Calcrete aquifers from the Yilgarn region of arid central Western Australia contain an assemblage of obligate groundwater invertebrate species that are each endemic to single aquifers. Fine-scale phylogeographic and population genetic analyses of three sympatric and independently derived species of amphipod (Chiltoniidae) were carried out to determine whether there were common patterns of population genetic structure or evidence for past geographic isolation of populations within a single calcrete aquifer. Genetic diversity in amphipod mitochondrial DNA (cytochrome c oxidase subunit I gene) and allozymes were examined across a 3.5 km2 region of the Sturt Meadows calcrete, which contains a grid of 115 bore holes (=wells). Stygobiont amphipods were found to have high levels of mitochondrial haplotype diversity coupled with low nucleotide diversity. Mitochondrial phylogeographic structuring was found between haplogroups for one of the chiltoniid species, which also showed population structuring for nuclear markers. Signatures of population expansion in two of the three species, match previous findings for diving beetles at the same site, indicating that the system is dynamic. We propose isolation of populations in refugia within the calcrete, followed by expansion events, as the most likely source of intraspecific genetic diversity, due to changes in water level influencing gene flow across the calcrete.
Stygofauna; mitochondrial DNA; allozymes; population genetic structure
Reasons for performing study
The 2 sites of bone marrow harvest for isolation of mesenchymal stromal cells (MSC) in the horse are the sternum and ilium. The technical procedure is based on practitioner preference but no studies have compared MSC concentrations and growth rates between each site in horses aged 2–5 years.
The objective of this study was to compare nucleated cell counts and growth rates between the sternum and ilium and between consecutive 5 ml bone marrow aspirates. We hypothesised that there would be a higher concentration of MSCs in the sternum than the ilium, and that the first sequential aspirate from either site would yield the greatest concentration of MSCs. We hypothesised that growth rates of cells from each site would not differ.
Seven horses, aged 2–5 years old, had 2 sequential 5 ml marrow aspirates taken from the sternum and ilium. Nucleated cell counts (NCCs) were obtained pre- and post- marrow processing. Cells were expanded in culture for 3 passages and growth rate characteristics compared for all aspirates.
The NCCs of the first 5 ml aspirate were higher than the second 5 ml aspirate for both sites (p<0.05). There was no difference between growth rates for any of the groups (p>0.05).
The NCCs and growth rates of progenitor cells in the ilium and sternum are similar for horses in the 2–5 year age category. The first 5 ml bone marrow aspirate has a higher concentration of NCCs and resulting bone marrow-derived MSC population than subsequent aspirates.
The first 5 ml aspirates from the sternum and ilium offer a rich supply of bone marrow-derived MSCs with similar growth rate characteristics. The harvesting procedure of only a 5 ml draw from either the sternum or ilium should result in adequate numbers of MSCs.
horse; bone marrow; stem cells; mesenchymal
Anthracycline-treated childhood cancer survivors suffer cardiac damage that results in decreased left ventricular (LV) mass, leading to increased LV wall stress, which underlies their greater risk of cardiomyopathy. Many of these survivors are also at risk of growth hormone (GH) abnormalities from cranial irradiation exposure though it is unknown if such exposure is associated with cardiotoxicity.
Echocardiograms and insulin-like growth factor-1 (IGF-1), a marker of GH, were measured in 130 anthracycline-treated childhood cancer survivors, 59 of whom had been exposed to cranial irradiation, a mean 10 years from cancer diagnosis. Echocardiographic parameters and IGF-1 were standardized relative to age or body-surface area using data from sibling controls and expressed as the percent difference from normal.
After adjusting for other risk factors, survivors exposed to cranial irradiation had an additional 12% decrease in LV mass compared to unexposed survivors (P<.01), and an additional 3.6% decrease in LV dimension (P=.03). Survivors exposed to cranial irradiation also had a 30.8% decrease in IGF-1 relative to normal, which was greater than the 10.5% decrease in unexposed survivors (P<.01).
In anthracycline-treated childhood cancer survivors, a mean 10 years from diagnosis, those with cranial irradiation exposure had significantly greater decreases in LV mass and dimension. Because cranial irradiation was also associated with decreased IGF-1, it is possible that GH deficiencies mediated this effect suggesting that GH replacement therapy may help prevent the development of cardiotoxicity.
Cardiomyopathy; Anthracyclines; Cranial Irradiation; Cancer; Survivors
Childhood cancer five-year survival now exceeds 70–80%. Childhood exposure to radiation is a known thyroid carcinogen; however, data are limited for the evaluation of radiation dose-response at high doses, modifiers of the dose-response relationship and joint effects of radiotherapy and chemotherapy. To address these issues, we pooled two cohort and two nested case-control studies of childhood cancer survivors including 16,757 patients, with 187 developing primary thyroid cancer. Relative risks (RR) with 95% confidence intervals (CI) for thyroid cancer by treatment with alkylating agents, anthracyclines or bleomycin were 3.25 (0.9–14.9), 4.5 (1.4–17.8) and 3.2 (0.8–10.4), respectively, in patients without radiotherapy, and declined with greater radiation dose (RR trends, P = 0.02, 0.12 and 0.01, respectively). Radiation dose-related RRs increased approximately linearly for <10 Gy, leveled off at 10–15-fold for 10–30 Gy and then declined, but remained elevated for doses >50 Gy. The fitted RR at 10 Gy was 13.7 (95% CI: 8.0–24.0). Dose-related excess RRs increased with decreasing age at exposure (P < 0.01), but did not vary with attained age or time-since-exposure, remaining elevated 25+ years after exposure. Gender and number of treatments did not modify radiation effects. Thyroid cancer risks remained elevated many decades following radiotherapy, highlighting the need for continued follow up of childhood cancer survivors.
To determine whether cardiovascular abnormalities in childhood cancer survivors are restricted to patients exposed to cardiotoxic anthracyclines and cardiac irradiation and how risk factors for atherosclerotic disease and systemic inflammation contribute to global cardiovascular status.
We assessed echocardiographic characteristics and atherosclerotic disease risk in 201 survivors of childhood cancer with and without exposure to cardiotoxic treatments at a median of 11 years after diagnosis (range, 3 to 32 years) and in 76 sibling controls.
The 156 exposed survivors had below normal left ventricular (LV) mass, wall thickness, contractility, and fractional shortening and above normal LV afterload. The 45 unexposed survivors also had below normal LV mass overall, and females had below normal LV wall thickness. Exposed and unexposed survivors, compared with siblings, had higher levels of N-terminal pro-brain natriuretic peptide (81.7 and 69.0 pg/mL, respectively, v 39.4 pg/mL), higher mean fasting serum levels of non–high-density lipoprotein cholesterol (126.5 and 121.1 mg/dL, respectively, v 109.8 mg/dL), higher insulin levels (10.4 and 10.5 μU/mL, respectively, v 8.2 μU/mL), and higher levels of high-sensitivity C-reactive protein (2.7 and 3.1 mg/L, respectively, v 0.9 mg/L; P < .001 for all comparisons). Age-adjusted, predicted-to-ideal 30-year risk of myocardial infarction, stroke, or coronary death was also higher for exposed and unexposed survivors compared with siblings (2.16 and 2.12, respectively, v 1.70; P < .01 for both comparisons).
Childhood cancer survivors not receiving cardiotoxic treatments nevertheless have cardiovascular abnormalities, systemic inflammation, and an increased risk of atherosclerotic disease. Survivorship guidelines should address cardiovascular concerns, including the risk of atherosclerotic disease and systemic inflammation, in exposed and unexposed survivors.
Childhood cancer survivors are at increased risk of cardiovascular disease (CVD), which may be associated with traditional CVD risk factors. We used CVD risk aggregation instruments to describe survivor cardiometabolic health and compared their results with sibling controls.
Traditional CVD risk factors measured in 110 survivors and 31 sibling controls between 15 and 39 years old were aggregated using Pathobiological Determinants of Atherosclerosis in Youth (PDAY) scores and the Framingham Risk Calculator (FRC) and expressed as ratios. The PDAY odds ratio represents the increased odds of currently having an advanced coronary artery lesion, and the FRC risk ratio represents the increased risk of having a myocardial infarction, stroke, or coronary death in the next 30 years. Ratios are relative to an individual of similar age and sex without CVD risk factors.
The median PDAY odds ratio for survivors was 2.2 (interquartile range 1.3-3.3), with 17% N4. The median FRC risk ratio was 1.7 (interquartile range 1.0-2.0), with 12% N4. Survivors and siblings had similar mean PDAY odds ratios (2.33 vs 2.29, P = .86) and FRC risk ratios (1.72 vs 1.53, P = .24). Cancer type and treatments were not associated with cardiometabolic health. There was a suggested association for physical inactivity with PDAY odds ratios (r = 0.17, P = .10) and FRC risk ratios (r = 0.19, P = .12).
Cardiometabolic health is poor in childhood cancer survivors but not different than that of their siblings, highlighting the importance of managing traditional CVD risk factors and considering novel exposures in survivors.
Most smokers begin smoking during adolescence, a period during which social reward is highly influential. Initial exposure to nicotine can produce anxiogenic effects that may be influenced by social context. This study examined play behavior and plasma corticosterone following nicotine administration (0.6 mg/kg, s.c.) in both male and female adolescent (PND39) Sprague-Dawley rats in either isolate or social contexts. In blood samples collected immediately following the 15-min test session, nicotine increased plasma corticosterone relative to saline in both male and female isolate rats, but failed to do so in both males and females placed together in same-sex pairs. Nicotine also attenuated several indices of play behavior including nape attacks, pins and social contact. In isolate rats, nicotine selectively increased locomotor activity in females; however, when administered to social pairs, nicotine decreased locomotion in both sexes. These findings suggest that the presence of a social partner may decrease the initial negative, stress-activating effects of nicotine, perhaps leading to increased nicotine reward.
Nicotine; Corticosterone; Sociality; Sex differences; Stress; Play behavior
Women with diabetes have elevated gestational risks for severe hemodynamic complications, including preeclampsia in mid- to late pregnancy. This study employed continuous, chronic radiotelemetry to compare the hemodynamic patterns in non-obese diabetic (NOD) mice who were overtly diabetic or normoglycemic throughout gestation. We hypothesized that overtly diabetic, pregnant NOD mice would develop gestational hypertension and provide understanding of mechanisms in progression of this pathology.
Telemeter-implanted, age-matched NOD females with and without diabetes were assessed for six hemodynamic parameters (mean, systolic, diastolic, pulse pressures, heart rate and activity) prior to mating, over pregnancy and over a 72 hr post-partum interval. Urinalysis, serum biochemistry and renal histopathology were also conducted.
Pregnant, normoglycemic NOD mice had a hemodynamic profile similar to other inbred strains, despite insulitis. This pattern was characterized by an interval of pre-implantation stability, post implantation decline in arterial pressure to mid gestation, and then a rebound to pre-pregnancy baseline during later gestation. Overtly diabetic NOD mice had a blood pressure profile that was normal until mid-gestation then become mildly hypotensive (−7mmHg, P<0.05), severely bradycardic (−80bpm, P<0.01) and showed signs of acute kidney injury. Pups born to diabetic dams were viable but growth restricted, despite their mothers’ failing health, which did not rebound post-partum (−10% pre-pregnancy pressure and HR, P<0.05).
Pregnancy accelerates circulatory and renal pathologies in overtly diabetic NOD mice and is characterized by depressed arterial pressure from mid-gestation and birth of growth 45 restricted offspring.
PMID: 22014504 CAMSID: cams2033
Type 1 Diabetes Mellitus; Pregnancy; Radiotelemetry; Hemodynamics
African American men have the highest rates of prostate cancer of any racial group, but very little is known about the psychological functioning of African American men in response to prostate cancer diagnosis and treatment.
In this secondary analysis of a national trial testing a psychological intervention for prostate cancer patients, we report on the traumatic stress symptoms of African American and non-African American men.
A total of 329 men were enrolled in the intervention trial, which included 12 weeks of group psychotherapy and 24 months of follow-up. Using mixed model analysis, total score on the Impact of Events Scale (IES) and its Intrusion and Avoidance subscales were examined to determine mean differences in traumatic stress across all time points (0, 3, 6, 12, 18, and 24 months). In an additional analysis, relevant psychosocial, demographic, and clinical variables were added to the model.
Results showed significantly higher levels of traumatic stress for African American men compared to non-African American men in all models independently of the intervention arm, demographics and relevant clinical variables. African Americans also had a consistently higher prevalence of clinically significant traumatic stress symptoms (defined as IES total score ≥ 27). These elevations remained across all time points over 24 months.
This is the first study to show a racial disparity in traumatic stress specifically as an aspect of overall psychological adjustment to prostate cancer. Recommendations are made for appropriate assessment, referral, and treatment of psychological distress in this vulnerable population.
Prostate cancer; African American; PTSD; traumatic stress; health disparities
Background and purpose:
Apremilast is an orally administered phosphodiesterase-4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro-inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis.
Apremilast was tested in vitro against endotoxin- and superantigen-stimulated PBMC, bacterial peptide and zymosan-stimulated polymorphonuclear cells, immunonoglobulin and cytokine-stimulated NK cells, and ultraviolet B light-activated keratinocytes. Apremilast was orally administered to beige-severe combined immunodeficient mice, xenotransplanted with normal human skin and triggered with human psoriatic NK cells. Epidermal skin thickness, proliferation index and inflammation markers were analysed.
Apremilast inhibited PBMC production of the chemokines CXCL9 and CXCL10, cytokines interferon-γ and tumour necrosis factor (TNF)-α, and interleukins (IL)-2, IL-12 and IL-23. Production of TNF-α by NK cells and keratinocytes was also inhibited. In vivo, apremilast significantly reduced epidermal thickness and proliferation, decreased the general histopathological appearance of psoriasiform features and reduced expression of TNF-α, human leukocyte antigen-DR and intercellular adhesion molecule-1 in the lesioned skin.
Conclusions and implications:
Apremilast displayed a broad pattern of anti-inflammatory activity in a variety of cell types and decreased the incidence and severity of a psoriasiform response in vivo. Inhibition of TNF-α, IL-12 and IL-23 production, as well as NK and keratinocyte responses by this phosphodiesterase-4 inhibitor suggests a novel approach to the treatment of psoriasis.
immunopharmacology; skin pharmacology; chemokines; anti-inflammatory drugs; PDE inhibitor; inflammation
Cancer patients often report impaired sleep quality. Impaired sleep quality may be due to increased levels of sleep-mediating cytokines resulting from cancer treatment. Exercise may have a positive influence on sleep-mediating cytokines, such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and soluble tumor necrosis factor-alpha receptor (sTNF-R), which may improve sleep quality. This two-arm pilot study compared the influence of a home-based exercise intervention with standard care/control on sleep quality and mediators of sleep. Breast and prostate cancer patients (n = 38) beginning radiation therapy were randomized to a 4-week exercise program or no exercise arm. Global sleep quality, subjective sleep quality, sleep latency, sleep duration, sleep efficiency, sleep disturbances, use of sleep medication, and daytime dysfunction were assessed with the Pittsburgh Sleep Quality Index. IL-6, TNF-α, and sTNF-R were measured before and after intervention. There was a greater improvement in sleep quality in the exercise group from pre- to postintervention, although the difference was not significant. Additionally, there were associations between IL-6 and sleep efficiency and duration, suggesting that regulation of sleep-mediating cytokines by exercise may mediate improvements in sleep-quality components.
We conducted a randomized clinical trial examining the effects of modafinil in reducing persistent fatigue in patients following treatment for cancer and performed secondary analyses to assess the effect of modafinil on cognitive function.
Breast cancer patients who reported a score of ≥ 2 on the Brief Fatigue Inventory (BFI) were enrolled in the study. In Phase 1 (P1), patients received 200mg modafinil open-label once daily for 4 weeks. In Phase 2 (P2), patients with a positive response following P1 were randomized either to an additional 4 weeks of modafinil or to placebo. Tests of memory and attention selected from the Cognitive Drug Research (CDR) computerized cognitive assessment were performed at baseline (before modafinil) and after completing Phase 1 and 2. The paired differences for each test score were subjected to a Wilcoxon's signed rank test.
Of the 82 women who were enrolled, 76 completed P1 and 68 completed all assessments in the study. Modafinil had a significant effect on the Speed of Memory (p=0.0073) and Quality of Episodic Memory (p<0.0001) during P1 of the study. After randomization at week 8, those patients who continued modafinil demonstrated significantly greater improvement in Speed of Memory (p=0.029), Quality of Episodic Memory (p=0.0151) and mean Continuity of Attention (p=0.0101) relative to the group switched to placebo.
We found that modafinil improved cognitive performance in breast cancer survivors by enhancing some memory and attention skills. Although confirmation is needed, these findings suggest that modafinil may enhance quality of life in this patient population.
modafinil; cognitive function; memory; attention; breast cancer
In this study we report that primary cultures of rat fetal neurons contain subpopulations of cells that may be sensitive or resistant to HIV-1 Tat neurotoxicity. We demonstrate that rapid binding/uptake of Tat 1–86 for 2 hours was sufficient to trigger caspase activation and neurodegeneration in rat fetal midbrain cell cultures. The uptake of Tat was followed by an increase in MCP1 (CCL2) immunoreactivity. Approximately 70% of neurons were able to survive transient or continuous (7 days) Tat exposure. The surviving neurons did not contain bound/internalized Tat, but were able to interact with Tat after medium replacement. These neurons were resistant to Tat toxicity. In neurons that resisted the toxic effects of continuous and repeated Tat treatment, levels of NR2A subunit of the NMDA receptor complex were significantly lower than in controls. We suggest that the subunit composition of NMDAR complexes may be important for the sensitivity of neurons to Tat toxicity.
Structural genomics (or proteomics) activities are critically dependent
on the availability of high-throughput structure determination methodology.
Development of such methodology has been a particular challenge for NMR based
structure determination because of the demands for isotopic labeling of proteins
and the requirements for very long data acquisition times. We present here a
methodology that gains efficiency from a focus on determination of backbone
structures of proteins as opposed to full structures with all side chains in
place. This focus is appropriate given the presumption that many protein
structures in the future will be built using computational methods that start
from representative fold family structures and replace as many as
70% of the side chains in the course of structure determination. The
methodology we present is based primarily on residual dipolar couplings (RDCs),
readily accessible NMR observables that constrain the orientation of backbone
fragments irrespective of separation in space. A new software tool is described
for the assembly of backbone fragments under RDC constraints and an application
to a structural genomics target is presented. The target is an 8.7 kDa protein
from Pyrococcus furiosus, PF1061, that was previously not well
annotated, and had a nearest structurally characterized neighbor with only
33% sequence identity. The structure produced shows structural
similarity to this sequence homologue, but also shows similarity to other
proteins that suggests a functional role in sulfur transfer. Given the backbone
structure and a possible functional link this should be an ideal target for
development of modeling methods.
NMR; partial alignment; pyrococcus furiosus; RDC; structure determination; C12E5, Pentaethylene Glycol Monododecyl Ether; CTAB, Hexadecyltrimethylammonium bromide; DSS, 2,2-dimethyl-2-silapentane- 5-sulfonic acid DTT, dithiothreitol
rates in children with insulin dependent diabetes (IDDM) in the UK are
unknown and the causes of death not well documented.
AIM—To determine the
mortality rate and causes of death in children with IDDM.
METHODS—The Office of
National Statistics (England and Wales) and the General Register Office
(Scotland) notified all deaths under 20 years of age from 1990 to 1996 with diabetes on the certificate. Further details were provided by
coroners, pathologists, and clinicians.
deaths were notified and 83 were caused by diabetes. The standardised
mortality ratio was 2.3 (95% confidence interval (CI), 1.9 to 2.9),
being highest in the age group 1-4 years, at 9.2 (95% CI, 5.4 to
14.7). Of the 83 diabetic deaths, hyperglycaemia/diabetic ketoacidosis
(DKA) was implicated in 69 and hypoglycaemia in 7.Cerebral oedema was
the most common cause of death in young children (25 of 36 diabetes
related deaths in children under 12 years of age). 34 young people
(10-19 years; 24 male) were either found dead at home (n = 26) or
moribund on arrival at hospital (n = 8). In 24 of these, it appeared
that DKA was the cause of death, in four hypoglycaemia was likely. Nine
of these were found "dead in bed".
with IDDM have a higher mortality than the general population. Cerebral
oedema accounts for most hospital deaths in young children. There are a
large number of young men dying at home from neglected IDDM. Early
diagnosis of IDDM in children and closer supervision of young people
might prevent some of these deaths.
OBJECTIVE: To examine regional changes in the fluid content of human intervertebral discs by comparing sagittal plane "profiles" of hydration before and after mechanical loading. METHODS: Cadaveric lumbar intervertebral discs were loaded to simulate a typical day's loading in vivo. Ten motion segments were subjected to a 1500 N compressive load for a period of 6 h with the superior vertebrae inclined by 4-8 degrees to simulate a slightly flexed posture. Immediately after loading the discs were frozen at -80 degrees C. Subsequently they were cut into slices perpendicular to the sagittal midline of the disc, and each slice was weighed before and after freeze drying. This enabled a profile of fluid content across the disc to be constructed. Fluid loss due to loading was estimated by comparing the water content of each loaded disc with that of an adjacent unloaded disc from the same spine. RESULTS: After 6 h of creep loading, disc height approached, but did not quite reach, an equilibrium. The mean fluid loss from all discs was 18%. All regions except the outer 2 mm experienced a significant loss of fluid (P < 0.01). The posterior mid-annulus showed the greatest fluid loss (30%), while the nucleus lost 15%. CONCLUSIONS: A comparison with previously published work suggests that fluid exchange of this magnitude will have a considerable effect on disc cell metabolism and on metabolite transport.
Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.
BACKGROUND: The training provided for senior house officers (SHOs) has been the subject of debate, and variable satisfaction with training has been reported. The reliability of the instruments used for measuring satisfaction has not been adequately addressed. AIM: To develop a reliable questionnaire to measure SHO satisfaction with hospital training. METHOD: A 42-item questionnaire with eight scales was developed using criteria from the joint hospital visiting guidelines of the Royal College of General Practitioners. The questionnaire was sent to SHOs in Anglia before monitoring visits from the royal colleges, the postgraduate dean and the Joint Committee on Postgraduate Training for General Practice. RESULTS: Response rates varied from 37.0% to 100%, with an overall response rate of 58.8%. The internal reliability of the whole questionnaire was 0.82. Levels of internal reliability for the individual scales were satisfactory, Cronbach's alpha coefficient being 0.75 or more in all but two of the scales. Test-retest reliability using Pearson's product moment correlation coefficient was greater than 0.82 for six of the scales. There were significant differences in total satisfaction between SHOs reporting on posts accredited by the different royal colleges and also between SHOs training for general practice and those training to be specialists. CONCLUSION: A reliable questionnaire has been developed to measure SHO satisfaction with hospital training that is acceptable to doctors and feasible to administer. National acceptance of a single questionnaire for monitoring SHO posts would enable standards to be monitored regularly at a time of considerable change in hospital training.
Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.
Butylated hydroxytoluene (BHT) can inhibit experimental atherosclerosis in animals. Although the agent is an antioxidant, the exact mechanism of the reaction in atherosclerosis is still unknown. To investigate the effects of BHT on expression of P-selectin (PADGEM, GMP-140), intercellular adhesion molecule-1 (ICAM-1) and class II MHC (Ia) antigen, we proposed an experiment on rats. Male rats (n=18 per group) were fed either a normal cholesterol control diet, a normal cholesterol diet containing 0.5% BHT (BD), a high cholesterol diet containing 1.5% cholesterol and 0.1% sodium cholate (CD), or the CD diet containing 0.5% BHT (BCD). Rats were sacrificed after 3 days, and after 1, 2, 4, 10, and 17 weeks of dietary treatment. Although there was no gross or light microscopic atherosclerotic lesions, scanning electron microscopy revealed monocytic adhesion to aortic endothelium and mild endothelial injuries in CD and BCD groups. Immunohistochemically, the addition of BHT to a high cholesterol diet inhibited P-selectin expression but not in ICAM-1 and Ia antigen. These findings suggest that in rats, high cholesterol diets induce expression of ICAM-1, P-selectin and Ia antigen. In addition, the antiatherogenic effect of BHT may play a role in the inhibition of P-selectin.