In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells.

Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.

It still hurts: altered endogenous opioid activity in the brain during social rejection and acceptance in major depressive disorder.

The µ-opioid receptor (MOR) system, well known for dampening physical pain, is also hypothesized to dampen 'social pain.' We used positron emission tomography scanning with the selective MOR radioligand [(11)C]carfentanil to test the hypothesis that MOR system activation (reflecting endogenous opioid release) in response to social rejection and acceptance is altered in medication-free patients diagnosed with current major depressive disorder (MDD, n=17) compared with healthy controls (HCs, n=18). During rejection, MDD patients showed reduced endogenous opioid release in brain regions regulating stress, mood and motivation, and slower emotional recovery compared with HCs. During acceptance, only HCs showed increased social motivation, which was positively correlated with endogenous opioid release in the nucleus accumbens, a reward structure. Altered endogenous opioid activity in MDD may hinder emotional recovery from negative social interactions and decrease pleasure derived from positive interactions. Both effects may reinforce depression, trigger relapse and contribute to poor treatment outcomes.

Response of the µ-opioid system to social rejection and acceptance.

The endogenous opioid system, which alleviates physical pain, is also known to regulate social distress and reward in animal models. To test this hypothesis in humans (n=18), we used an µ-opioid receptor (MOR) radiotracer to measure changes in MOR availability in vivo with positron emission tomography during social rejection (not being liked by others) and acceptance (being liked by others). Social rejection significantly activated the MOR system (i.e., reduced receptor availability relative to baseline) in the ventral striatum, amygdala, midline thalamus and periaqueductal gray (PAG). This pattern of activation is consistent with the hypothesis that the endogenous opioids have a role in reducing the experience of social pain. Greater trait resiliency was positively correlated with MOR activation during rejection in the amygdala, PAG and subgenual anterior cingulate cortex (sgACC), suggesting that MOR activation in these areas is protective or adaptive. In addition, MOR activation in the pregenual ACC was correlated with reduced negative affect during rejection. In contrast, social acceptance resulted in MOR activation in the amygdala and anterior insula, and MOR deactivation in the midline thalamus and sgACC. In the left ventral striatum, MOR activation during acceptance predicted a greater desire for social interaction, suggesting a role for the MOR system in social reward. The ventral striatum, amygdala, midline thalamus, PAG, anterior insula and ACC are rich in MORs and comprise a pathway by which social cues may influence mood and motivation. MOR regulation of this pathway may preserve and promote emotional well being in the social environment.

Consistency of predictive signature genes and classifiers generated using different microarray platforms.

Microarray-based classifiers and associated signature genes generated from various platforms are abundantly reported in the literature; however, the utility of the classifiers and signature genes in cross-platform prediction applications remains largely uncertain. As part of the MicroArray Quality Control Phase II (MAQC-II) project, we show in this study 80-90% cross-platform prediction consistency using a large toxicogenomics data set by illustrating that: (1) the signature genes of a classifier generated from one platform can be directly applied to another platform to develop a predictive classifier; (2) a classifier developed using data generated from one platform can accurately predict samples that were profiled using a different platform. The results suggest the potential utility of using published signature genes in cross-platform applications and the possible adoption of the published classifiers for a variety of applications. The study reveals an opportunity for possible translation of biomarkers identified using microarrays to clinically validated non-array gene expression assays.

Functional muscle analysis of the Tcap knockout mouse.

Autosomal recessive limb-girdle muscular dystrophy type 2G (LGMD2G) is an adult-onset myopathy characterized by distal lower limb weakness, calf hypertrophy and progressive decline in ambulation. The disease is caused by mutations in Tcap, a z-disc protein of skeletal muscle, although the precise mechanisms resulting in clinical symptoms are unknown. To provide a model for preclinical trials and for mechanistic studies, we generated knockout (KO) mice carrying a null mutation in the Tcap gene. Here we present the first report of a Tcap KO mouse model for LGMD2G and the results of an investigation into the effects of Tcap deficiency on skeletal muscle function in 4- and 12-month-old mice. Muscle histology of Tcap-null mice revealed abnormal myofiber size variation with central nucleation, similar to findings in the muscles of LGMD2G patients. An analysis of a Tcap binding protein, myostatin, showed that deletion of Tcap was accompanied by increased protein levels of myostatin. Our Tcap-null mice exhibited a decline in the ability to maintain balance on a rotating rod, relative to wild-type controls. No differences were detected in force or fatigue assays of isolated extensor digitorum longus (EDL) and soleus (SOL) muscles. Finally, a mechanical investigation of EDL and SOL indicated an increase in muscle stiffness in KO animals. We are the first to establish a viable KO mouse model of Tcap deficiency and our model mice demonstrate a dystrophic phenotype comparable to humans with LGMD2G.

TP53 mutations in malignant and premalignant Barrett's esophagus.

In order to improve the efficacy of endoscopic surveillance of Barrett's esophagus, markers of neoplastic progression in addition to dysplasia are required. The aim of the present study was to assess TP53 mutational analysis as a method of identifying patients with Barrett's esophagus who are at greatest risk of adenocarcinoma, for whom endoscopic surveillance is most appropriate. TP53 mutational analysis was initially performed on premalignant and malignant tissue from 30 patients undergoing esophagectomy for adenocarcinoma, and on premalignant biopsies from 48 patients participating in a Barrett's surveillance program. Surveillance patients were followed up endoscopically and histologically for a median of 5 years following TP53 assessment. Mutational analysis was performed by single-strand conformation polymorphism analysis and direct DNA sequencing. TP53 mutations were detected in 10 of 30 esophageal adenocarcinomas, and were more common in well-differentiated carcinomas. An identical TP53 mutation was detected in carcinoma and adjacent dysplasia. Two patients with premalignant Barrett's esophagus had TP53 mutations and one of these patients developed adenocarcinoma on follow up whilst the other has not yet progressed beyond metaplasia. No patient without TP53 mutation developed high-grade dysplasia or adenocarcinoma. TP53 mutations are detected in 33% of esophageal adenocarcinomas and in 4% of premalignant Barrett's esophagus in patients undergoing endoscopic surveillance. TP53 mutation can be detected before the development of high-grade dysplasia or carcinoma, and may be useful in stratifying the risk of adenocarcinoma in patients with Barrett's esophagus.

Mucosal ablation in Barrett's esophagus.

Barrett's esophagus is a prevalent, premalignant condition affecting the gastroesophageal junction and distal esophagus. Ablation plus antireflux therapy has recently been advocated to prevent the development of adenocarcinoma or to treat those unfit or unwilling to undergo esophagectomy. The present article, based on a search of Medline/ISI databases and cross-referencing of relevant articles, reviews the literature on this subject. A number of techniques have been used to remove the affected mucosa, including laser, electrocoagulation, argon plasma coagulation and photodynamic therapy but, as yet, none has been shown to be superior. Depending on the method used, ablation results in complete removal of Barrett's esophagus in approximately one third of patients and a partial response in nearly two-thirds. The resultant squamous mucosa is apparently 'normal' but may regress. To promote and maintain regeneration, antireflux therapy must be sufficient to reduce repetitive injury to the esophageal mucosa. Whether ablation reduces the cancer risk or delays its occurrence is unknown, though recent data suggests benefit. Complications are infrequent and usually mild. Regular follow-up endoscopy and deep biopsies continue to be necessary. Careful data from much larger populations with long-term follow-up is required before ablation reaches the stage of broad clinical application.

Examination of a CYP2E1 repeat polymorphism in a monkey model of alcohol abuse.

BACKGROUND: Cytochrome P-450 2E1 (CYP2E1) is involved in alcohol metabolism, and the expression of this enzyme displays wide phenotypic variability in humans. It has been proposed that some of this variability in expression may be a consequence of the size of a repeat polymorphism in the 5" regulatory region of the gene and that the polymorphism may segregate with alcoholism. This study examined whether the repeat polymorphism exists in macaque monkeys and whether it associates with excessive alcohol consumption in this animal model. METHODS: Ten outbred cynomolgus monkeys (Macaca fascicularis) that displayed a voluntary alcohol consumption ranging from 1.0 to 3.6 g/kg/day were genotyped for a CYP2E1 repeat polymorphism. This polymorphism has been documented in the region from -2519 base pair (bp) to -1953 bp of the human CYP2E1 gene 5" distal promoter. RESULTS: Individual polymerase chain reaction amplification of genomic DNA from each of the 10 monkey samples revealed a single band of approximately 400 bp in the region corresponding to the human CYP2E1 polymorphism. Polymerase chain reaction amplicons from the 10 individuals were sequenced, and each one generated a 370 bp sequence that is 90% identical to the human gene sequence. However, unlike human alleles that contain five to eight repeats, the cynomolgus monkey is homozygous for a single copy of the repeat most closely resembling repeat 8 (88% identical) in the human gene. CONCLUSIONS: These data demonstrate that the CYP2E1 distal promoter region in monkeys is very similar to the human sequence yet lacks the extensive repeated DNA found in humans. This includes the rare repeats 3 and 4, which have been postulated to play a role in transcription regulation and to associate with alcohol abuse liability in humans. These data suggest that the CYP2E1 polymorphism arose late in evolution and that the regulation of the gene by this genetic region is not associated with a heavy alcohol drinking phenotype in the cynomolgus monkey.

Photodynamic therapy and the alimentary tract.

Photodynamic therapy offers the possibility of relatively selective tumour necrosis and normal tissue healing. It has many potential applications but as yet no clear role. Articles, editorials and case reports published primarily in English and listed in Medline/ISI up to April 2000 or identified by a manual search have been reviewed in an attempt to provide a comprehensive overview of the use of photodynamic therapy in the alimentary tract. It is concluded that photodynamic therapy can be an effective treatment for superficial pre-malignant mucosal lesions and early cancers, especially in diffuse disease. Suitable patients include those wishing to avoid surgery, high risk subjects or those in whom other forms of treatment have failed. Superiority over other methods of ablation has not so far been demonstrated. Cheaper and more effective photosensitizers and improved techniques of light delivery are likely to increase the application of photodynamic therapy.

Cognitive, linguistic, and social aspects of adults' noun definitions.

Low-income, rural adults and middle-income, urban adults provided oral definitions for eight common nouns. Two general issues were addressed: (1) whether the rural adults' definitions would conform to the well-documented Aristotelian form typically found among middle-class, well-educated adults; and (2) whether different definitional types would emerge for two different noun classes, social vs. object nouns. Participants' definitions were examined for conceptual content and linguistic form. Among rural participants, the mean proportion of definitions conforming to the Aristotelian model was .13, contrasted with .69 for the urban participants. Also, rural adults were significantly less likely to cast definitions in the conventional linguistic form than were urban adults. On other measures of definitional skill as well, rural participants demonstrated less mastery. There were no significant differences in definitional form between social and object nouns. Various explanations for the findings are considered.

Interaction of phospholipase D1 with a casein-kinase-2-like serine kinase.

Phospholipase D (PLD)1 was phosphorylated in vivo and by an associated kinase in vitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the serine residue at position 911 was converted into alanine (S911A). The kinase could be enriched from detergent-extracted brain membranes and bind and phosphorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel kinase assays we determined that the size of the kinase is approximately 40 kDa and that PLD1 is more effective than S911A in binding the kinase. Preliminary analysis of the phosphorylation sites on PLD1 suggested that the kinase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Membrane fractions from Chinese hamster ovary (CHO) cell lines that inducibly express PLD1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came from observations that immunoprecipitates using PLD1 antibodies contained immunoreactive CK2alpha, and immunoprecipitates using CK2alpha antibodies contained immunoreactive PLD1. Co-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, alpha or beta, suggests that the association of PLD1 with the kinase is through the beta subunit. Supporting this, phosphorylation of PLD1 by purified recombinant CK2alpha was enhanced by purified recombinant CK2beta. Assays measuring PLD1 catalytic activity following phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine in vitro.

Prevalence of aneuploidy and additional anatomic abnormalities in fetuses and neonates with cleft lip with or without cleft palate: a population-based study in Utah.

OBJECTIVE: To determine the prevalence of aneuploidy and additional major anatomic abnormalities in fetuses and neonates with cleft lip with or without cleft palate. METHODS: All cases of cleft lip with or without cleft palate (cleft lip/cleft palate) occurring in Utah from 1995 through 1999 were reviewed by using the Utah Birth Defect Network population-based surveillance system. All pregnancy outcomes are included (stillborn, live born, and termination) in this analysis. RESULTS: Of 263 cases of cleft lip/cleft palate, 72 (27.4%) were unilateral cleft lip, 112 (42.6%) were unilateral cleft lip and cleft palate, 12 (4.6%) were bilateral cleft lip, and 67 (25.5%) were bilateral cleft lip and cleft palate. Fifteen (5.7%) of the 263 fetuses and neonates were aneuploid. One (1.2%) with cleft lip (unilateral and bilateral combined) was aneuploid. Five (4.5%) of the fetuses and neonates with unilateral cleft lip and cleft palate were aneuploid compared with 9 (13.4%) of fetuses and neonates with bilateral cleft lip and cleft palate. In known or presumed euploid fetuses and neonates, additional sonographically occult major anatomic abnormalities occurred in 5 (7.0%) of 71 with unilateral cleft lip, 18 (16.8%) of 107 with unilateral cleft lip and cleft palate, 1 (8.3%) of 12 with bilateral cleft lip, and 12 (20.7%) of 58 with bilateral cleft lip and cleft palate. These abnormalities primarily involved the heart and the central nervous system. CONCLUSIONS: Amniocentesis for karyotype should be offered in all cases of cleft lip/cleft palate because of the risk of aneuploidy. Patients should be counseled that sonographically occult additional anatomic abnormalities might be present with all clefts.

Sample sizes for cancer trials where Health Related Quality of Life is the primary outcome.

Health Related Quality of Life (HRQoL) instruments are increasingly important in evaluating health care, especially in cancer trials. When planning a trial, one essential step is the calculation of a sample size, which will allow a reasonable chance (power) of detecting a pre-specified difference (effect size) at a given level of statistical significance. It is almost mandatory to include this calculation in research protocols. Many researchers quote means and standard deviations to determine effect sizes, and assume the data will have a Normal distribution to calculate their required sample size. We have investigated the distribution of scores for two commonly used HRQoL instruments completed by lung cancer patients, and have established that scores do not have the Normal distribution form. We demonstrate that an assumption of Normality can lead to unrealistically sized studies. Our recommendation is to use a technique that is based on the fact that the HRQoL data are ordinal and makes minimal but realistic assumptions.

Genetic polymorphisms of the IL-1alpha and IL-1beta genes in African-American LJP patients and an African-American control population.

BACKGROUND: A functional polymorphism of the interleukin-1 beta (IL-1beta) gene has been proposed to be a risk factor for periodontitis. In adult forms of periodontitis, non-smokers of northern European heritage carrying the "2" allele of the IL-1alpha-889 and the IL-1beta +3953 RFLPs in either the heterozygous or the homozygous state at both loci were observed to have a greater risk for developing severe periodontitis. Studies of early-onset periodontitis (EOP) found that allele "1" of both IL-1alpha-889 and IL-1beta +3953 was transmitted more frequently with the EOP phenotype. The purpose of the present study was to determine the prevalence of the IL-1alpha and IL-1beta genotype polymorphisms in an African-American (AA) control population and in 37 African-Americans with localized juvenile periodontitis (LJP). METHODS: The IL-1alpha +4845 and IL-1beta +3953 loci were genotyped by PCR amplification, followed by restriction enzyme digestion and gel electrophoresis. The IL-1alpha +4845 locus, in linkage disequilibrium (>99%) with IL-1alpha-889, was genotyped because it is technically easier. Data were analyzed using r x c contingency tables. RESULTS: The IL-1beta +3953 allele "1" was carried by >99% of the AA control population and by 100% of the AA LJP group, with most individuals being homozygous 1,1. The prevalence of the composite genotype with at least one allele "2" at each of the IL-1beta +3953 and IL-1alpha +4845 loci was 14% (AA control group) and 8% (AA LJP group). CONCLUSIONS: Given the high frequency of the IL-1beta allele "1" in the African-American population, it would appear that knowledge of this +3953 polymorphism would provide little diagnostic or predictive information for LJP.

Histological and molecular mapping of adenocarcinoma of the oesophagus and gastroesophageal junction: loss of heterozygosity occurs in histologically normal epithelium in the oesophagus and stomach.

Seventy malignant, premalignant and histologically normal biopsies from 7 oesophagogastrectomy specimens of adenocarcinomas of the lower oesophagus and gastroesophageal junction were analysed for loss of heterozygosity (LOH) at 9 known or putative gene loci. LOH was detected in 20 of 27 (74%) malignant biopsies, 4 of 7 (57%) biopsies of dysplasia, 2 of 12 (25%) biopsies of histologically normal oesophagus adjacent to adenocarcinoma, and in 2 of 14 (14%) biopsies of histologically normal stomach adjacent to adenocarcinoma. LOH at the VHL, APC, CDKN2 and DCC tumour suppressor and MSH3 mismatch repair gene loci can be detected in histologically normal tissue and in adjacent adenocarcinoma, and are potential markers of early neoplastic progression.

Activation of phospholipase D1 by Cdc42 requires the Rho insert region.

Members of the Rho subfamily of GTP-binding proteins are implicated in the regulation of phospholipase D (PLD). In the present study, we demonstrate a physical association between a Rho family member, Cdc42, and PLD1. Binding of Cdc42 to PLD1 and subsequent activation are GTP-dependent. Although binding of Cdc42 to PLD1 does not require geranylgeranylation, activation of PLD1 is dependent on this lipid modification of Cdc42. Specific point mutations in the switch I region of Cdc42 abolish binding to and, therefore, activation of PLD1 by Cdc42. Deletion of the Rho insert region, which consists of residues 120-139, from Cdc42 does not interfere with binding to PLD1 but inhibits Cdc42 stimulated PLD1 activity. Interestingly, deletion of the insert region from Cdc42 also inhibits activation of PLD1 by Arf and protein kinase C. With the lack of specific inhibitors of PLD activity, the insert deletion mutant of Cdc42 (designated (DeltaL8)Cdc42) is a novel reagent for in vitro studies of PLD1 regulation, as well as for in vivo studies of Cdc42-mediated signaling pathways leading to PLD1 activation. Because the insert region is required for the transforming activity of Cdc42, regulation of PLD1 by this region on Cdc42 is of major interest.