent region of change identified by aCGH was gain of a 1-Mbp region at 20q11.1. In the validation arm of 59 cases the minimal common region of copy number gain at 20q11.1, seen in 54% of the samples, was BAC clone RP11-4O9, which contained the genes TPX2 and BCL2L1. For most cases, amplification was restricted to the mononuclear component and was not present in the multinucleated giant cells. Southern blot for TPX2 and BCL2L1 identified the former as the gene with the highest level of amplification for these two proposed candidate genes of importance. Immunohistochemistry for TPX2 expression correlated with amplification, while BCL2L1 expression was not identified. Kaplan-Meier curves for progression-free survival showed a statistically significant difference for cases with 20q11.1 amplification (P = 0.0001). Univariate analysis involving Cox proportional hazards models did not show a significant difference for initial treatment type (curettage versus resection) (P = 0.575), age (50) (P = 0.543), or sex (P = 0.268), but did correlate with 20q11.1 amplification (P = 0.001). By multivariate analysis, it was found that 20q11.1 amplification (P = 0.001) was the only factor to reach statistical significance. 20q11.1 amplification can be used as a marker of prognostic importance in GCTb. We propose TPX2 as a candidate oncogene in the core-amplified region at 20q11.1., ((c) 2006 Wiley-Liss, Inc.)

Reasons for Performing Study: Electrolyte mixtures given to counter sweat loss usually contain abundant potassium. However, increases in plasma [K+] occur with exercise and supplementation may further increase plasma levels, potentially increasing the risk of neuromuscular hyperexcitability and development of adverse clinical sequellae. This proposition requires study., Objectives: To compare effects of a K-rich electrolyte supplement (EM+K) to a K-free one (EM-K) on plasma [K+], [Ca++] and acid-base status during an endurance incremental exercise test on the treadmill., Methods: The test consisted of 3 bouts (simulating loops in an endurance race) of 12 km performed at 6, then 7, then 8 m/sec with 25 min rest stops (S1, S2) between loops on 13 endurance trained Arabian horses (7 EM-K, 6 EM+K). Electrolytes were supplied orally 60 mins before exercise (Pre) and at each stop. Blood samples were taken before exercise and during exercise, each S and 120 mins of recovery (R). Blood was analysed for pH, PCO2, packed cell volume (PCV), plasma [Na+], [K+], [Cl-], [Ca++], glucose, and lactate [La-]; plasma [H+] and osmolality (osm) were calculated. The dietary cation anion difference (DCAD) was calculated to be -27 meq/dose EM-K and 109 meq in EM+K, respectively., Results: Plasma [H+] decreased during the 6 and 7 m/sec loops, increased during the 8 m/sec loop, and returned to Pre at S1, S2 and R. Plasma [K+] was higher at 8 m/sec and plasma [Ca++] was overall lower in the EM+K group compared to EM-K. Other findings included higher overall PCV, overall glucose, and [La-] during the 8 m/sec loop (P<0.040) in EM+K compared to EM-K horses., Conclusions: EM+K supplementation leads to higher plasma [K+] increasing the risk of neuromuscular hyperexcitability during exercise. Acute effects of a lower DCAD in EM-K may have led to higher plasma [Ca++]. Potassium-rich electrolytes may have triggered the release of epinephrine, contributing to higher PCV, glucose release and increased lactate production., Potential Relevance: Lower plasma [K+] and higher plasma [Ca++] with EM-K supplementation may help reduce the risk of conditions associated with neuromuscular hyperexcitability occurring especially during higher speeds in endurance races.

In the post-genome era, attention has focused on the functions of genome sequences and how they are regulated. The emerging epigenomic changes and the interactions between cis-acting elements and protein factors may play a central role in gene regulation. To understand the crosstalk between DNA and protein on a genome-wide scale, one emerging technique, called ChIP-chip, takes the strategy of combining chromatin immunoprecipitation with microarray. This new high-throughput strategy helps screen the targets of critical transcription factors and profile the genome-wide distribution of histone modifications, which will enable the feasibility of conducting a large-scale study, such as the Human Epigenome Project.

Washington State's Healthy Communities pilot projects were developed to test approaches and recommendations of the Washington State Nutrition and Physical Activity Plan and to provide a statewide model for implementation. The Healthy Communities program included plans for ongoing process evaluation to ensure implementation. Two years into the first project, however, the evaluation team recognized that data for evaluation were inadequate to explain the experiences of the pilot community partnership. The team sought a framework through which to better understand how the community partnership functioned, including what worked well and how guidance and technical assistance could best be provided. The evaluation team identified the community health governance model of Lasker and Weiss through a literature search and applied this model to existing Healthy Communities project evaluation data. The team also designed a new survey tool based on the model and used it in the second pilot community. The new tool provides feedback to community partners to help guide project implementation and tests the applicability of a theoretical model to public health practice.

Background: The Washington State Nutrition and Physical Activity Plan provides a framework in which policy makers can work together to build and support healthy environments for nutrition and physical activity. The city of Moses Lake, Wash, was chosen to serve as a pilot site to test the conceptual approaches and recommendations of the plan and to develop a model for healthy communities elsewhere in the state., Context: Moses Lake is an ethnically diverse, geographically isolated town with a population of about 15,000., Methods: An advisory committee used data from an inventory of local policies and environments, along with the recommendations from the state plan, to develop a plan for Healthy Communities Moses Lake. Three initiatives were chosen for the first actions: a connected system of trails and paths, enhanced facilities for breastfeeding in the community, and a community garden., Consequences: Records of cumulative actions demonstrated that Healthy Communities Moses Lake continued to be an active and productive project. Initial measures of success were collected by each of the three first action teams. Environmental changes will be monitored by comparison with the initial inventory of local policies. Long-term health outcomes in Moses Lake will be monitored by the Washington State Department of Health., Interpretation: Healthy Communities Moses Lake was successful because the city had leaders and volunteers who were committed to making the city a healthier place. Lessons learned about community-based planning and evaluation are now being applied to Healthy Communities initiatives.

Gene amplification, a common mechanism for oncogene activation in cancer, has been used as a tag for the identification of novel oncogenes. DNA amplification is frequently observed in head and neck squamous cell carcinoma (HNSCC) and potential oncogenes have already been reported. We applied restriction landmark genome scanning (RLGS) to study gene amplifications and low-level copy number changes in HNSCC in order to locate previously uncharacterized regions with copy number gains in primary tumor samples. A total of 63 enhanced RLGS fragments, indicative of DNA copy number changes, including gains of single alleles, were scored. Enhanced sequences were identified from 33 different chromosomal regions including those previously reported (e.g. 3q26.3 and 11q13.3) as well as novel regions (e.g. 3q29, 8q13.1, 8q22.3, 9q32, 10q24.32, 14q32.32, 17q25.1 and 20q13.33). Furthermore, our data suggest that amplicons 11q13.3 and 3q26.3-q29 may be divided into possibly two and three independent amplicons, respectively, an observation supported by published microarray expression data.

In breast cancer and normal estrogen target tissues, estrogen receptor-alpha (ERalpha) signaling results in the establishment of spatiotemporal patterns of gene expression. Whereas primary target gene regulation by ERalpha involves recruitment of coregulatory proteins, coactivators, or corepressors, activation of these downstream promoters by receptor signaling may also involve partnership of ERalpha with other transcription factors. By using an integrated, genome-wide approach that involves ChIP-chip and computational modeling, we uncovered 13 ERalpha-responsive promoters containing both ERalpha and c-MYC binding elements located within close proximity (13-214 bp) to each other. Estrogen stimulation enhanced the c-MYC-ERalpha interaction and facilitated the association of ERalpha, c-MYC, and the coactivator TRRAP with these estrogen-responsive promoters, resulting in chromatin remodeling and increased transcription. These results suggest that ERalpha and c-MYC physically interact to stabilize the ERalpha-coactivator complex, thereby permitting other signal transduction pathways to fine-tune estrogen-mediated signaling networks.

The identification of tumor suppressor genes has classically depended on their localization within recurrent regions of loss of heterozygosity. According to Knudson's two-hit hypothesis, the remaining allele is lost, either genetically or, more recently identified, through epigenetic events. To date, retrospective analyses have determined promoter methylation as a common alternative alteration in cancer cells to silence cancer-related genes. Here we report an application of restriction landmark genomic scanning that allows for DNA methylation profiling along a region of recurrent loss of heterozygosity at chromosome 6q23-q24. This approach resulted in the identification of a tumor suppressor gene, TCF21, which is frequently lost in human malignancies. We demonstrate that TCF21 is expressed in normal lung airway epithelial cells and aberrantly methylated and silenced in the majority of head and neck squamous cell carcinomas and non-small-cell lung cancers analyzed. TCF21 is known to regulate mesenchymal cell transition into epithelial cells, a property that has been shown to be deficient in carcinomas. We further demonstrate that exogenous expression of TCF21 in cells that have silenced the endogenous TCF21 locus resulted in a reduction of tumor properties in vitro and in vivo.

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random. We also identified Idb4 as a putative tumor-suppressor gene that is methylated in most mouse and human leukemias but in only a minority of other human cancers.

Introduction: Diets that are high in fruits and vegetables lower an individual's risk of chronic disease and contribute to healthy aging. Homebound seniors often have low intake of fruits and vegetables and limited access to fruits and vegetables with the most protective nutrients and phytochemicals. From June through October 2001, the Seattle Senior Farmers' Market Nutrition Pilot Program delivered bi-weekly market baskets that included a variety of fresh, locally grown produce to 480 low-income Meals on Wheels participants. The purpose of this study was to determine if the program increased fruit and vegetable intake in individuals who received the baskets., Methods: One hundred basket recipients were recruited to complete a telephone survey before and at the end of the farmers' market basket season. Fifty-two low-income homebound seniors who lived outside the project service area were recruited to serve as control respondents. Fruit and vegetable intake was determined with modified versions of the 6 fruits and vegetables questions in the Behavior Risk Factor Surveillance System., Results: Seniors who received the baskets reported consuming an increase of 1.04 servings of fruits and vegetables. The difference between the mean servings in the seniors who received the baskets compared to the controls was 1.31 (95% CI, 0.68-1.95, P < .001). At baseline, 22% of the basket recipients were consuming 5 or more servings of fruits and vegetables per day, but by the end of the season, 39% reported consuming 5 or more per day., Conclusion: Home delivery of fruits and vegetables is an effective way to increase fruit and vegetable intake in homebound seniors.

Introduction: The Seattle Senior Farmers' Market Nutrition Pilot Program delivered fresh fruits and vegetables to homebound seniors in King County, Washington, from June through October 2001. A primary objective of the program was to increase participants' intake of fruits and vegetables. A qualitative study was conducted to examine the impact of the program on participating homebound seniors., Methods: Semi-structured interviews were performed with 27 participants in their homes to identify benefits and barriers they encountered and to measure their use and sense of satisfaction with the program., Results: Analysis of the transcribed interviews revealed several common themes: Participants appreciated the variety and quality of the fresh fruits and vegetables. Some participants would not have had access to fresh fruits and vegetables without the program. Home-delivered baskets of fresh fruits and vegetables brought participants joy, stimulated interest in healthy foods, and improved quality of life. The program newsletter supported consumption of fresh produce., Conclusion: Program success was rooted in the multiple ways the program addressed potential barriers and reinforced behavioral intent.

There are only a few reports on the measurement of the energy of the low-lying (3)deltadelta state of quadruply bonded bimetallic complexes, and the direct observation of the (1)deltadelta excited electronic state was only recently reported. In the quadruply bonded bimetallic complexes reported to date, luminescence arises from their (1)deltadelta excited state, and the (3)deltadelta state is nonemissive. Here we report the luminescence of Re(2)Cl(2)(p-OCH(3)form)(4) [p-OCH(3)form = (p-CH(3)OC(6)H(4))NCHN(p-CH(3)OC(6)H(4))(-)] observed upon 400-460 nm excitation with maxima at 820 nm (CH(2)Cl(2), tau = 1.4 micros) and 825 nm (CH(3)CN, tau = 1.3 micros) at 298 K. From the large Stokes shift, the vibronic progression at 77 K, the quenching by O(2), the long lifetime, and the calculated energy of the (3)deltadelta state, the luminescence of Re(2)Cl(2)(p-OCH(3)form)(4) and the corresponding transient absorption signal are assigned as arising from the (3)deltadelta ((3)A(2u)) excited state of the complex.

Head and neck squamous cell carcinomas (HNSCC) often metastasise to the cervical lymph nodes. It is known for HNSCC as well as other cancers that progression from normal tissue to primary tumour and finally to metastatic tumour is characterised by an accumulation of genetic mutations. DNA methylation, an epigenetic modification, can result in loss of gene function in cancer, similar to genetic mutations such as deletions and point mutations. We have investigated the DNA methylation phenotypes of both primary HNSCC and metastatic tumours from 13 patients using restriction landmark genomic scanning (RLGS). With this technique, we were able to assess the methylation status of an average of nearly 1300 CpG islands for each tumour. We observed that the number of CpG islands hypermethylated in metastatic tumours is significantly greater than what is found in the primary tumours overall, but not in every patient. Interestingly, the data also clearly show that many loci methylated in a patient's primary tumour are no longer methylated in the metastatic tumour of the same patient. Thus, even though metastatic HNSCC methylate a greater proportion of CpG islands than do the primary tumours, they do so at different subsets of loci. These data show an unanticipated variability in the methylation state of loci in primary and metastatic HNSCCs within the same patient. We discuss two possible explanations for how different epigenetic events might arise between the primary tumour and the metastatic tumour of a person.

The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K(m) of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 microM. This porter has a K(m) for glycine betaine uptake of about 6 micro M. The dedicated carnitine porter, OpuC, has a K(m) for carnitine uptake of 1 to 3 microM and a V(max) of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by gamma-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.

Ehlers-Danlos syndrome (EDS) type I (the classical variety) is a dominantly inherited, genetically heterogeneous connective-tissue disorder. Mutations in the COL5A1 and COL5A2 genes, which encode type V collagen, have been identified in several individuals. Most mutations affect either the triple-helical domain of the protein or the expression of one COL5A1 allele. We identified a novel splice-acceptor mutation (IVS4-2A-->G) in the N-propeptide-encoding region of COL5A1, in one patient with EDS type I. The outcome of this mutation was complex: In the major product, both exons 5 and 6 were skipped; other products included a small amount in which only exon 5 was skipped and an even smaller amount in which cryptic acceptor sites within exon 5 were used. All products were in frame. Pro-alpha1(V) chains with abnormal N-propeptides were secreted and were incorporated into extracellular matrix, and the mutation resulted in dramatic alterations in collagen fibril structure. The two-exon skip occurred in transcripts in which intron 5 was removed rapidly relative to introns 4 and 6, leaving a large (270 nt) composite exon that can be skipped in its entirety. The transcripts in which only exon 5 was skipped were derived from those in which intron 6 was removed prior to intron 5. The use of cryptic acceptor sites in exon 5 occurred in transcripts in which intron 4 was removed subsequent to introns 5 and 6. These findings suggest that the order of intron removal plays an important role in the outcome of splice-site mutations and provide a model that explains why multiple products derive from a mutation at a single splice site.

The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress.

Listeria monocytogenes accumulates low molecular weight compounds (osmolytes, or compatible solutes) in response to chill stress. This response has been shown to be responsible, in part, for the chill tolerance of the species. Among the osmolytes tested to date, glycine betaine, gamma-butyrobetaine and carnitine display the strongest cryoprotective effect. These osmolytes are not synthesized in the cell and must be transported from the medium. In this study, the compatible solute accumulation profile of the food-borne pathogen L. monocytogenes was determined in balanced growth and stationary phase cultures grown in milk whey at 7 and 30 degrees C. In balanced growth cultures at 7 degrees C, glycine betaine (720 nmol/10(10) cfu) and carnitine (130 nmol/10(10) cfu) were the major osmolytes accumulated by wild-type L. monocytogenes 10403S, whereas carnitine (490 nmol/10(10) cfu) was the dominant osmolyte and glycine betaine was present in smaller amounts (270 nmol/10(10) cfu) in a mutant (L. monocytogenes LTG59) blocked in the major glycine betaine uptake system, glycine betaine porter II. In strain 10403S, glycine betaine and carnitine were present in eightfold and twofold excess at 7 degrees C compared to 30 degrees C; the respective ratios for strain LTG59 were 6 and 8. The intracellular concentration of osmolytes in stationary phase cultures at 7 degrees C was markedly reduced compared to that during balanced growth. Furthermore, at 4 degrees C, small but highly significant differences in growth were observed between strains. Strain LTG59 grew with a lag phase that was significantly longer, a generation time that was significantly greater and reached a final cell yield that was significantly lower than that of strain 10403S. The elevated accumulation of carnitine in the absence of glycine betaine porter II was insufficient to confer the magnitude of the cryoprotective effect displayed by the wild type.

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na(+)-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12 degrees C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and gamma-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.

In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes or by activating growth-stimulating genes. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive, little is known about how DNA methylation is controlled. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated domain (DMD) 30 kbp 5' of the promoter. Expression is exclusively from the paternal allele in neonatal brain. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3' of the DMD. This sequence is present in organisms in which Rasgrf1 is imprinted. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates imprinting at the locus.

Bacteria inhabit natural and artificial environments with diverse and fluctuating osmolalities, salinities and temperatures. Many maintain cytoplasmic hydration, growth and survival most effectively by accumulating kosmotropic organic solutes (compatible solutes) when medium osmolality is high or temperature is low (above freezing). They release these solutes into their environment when the medium osmolality drops. Solutes accumulate either by synthesis or by transport from the extracellular medium. Responses to growth in high osmolality medium, including biosynthetic accumulation of trehalose, also protect Salmonella typhimurium from heat shock. Osmotically regulated transporters and mechanosensitive channels modulate cytoplasmic solute levels in Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Lactobacillus plantarum, Lactococcus lactis, Listeria monocytogenes and Salmonella typhimurium. Each organism harbours multiple osmoregulatory transporters with overlapping substrate specificities. Membrane proteins that can act as both osmosensors and osmoregulatory transporters have been identified (secondary transporters ProP of E. coli and BetP of C. glutamicum as well as ABC transporter OpuA of L. lactis). The molecular bases for the modulation of gene expression and transport activity by temperature and medium osmolality are under intensive investigation with emphasis on the role of the membrane as an antenna for osmo- and/or thermosensors.

Epidermolysis bullosa simplex are dominant disorders of skin fragility characterized by intraepidermal blistering upon mild mechanical trauma. Skin fragility is caused by expression of either an abnormal keratin 5 or an abnormal keratin 14 protein, which compromises the structure and function of the keratin cytoskeleton of basal cells. We report an epidermolysis bullosa simplex patient with a novel single base substitution (A-->T1414) that changes the lysine residue at amino acid 472 to a non-sense codon (K472X). This change predicts the synthesis of a truncated keratin 5, missing 119 amino acids, including the entire tail domain and the highly conserved KLLEGE motif at the carboxy terminus of the 2B domain of the central rod. Expression of an altered keratin 5, of predicted mass and pI for the product of the K472X allele, was documented by one- and two-dimensional western blots of protein extracts from patient skin. Ultrastructural analysis of the patient's nonhyperkeratotic skin was remarkable for basal keratinocytes with dense and irregular keratin filaments proximal to the basement membrane. Keratinocytes, transfected with a cDNA carrying the A-->T1414 non-sense mutation, overexpressed a truncated keratin 5, and showed a disorganized and collapsed keratin filament cytoskeleton. This is the second epidermolysis bullosa simplex patient reported with a premature termination mutation in the KLLEGE motif. The remarkable occurrence of severe palmar--plantar hyperkeratosis in both patients suggests that the keratin 5 tail domain may have unrecognized, but important, normal functions in palmar-plantar tissues.

Information on the morphology of mitochondria during embryogenesis is scattered in the literature, but there appears to be a consistent pattern. During early organogenesis, the embryo is in a state of relative hypoxia associated with a major decrease in terminal electron transport system activity and a marked increase in anaerobic glycolysis. Ultrastructural studies of a 14-somite monkey embryo and day 10 and 12 rat embryos, together with a review of the literature, led us to determine that this hypoxic stage is characterized by vesiculation of the mitochondrial inner membranes, or cristae. Starting in the late morula stage and continuing during early postimplantation embryogenesis, the cristae increase but appear tubular or vesicular. After the end of neurulation, and with the onset of vascular perfusion of embryonic tissues, the cristae gradually become lamellated; by the limb bud stage they appear more mature. We suggest that new cristae derive from blebs of the inner mitochondrial membrane and that with maturation these blebs collapse, giving them a lamelliform appearance. The delamellated state of the cristae might inactivate oxidative phosphorylation to protect the embryo from toxic respiratory end-products that could accumulate in an embryo before there is vascular perfusion. Consistent with this hypothesis, mitochondrial diameters in the developing heart of monkey and rat embryos were approximately twice those found in skin and neural tube.

Epidermal transglutaminase (TGase) is known to catalyse cross-linking of several precursor proteins in the formation of cornified cell envelope at the terminal differentiation of keratinocytes. Expression of TGase activity was studied using an in situ TGase activity assay in human fetal skin samples of 49-163 days estimated gestational age (EGA). In the early two-layered epidermis (49-56 days EGA), in situ TGase activity was not observed in the periderm cells or the basal cells. In the late two-layered epidermis (57-65 days EGA), in situ TGase activity became weakly positive in the periderm cells, but not in the basal cells. In the three-layered (66-95 days EGA) and in four- or more layered (96-135 days EGA) stratified epidermis, in situ TGase activity was still restricted only to the periderm cells. After keratinization occurred in the interfollicular epidermis (163 days EGA), in situ TGase activity was expressed in the granular and cornified layers. This unique localization of TGase activity further support the hypothesis that periderm cells form cornified cell envelope during their regression process in human fetal skin development.

In rodents, the hair follicle stem cells lie in a well-defined bulge in the outer root sheath; however, the bulge as a stem cell site of human hair follicle epithelium is still controversial. Epidermal stem cells are thought to express high levels of beta1 integrin and low levels of E-cadherin and beta- and gamma-catenin. In order to clarify the ontogenic distribution of possible stem cells during hair follicle development, the expression patterns of beta1 integrin subunits, E-cadherin, and beta- and gamma-catenins in the skin samples from human fetuses of a series of estimated gestational ages (EGA) were examined. beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells, possible stem cells, were localized to the entire hair germ (65-84 d EGA) and later to the outermost cells of hair peg (85-104 d EGA). In the bulbous hair peg (105-135 d EGA) and in the differentiated lanugo hair follicle (>135 d EGA), they were settled in the bulge and the outermost layer of the outer root sheath. This sequential localization was similar to that of cells rich in epidermal growth factor receptor expression and positive with keratin 19, a putative marker of epidermal stem cells. In addition, these beta1 integrin-rich, E-cadherin-, and beta- and gamma-catenin-poor cells showed similar, undifferentiated morphologic features by electron microscopy. This information of ontogenic localization of possible hair follicle stem cells contributes to the further understanding of mechanisms of human hair follicle morphogenesis and supports the idea that the human fetal hair follicle bulge is a site of stem cells for follicular epithelium.

Type I collagen is the most abundant structural protein in the mammalian body. It exists as a heterotrimer of two subunits in the form [alpha1(I)]2alpha2(I). Pathogenic mutations in COL1A1 and COL1A2, the genes that encode the two subunits, cause a range of phenotypes including mild to lethal forms of osteogenesis imperfecta and a restricted set of Ehlers-Danlos syndrome phenotypes. Lethal mutations usually result from missense mutations that disrupt the normal triple helical structure of the molecule. Multi-exon duplication or deletion in type I collagen genes has rarely been observed and has generally resulted in a lethal or severe phenotype. We report a partial duplication in the COLIA2 gene that causes a relatively mild phenotype, despite the addition of 477 amino acids to the triple helical domain of the proalpha2(I) chain. The abnormal molecule is synthesized and secreted by cultured dermal fibroblasts in a normal fashion. Electron microscopy of dermal tissue reveals small but otherwise near normal collagen fibrils. The gene duplication occurred by mitotic sister chromatid exchange in the mother who is mosaic for the duplication allele. Examination of the abnormal sequence suggests a means by which the duplicated molecule could be processed and properly incorporated into mature collagen fibrils.

Calcium concentration is a critical factor for epidermal differentiation and is implicated in the expression and post-translational modification of numerous proteins in suprabasal cells of the epidermis. Calpains (calcium-activated neutral proteases) are believed to participate in signal transduction via highly regulated cytoplasmic protease activity. Here we investigate the expression of calpain I in normal human skin development and in neonatal harlequin ichthyosis (HI), a disorder of altered epidermal differentiation, especially the transition from the granular to the fully differentiated cornified layer. Calpain I was detected in developing foetal epidermis at 54 days estimated gestational age in the basal layer and the periderm of the developing foetal epidermis. By 125 days, calpain I was also detected in the granular layer. This pattern was maintained in newborn skin, but expression was significantly weaker in HI biopsies (n = 7). Reduced expression of calpain was specific to HI and was not observed in other skin diseases. Calpain was also normally expressed in the outer root sheath of hair follicles, in sebaceous glands and in sweat ducts and glands. Immunoblots of epidermal and keratinocyte extracts showed that the 78-kDa and 76-kDa active forms were generated via limited proteolysis of the 80-kDa inactive subunit; however, all forms were diminished in HI, consistent with findings in tissue sections. Our results show that calpain is present throughout the epidermis and is expressed from the early stages of development. These findings implicate calcium-mediated signalling events in the alteration of differentiation that occurs in HI.