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1.  CIAPIN1 gene silencing enhances chemosensitivity in a drug-resistant animal model in vivo  
Overexpression of cytokine-induced apoptosis inhibitor 1 (CIAPIN1) contributes to multidrug resistance (MDR) in breast cancer. This study aimed to evaluate the potential of CIAPIN1 gene silencing by RNA interference (RNAi) as a treatment for drug-resistant breast cancer and to investigate the effect of CIAPIN1 on the drug resistance of breast cancer in vivo. We used lentivirus-vector-based RNAi to knock down CIAPIN1 in nude mice bearing MDR breast cancer tumors and found that lentivirus-vector-mediated silencing of CIAPIN1 could efficiently and significantly inhibit tumor growth when combined with chemotherapy in vivo. Furthermore, Western blot analysis showed that both CIAPIN1 and P-glycoprotein expression were efficiently downregulated, and P53 was upregulated, after RNAi. Therefore, we concluded that lentivirus-vector-mediated RNAi targeting of CIAPIN1 is a potential approach to reverse MDR of breast cancer. In addition, CIAPIN1 may participate in MDR of breast cancer by regulating P-glycoprotein and P53 expression.
doi:10.1590/1414-431X20133356
PMCID: PMC4075290  PMID: 24676475
CIAPIN1 gene; Multidrug resistance; RNA interference; MDR1 gene; Breast neoplasms
2.  Interleukin-1β promotes hypoxia-induced apoptosis of glioblastoma cells by inhibiting hypoxia-inducible factor-1 mediated adrenomedullin production 
Cell Death & Disease  2014;5(1):e1020-.
Glioblastoma is the most common brain tumor in adults. Advanced glioblastomas normally contain hypoxic areas. The primary cellular responses to hypoxia are generally mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1). Interleukin-1β (IL-1β) is a cytokine that is often present in the glioblastoma microenvironment and is known to be a modulator of glioblastoma progression. However, the role of IL-1β in regulating glioblastoma progression is still controversial. In this study, we found that in the human glioblastoma cell lines U87MG and U138MG, IL-1β inhibits the transactivation activity of HIF-1 by promoting the ubiquitin-independent proteasomal degradation of the oxygen-labile α-subunit of HIF-1 and downregulates the expression of the HIF-1 target gene adrenomedullin (AM). Apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays showed that AM protects glioblastoma cells against hypoxia-induced apoptosis in a dose-dependent manner. Thus, in the presence of IL-1β more glioblastoma cells undergo hypoxia-induced cell death. Our findings suggest that when estimating the influence of IL-1β on the prognosis of glioblastoma patients, factors such as the degree of hypoxia, the expression levels of HIF-1 and AM should be taken into consideration. For the AM-producing glioblastoma cells, IL-1β represents a potent apoptosis inducer.
doi:10.1038/cddis.2013.562
PMCID: PMC4040669  PMID: 24457964
apoptosis; glioblastoma; HIF-1; interleukin-1β
3.  (ADP-ribose) polymerase 1 and AMP-activated protein kinase mediate progressive dopaminergic neuronal degeneration in a mouse model of Parkinson's disease 
Cell Death & Disease  2013;4(11):e919-.
Genetic and epidemiologic evidence suggests that cellular energy homeostasis is critically associated with Parkinson's disease (PD) pathogenesis. Here we demonstrated that genetic deletion of Poly (ADP-ribose) polymerase 1 completely blocked 6-hydroxydopamine-induced dopaminergic neurodegeneration and related PD-like symptoms. Hyperactivation of PARP-1 depleted ATP pools in dopaminergic (DA) neurons, thereby activating AMP-activated protein kinase (AMPK). Further, blockade of AMPK activation by viral infection with dominant-negative AMPK strongly inhibited DA neuronal atrophy with moderate suppression of nuclear translocation of apoptosis-inhibiting factor (AIF), whereas overactivation of AMPK conversely strengthened the 6-OHDA-induced DA neuronal degeneration. Collectively, these results suggest that manipulation of PARP-1 and AMPK signaling is an effective therapeutic approach to prevent PD-related DA neurodegeneration.
doi:10.1038/cddis.2013.447
PMCID: PMC3847323  PMID: 24232095
PARP-1; ATP; AMPK; 6-OHDA; Parkinson's disease
4.  Diffusion Tensor Imaging Reveals White Matter Reorganization in Early Blind Humans 
Multiple functional methods including functional magnetic resonance imaging, transcranial magnetic stimulation, and positron emission tomography have shown cortical reorganization in response to blindness. We investigated microanatomical correlates of this reorganization using diffusion tensor imaging and diffusion tensor tractography (DTT). Five early blind (EB) were compared with 7 normally sighted (NS) persons. DTT showed marked geniculocalcarine tract differences between EB and NS participants. All EB participants showed evidence of atrophy of the geniculocortical tracts. Connections between visual cortex and the orbital frontal and temporal cortices were relatively preserved in the EB group. Importantly, no additional tracts were found in any EB participant. Significant alterations of average diffusivity and relative anisotropy were found in the white matter (WM) of the occipital lobe in the EB group. These observations suggest that blindness leads to a reorganization of cerebral WM and plausibly support the hypothesis that visual cortex functionality in blindness is primarily mediated by corticocortical as opposed to thalamocortical connections.
doi:10.1093/cercor/bhj102
PMCID: PMC3789517  PMID: 16400157
blindness; human; magnetic resonance imaging; visual cortex/*physiology
5.  Different cellular p16INK4a localisation may signal different survival outcomes in head and neck cancer 
British Journal of Cancer  2012;107(3):482-490.
Background:
Recently, the management of head and neck squamous cell carcinoma (HNSCC) has focused considerable attention on biomarkers, which may influence outcomes. Tests for human papilloma infection, including direct assessment of the virus as well as an associated tumour suppressor gene p16, are considered reproducible. Tumours from familial melanoma syndromes have suggested that nuclear localisation of p16 might have a further role in risk stratification. We hypothesised p16 staining that considered nuclear localisation might be informative for predicting outcomes in a broader set of HNSCC tumours not limited to the oropharynx, human papilloma virus (HPV) status or by smoking status.
Methods:
Patients treated for HNSCC from 2002 to 2006 at UNC (University of North Carolina at Chapel Hill) hospitals that had banked tissue available were eligible for this study. Tissue microarrays (TMA) were generated in triplicate. Immunohistochemical (IHC) staining for p16 was performed and scored separately for nuclear and cytoplasmic staining. Human papilloma virus staining was also carried out using monoclonal antibody E6H4. p16 expression, HPV status and other clinical features were correlated with progression-free (PFS) and overall survival (OS).
Results:
A total of 135 patients had sufficient sample for this analysis. Median age at diagnosis was 57 years (range 20–82), with 68.9% males, 8.9% never smokers and 32.6% never drinkers. Three-year OS rate and PFS rate was 63.0% and 54.1%, respectively. Based on the p16 staining score, patients were divided into three groups: high nuclear, high cytoplasmic staining group (HN), low nuclear, low cytoplasmic staining group (LS) and high cytoplasmic, low nuclear staining group (HC). The HN and the LS groups had significantly better OS than the HC group with hazard ratios of 0.10 and 0.37, respectively, after controlling for other factors, including HPV status. These two groups also had significantly better PFS than the HC staining group. This finding was consistent for sites outside the oropharynx and did not require adjustment for smoking status.
Conclusion:
Different p16 protein localisation suggested different survival outcomes in a manner that does not require limiting the biomarker to the oropharynx and does not require assessment of smoking status.
doi:10.1038/bjc.2012.264
PMCID: PMC3405208  PMID: 22735904
p16 cellular localisation; tissue microarray; HPV infection
6.  Brief exposures of human body lice to sub-lethal amounts of ivermectin over transcribes detoxification genes involved in tolerance 
Insect molecular biology  2011;20(6):687-699.
Transcriptional profiling results, using our non-invasive induction assay [short exposure intervals (2–5 h) to sub-lethal amounts of insecticides (
Of the cytochrome P450 monooxygenase and ATP binding cassette transporter genes induced by ivermectin, CYP6CJ1, CYP9AG1, CYP9AG2 and PhABCC4 were respectively most significantly over-expressed, had high basal expression levels and were most closely related to genes from other organisms that metabolized insecticides, including ivermectin.
Injection of dsRNAs against either CYP9AG2 or PhABCC4 into non-induced female lice reduced their respective transcript level and resulted in increase sensitivity to ivermectin, indicating that these two genes are involved in the xenobiotic metabolism of ivermectin and in the production of tolerance.
doi:10.1111/j.1365-2583.2011.01097.x
PMCID: PMC3208734  PMID: 21895817
Pediculus humanus humanus; ivermectin; tolerance; transcriptional profiling; RNA interference; P450; ABC transporters
Neuroscience  2011;197:339-347.
Previous studies have shown the feasibility of using diffusion tensor imaging (DTI) as a noninvasive imaging modality to evaluate neurodegeneration in humans and animals. The axial and radial diffusivities derived from DTI were demonstrated to be sensitive markers for axonal and myelin damage, respectively. This study used DTI to evaluate optic nerve degeneration in wild-type and slow Wallerian degeneration (WldS) mutant mice. Longitudinal DTI was performed on optic nerves following high intraocular pressure-induced transient retinal ischemia. The axial diffusivity of wild-type nerves decreased 30% (P<0.05) at 3 days and 40% (P<0.05) at 5–30 days after transient elevation of intraocular pressure. In contrast, the axial diffusivity of WldS nerves did not change at 3 days; decreased by 20% (P<0.05) at 5 days, and continued to decrease by 30% (P<0.05) at 15 days and 40% (P<0.05) at 30 days after transient intraocular pressure elevation, suggesting delayed axonal damage in WldS mice. Radial diffusivity increased 200% (P<0.05) at 15–30 days in the wild-type mice and 100% (P<0.05) at 30 days in the WldS mice after transient intraocular pressure elevation, suggesting delayed myelin damage in WldS mice. DTI detected damage was confirmed with immunohistochemistry using phosphorylated neurofilament and myelin basic protein for assessing axonal and myelin integrity, respectively. These findings support the use of DTI not only to evaluate the progression of neurodegeneration but also to noninvasively demonstrate WldS mutation to delay the Wallerian degeneration.
doi:10.1016/j.neuroscience.2011.09.042
PMCID: PMC3282589  PMID: 21964470
diffusion tensor imaging; axial diffusivity; radial diffusivity; WldS mouse; axon degeneration; myelin damage
Age  2010;32(4):497-507.
Increasing numbers of Americans are reaching 85 years of age or older, yet there are no reliable biomarkers to predict who will live this long. The goal of this pilot study therefore was: (1) to identify a potential serum pattern that could identify proteins involved in longevity and (2) to determine if this pattern was a marker of longevity in an independent sample of individuals. Serum samples were analyzed in three cohorts of individuals (n = 12 in each) aged 20–34, 60–74, and ≥90 years who participated in The Louisiana Healthy Aging Study. The 12 most abundant proteins were removed and the remaining proteins separated by two-dimensional gel electrophoresis. Gels were matched and the intensity of each spot quantified. Multivariate discriminant analysis was used to identify a serum pattern that could separate these three age cohorts. Seven protein spots were found that correctly distinguished the subjects into the three groups. However, these spots were not as successful in discriminating the ages in a second set of 15 individuals as only eight of these subjects were placed into their correct group. These preliminary results show that the proteomics approach can be used to identify potential proteins or markers that may be involved in the aging process and/or be important determinants of longevity.
doi:10.1007/s11357-010-9146-8
PMCID: PMC2980595  PMID: 20490702
Aging; Serum; Proteomics; Biomarkers; Longevity
Drugs of the future  2009;34(5):381-400.
Subjective tinnitus, the phantom ringing or buzzing sensation that occurs in the absence of sound, affects 12–14% of adults; in some cases the tinnitus is so severe or disabling that patients seek medical treatment. However, although the economic and emotional impact of tinnitus is large, there are currently no FDA-approved drugs to treat this condition. Clinical trials are now underway to evaluate the efficacy of N-methyl-d-aspartate (NMDA) and dopamine D2 antagonists, selective serotonin reuptake inhibitors (SSRIs), γ-aminobutyric acid (GABA) agonists and zinc dietary supplements. Previous off-label clinical studies, while not definitive, suggest that patients with severe depression may experience improvement in their tinnitus after treatment with antidepressants such as nortriptyline or sertraline. A small subpopulation of patients with what has been described as “typewriter tinnitus” have been shown to gain significant relief from the anticonvulsant carbamazepine. Preliminary studies with misoprostol, a synthetic prostaglandin E1 analogue, and sulpiride, a dopamine D2 antagonist, have shown promise. Animal behavioral studies suggest that GABA transaminase inhibitors and potassium channel modulators can suppress tinnitus. Additionally, improvements in tinnitus have also been noted in patients taking melatonin for significant sleep disturbances. Like other complex neurological disorders, one drug is unlikely to resolve tinnitus in all patients; therapies targeting specific subgroups are likely to yield the greatest success.
doi:10.1358/dof.2009.034.05.1362442
PMCID: PMC3136369  PMID: 21765586
Chicken eggs categorised as conventional, omega-3 enriched, free range and organic were collected at grading stations in three regions of Canada between 2005 and 2006. Free run eggs, which were only available for collection from two regions, were also sampled during this time frame. Egg yolks from each of these egg types (n = 162) were analysed to determine brominated flame retardant levels, specifically polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD). PBDEs were detected in 100% of the 162 samples tested, while HBCD was observed in 85% of the egg yolks. Total PBDE concentrations in egg yolks ranged from 0.018 to 20.9 ng g−1 lipid (median = 3.03 ng g−1 lipid), with PBDE 209 identified as being the major contributor to ΣPBDE concentrations. In addition to PBDE 209, PBDE 99, 47, 100, 183 and 153 were important contributors to ΣPBDE concentrations. Total HBCD concentrations ranged from below the limit of detection to a maximum concentration of 71.9 ng g−1 lipid (median = 0.053 ng g−1 lipid). The α-isomer was the dominant contributor to ΣHBCD levels in Canadian egg yolks and was the most frequently detected HBCD isomer. ΣPBDE levels exhibited large differences in variability between combinations of region and type. ΣHBCD concentrations were not significantly different among regions, although differences were observed between the different types of egg yolks analysed in the present study.
doi:10.1080/19440049.2010.545443
PMCID: PMC3118488  PMID: 21623506
gas chromatography/mass spectrometry (GC/MS); clean-up; exposure; environmental contaminants; eggs
PLoS ONE  2010;5(10):e13657.
Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45+ and CD11b+ cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin- Sca-1+c-Kit+ (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion.
doi:10.1371/journal.pone.0013657
PMCID: PMC2965090  PMID: 21048959
Sun, W. | Jin, Y. | He, L | Lu, W-C. | Li, M.
Reference genes are used as internal controls in gene expression studies, but their expression levels vary according to tissue types and experimental treatments. Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. In this study, the suitability of eight commonly used genes (β?-actin, 5.8SrRNA, α?-TUB, GAPDH, RPL13a, RPS18, TBP, SDHA) were cloned and investigated to find the most stable candidates for normalizing real-time PCR data generated from the four different strains (abamectin-resistant, fenpropathrin-resistant, omethoate-resistant, and susceptible strains) and different developmental stages (eggs, protonymphs, nymphs, and adults) of carmine spider mite, Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae). The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, RPS18 and 5.8SrRNA had the most stable expression regardless of the four different strains, whereas RPS18 and α?-TUB were expressed most stably in different developmental stages.
doi:10.1673/031.010.20801
PMCID: PMC3029232  PMID: 21265619
normalization
Neuroscience  2008;159(1):325-334.
High doses of salicylate, the anti-inflammatory component of aspirin, induce transient tinnitus and hearing loss. Systemic injection of 250 mg/kg of salicylate, a dose that reliably induces tinnitus in rats, significantly reduced the sound evoked output of the rat cochlea. Paradoxically, salicylate significantly increased the amplitude of the sound-evoked field potential from the auditory cortex (AC) of conscious rats, but not the inferior colliculus (IC). When rats were anesthetized with isoflurane, which increases GABA-mediated inhibition, the salicylate-induced AC amplitude enhancement was abolished, whereas ketamine, which blocks N-methyl-d-aspartate receptors, further increased the salicylate-induced AC amplitude enhancement. Direct application of salicylate to the cochlea, however, reduced the response amplitude of the cochlea, IC and AC, suggesting the AC amplitude enhancement induced by systemic injection of salicylate does not originate from the cochlea. To identify a behavioral correlate of the salicylate-induced AC enhancement, the acoustic startle response was measured before and after salicylate treatment. Salicylate significantly increased the amplitude of the startle response. Collectively, these results suggest that high doses of salicylate increase the gain of the central auditory system, presumably by down-regulating GABA-mediated inhibition, leading to an exaggerated acoustic startle response. The enhanced startle response may be the behavioral correlate of hyperacusis that often accompanies tinnitus and hearing loss. Published by Elsevier Ltd on behalf of IBRO.
doi:10.1016/j.neuroscience.2008.12.024
PMCID: PMC2759817  PMID: 19154777
salicylate; tinnitus; hyperacusis; auditory cortex; inferior colliculus; GABA
Background/aims:
To demonstrate how spectral domain optical coherence tomography (SDOCT) can better evaluate drusen and associated anatomical changes in eyes with non-neovascular age-related macular degeneration (AMD) compared with time domain optical coherence tomography (TDOCT).
Methods:
Images were obtained from three eyes of three patients with AMD using an experimental SDOCT system. Both a titanium–sapphire (Ti:sapphire) laser and a superluminescent diode (SLD) were used as a broadband light source to achieve cross-sectional images of the retina. A qualitative and quantitative analysis was performed for structural changes associated with non-neovascular AMD. An automated algorithm was developed to analyse drusen area and volume from SDOCT images. TDOCT was performed using the fast macular scan (StratusOCT, Carl Zeiss Meditec, Dublin, California).
Results:
SDOCT images can demonstrate structural changes associated with non-neovascular AMD. A new SDOCT algorithm can determine drusen area, drusen volume and proportion of drusen.
Conclusions:
With new algorithms to determine drusen area and volume and its unprecedented simultaneous ultra-high speed ultra-high resolution imaging, SDOCT can improve the evaluation of structural abnormalities in non-neovascular AMD.
doi:10.1136/bjo.2008.137356
PMCID: PMC2628537  PMID: 18697811
Nucleic Acids Research  2007;36(Database issue):D884-D891.
The NIAID-funded Biodefense Proteomics Resource Center (RC) provides storage, dissemination, visualization and analysis capabilities for the experimental data deposited by seven Proteomics Research Centers (PRCs). The data and its publication is to support researchers working to discover candidates for the next generation of vaccines, therapeutics and diagnostics against NIAID's Category A, B and C priority pathogens. The data includes transcriptional profiles, protein profiles, protein structural data and host–pathogen protein interactions, in the context of the pathogen life cycle in vivo and in vitro. The database has stored and supported host or pathogen data derived from Bacillus, Brucella, Cryptosporidium, Salmonella, SARS, Toxoplasma, Vibrio and Yersinia, human tissue libraries, and mouse macrophages. These publicly available data cover diverse data types such as mass spectrometry, yeast two-hybrid (Y2H), gene expression profiles, X-ray and NMR determined protein structures and protein expression clones. The growing database covers over 23 000 unique genes/proteins from different experiments and organisms. All of the genes/proteins are annotated and integrated across experiments using UniProt Knowledgebase (UniProtKB) accession numbers. The web-interface for the database enables searching, querying and downloading at the level of experiment, group and individual gene(s)/protein(s) via UniProtKB accession numbers or protein function keywords. The system is accessible at http://www.proteomicsresource.org/.
doi:10.1093/nar/gkm903
PMCID: PMC2239001  PMID: 17984082
British Journal of Cancer  2005;92(10):1846-1849.
Inhibition of epidermal growth factor receptor (EGFR) signalling contributes to the therapy of colorectal cancer. Gefitinib, an oral EGFR tyrosine kinase inhibitor, shows supra-additive growth inhibition with irinotecan and fluoropyrimidines in xenograft models. We designed a study to determine the tolerability and efficacy of gefitinib in combination with irinotecan, infusional 5-fluorouracil (5-FU) and leucovorin (LV), on a 2-week schedule. Among 13 patients with advanced colorectal cancer, 10 required dose reductions of irinotecan and 5-FU because of dehydration, diarrhoea, and neutropenia, seven of whom required hospitalisation, three with neutropenic fever. One patient achieved partial response and seven had disease stabilisation. The combination of this standard chemotherapy regimen with gefitinib is associated with excessive toxicity, suggesting an interaction at a pharmacokinetic or pharmacodynamic level.
doi:10.1038/sj.bjc.6602569
PMCID: PMC2361767  PMID: 15870719
colon cancer; gefitinib; EGFR; irinotecan
British Journal of Cancer  2005;92(5):929-934.
To identify tumour and tumour-associated antigens in patients with hepatocellular carcinoma (HCC) one may find potential diagnostic markers and immunotherapeutic targets. In the current study, 30 distinct antigens reactive with serum IgG from HCC patients were identified by serological analysis of cDNA expression libraries (SEREX). The mRNA expression patterns of 14 of these 30 antigens were altered in cancer as further revealed by cDNA microarray, with upregulation for nine and downregulation for five antigens. One of the upregulated antigens was cancer-testis (CT) antigen (CAGE), which had been previously reported to be expressed exclusively in normal gametogenic tissues and aberrantly expressed in a variety of cancer cells. In our study, CAGE mRNA was expressed in 39.4% of HCC patients, 73.3% of patients with gastric cancer and 30.8% of patients with colorectal cancer. Antibodies against CAGE protein were detected in approximately 5.1% of the sera from HCC patients, 8.3% of that from gastric cancer patients and 7.3% of that from colorectal cancer patients. The relative high incidence of CAGE in cancer cells makes it a potential target for vaccine design. Another antigen of great interest is transgelin 2. The overexpression of transgelin 2 mRNA in a large per cent (69%) of HCC points to its potential as a diagnostic marker for HCC.
doi:10.1038/sj.bjc.6602460
PMCID: PMC2361901  PMID: 15756260
hepatocellular carcinoma; serological analysis of recombinant cDNA expression libraries; tumour antigens; cDNA microarray
Plasma from different species is the most accessible and valuable source for biomarker discovery in clinical and animal samples. However, due to the high abundance of some proteins such as albumin and immunoglobulins, low-abundant proteins are often undetectable in proteomic analysis of plasma. We have established a plasma depletion scheme using chicken antibodies against various abundant proteins. This immunoaffinity purification procedure is able to deplete albumin across multiple species. The high binding capacity and specificity of the chicken antibody enables the efficient capture of its ligand from microliter volumes of plasma sample. The resulting two-dimensional gel analyses of the depleted and captured samples show significant enhancement of the low-abundant proteins and specific capture of the abundant ligand. By utilizing this sample preparation scheme, it is now possible to analyze the plasma proteome from multiple species in a potentially rapid and large-scale capacity for biomarker discovery, drug target discovery, and toxicology studies.
PMCID: PMC2291691  PMID: 15331584
2-DE, 2-dimensional gel electrophoresis, HSA, human serum albumin; IgG, immunoglobulin
Gut  1998;43(1):123-127.
Aim—The incidence of anorectal symptoms after radiotherapy (RTH) for localised pelvic malignant disease is unclear. In addition, the effects of pelvic irradiation on both anorectal motility and sensory function are poorly defined. A prospective study was therefore performed on 35 patients (55-82 years of age) with localised prostatic carcinoma before and four to six weeks after RTH to assess its effects on anorectal function. 
Methods—Anorectal symptoms were assessed by questionnaire. Anorectal pressures at rest and in response to voluntary squeeze, rectal distension, and increases in intra-abdominal pressure were evaluated with perfused sleeve side hole manometry. Rectal sensation was tested during graded balloon distension. Rectal compliance was calculated by the pressure-volume relation obtained during the testing of rectal sensation. Ultrasound was used to determine anal sphincter structure and integrity. 
Results—RTH had no effect on anal sphincter morphology. The frequency of defecation increased after RTH (7 (3-21) v 10 (3-56) bowel actions a week; p<0.01). After RTH, 16 patients had faecal urgency and eight faecal incontinence, compared with five and one respectively before RTH (p<0.01 for each). Basal and squeeze sleeve recorded pressures were reduced after RTH (54 (3) v 49(3) mm Hg (p<0.05) and 111 (8) v 102 (8) mm Hg (p<0.01), before and after RTH respectively; means (SEM)). Rectal compliance was reduced after RTH (1.2 v 1.4 mm Hg/ml, p<0.05). After RTH, threshold volumes for perception of rectal distension were lower in the 16 patients who either experienced faecal urgency for the first time (13 patients) or reported worsening of this symptom (three patients) compared with the remaining patients (34 (4) v 48 (5) ml respectively, p<0.05). 
Conclusion—Faecal incontinence (23%) is a common problem four to six weeks after RTH for prostatic carcinoma and is associated with minor reductions in anal sphincter pressures. The high prevalence of faecal urgency in patients after RTH may be related to alterations in rectal perception of stool. 


Keywords: anorectal function; radiotherapy; motility; manometry; incontinence
PMCID: PMC1727195  PMID: 9771416
Environmental Health Perspectives  2000;108(10):967-972.
Whether exposure of humans to extremely low frequency electromagnetic fields (ELF-EMF) can cause cancer is controversial and therefore needs further research. We used a Friend erythroleukemia cell line that can be chemically induced to differentiate to determine whether ELF-EMF could alter proliferation and differentiation in these cells in a manner similar to that of a chemical tumor promoter. Exposure of this cell line to 60 Hz ELF-EMF resulted in a dose dependent inhibition of differentiation, with maximal inhibition peaking at 40% and 40 mG (4 microT). ELF-EMF at 10 mG (1.0 microT) and 25 mG (2.5 microT) inhibited differentiation at 0 and 20%, respectively. ELF-EMF at 1.0 (100) and 10.0 G (1,000 microT) stimulated cell proliferation 50% above the sham-treated cells. The activity of telomerase, a marker of undifferentiated cells, decreased 100[times] when the cells were induced to differentiate under sham conditions, but when the cells were exposed to 0.5 G (50 microT) there was only a 10[times] decrease. In summary, ELF-EMF can partially block the differentiation of Friend erythroleukemia cells, and this results in a larger population of cells remaining in the undifferentiated, proliferative state, which is similar to the published results of Friend erythroleukemia cells treated with chemical-tumor promoters.
PMCID: PMC1240130  PMID: 11049817
Gut  1997;41(4):494-499.
Background—The pathogenesis of anorectal dysfunction, which occurs frequently in patients with diabetes mellitus, is poorly defined. Recent studies indicate that changes in the blood glucose concentration have a major reversible effect on gastrointestinal motor function. 
Aims—To determine the effects of physiological changes in blood glucose and hyperglycaemia on anorectal motor and sensory function in normal subjects. 
Subjects—In eight normal subjects measurements of anorectal motility and sensation were performed on separate days while blood glucose concentrations were stabilised at 4, 8, and 12 mmol/l. 
Methods—Anorectal motor and sensory function was measured using a sleeve/sidehole catheter incorporating a balloon, and electromyography. 
Results—The number of spontaneous anal relaxations was greater at 12 mmol/l than at 8 and 4 mmol/l glucose (p<0.05 for both). Anal squeeze pressures were less at a blood glucose of 12 mmol/l when compared with 8 and 4 mmol/l (p<0.05 for both). During rectal distension, residual anal pressures were not significantly different between the three blood glucose concentrations. Rectal compliance was greater (p<0.05) at a blood glucose of 12 mmol/l when compared with 4 mmol/l. The threshold volume for initial perception of rectal distension was less at 12 mmol/l when compared with 4 mmol/l (40 (20-100) ml versus 10 (10-150) ml, p<0.05). 
Conclusions—An acute elevation of blood glucose to 12 mmol/l inhibits internal and external anal sphincter function and increases rectal sensitivity in normal subjects. In contrast, physiological changes in blood glucose do not have a significant effect on anorectal motor and sensory function. 


Keywords: hyperglycaemia; anorectum; motility; sensation; diabetes mellitus
PMCID: PMC1891535  PMID: 9391248
Journal of Clinical Investigation  1997;100(12):3014-3018.
The agent of human granulocytic ehrlichiosis (HGE) is a newly recognized tick-borne pathogen that resides within polymorphonuclear leukocytes. C3H/HeN mice can become infected with the agent of HGE (designated aoHGE) by syringe inoculation or tick-borne infection and develop transient neutropenia. They thereby partially mimic human disease and provide a model in which to study immunity to this microorganism. Mice vaccinated with lysates of purified aoHGE, or administered aoHGE antisera, were partially protected from both syringe- and tick-transmitted challenge with aoHGE. These data suggest that antibodies are sufficient to provide substantial, but not complete, immunity against aoHGE.
PMCID: PMC508513  PMID: 9399947
Nucleic Acids Research  1997;25(21):4385-4388.
Transcription of the bacteriophage Mu mom operon is strongly repressed by the host OxyR protein in dam - but not dam + cells. In this work we show that the extent of mom modification is sensitive to the relative levels of the Dam and OxyR proteins and OxyR appears to modulate the level of mom expression even in dam + cells. In vitro studies demonstrated that OxyR is capable of binding hemimethylated P mom , although its affinity is reduced slightly compared with unmethylated DNA. Thus, OxyR modulation of mom expression in dam + cells can be attributed to its ability to bind hemimethylated P mom DNA, the product of DNA replication.
PMCID: PMC147061  PMID: 9336472
Journal of Bacteriology  1996;178(23):6701-6705.
We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori(c)), leaving stem-loop I and the adjacent 5' CTG 3', the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson, J. Biol. Chem. 268:8026-8039, 1993). Using a small 278-nucleotide (nt) G4ori(c) single-stranded DNA fragment that supported primer RNA synthesis, we now demonstrate by gel shift that E. coli primase can stably interact with the SSB-G4ori(c) complex. This stable interaction requires Mg2+ for specificity. At 8 mM Mg2+, primase binds to an SSB-coated 278-nt G4ori(c) fragment but not to an SSB-coated control 285-nt LacZ ss-DNA fragment. In the absence of Mg2+, primase binds to both SSB-coated fragments and gives a gel shift. T4 gene 32 protein cannot substitute for E. coli SSB in this reaction. Stable interaction of primase with naked G4ori(c). single-stranded DNA was not observed. DNase I and micrococcal nuclease footprinting, of both 5' and 3' 32P-labeled DNA, demonstrated that primase interacts with two regions of G4ori(c): one covering stem-loop I and the 3' sequence flanking stem-loop I which contains the pRNA initiation site and another located on the 5' sequence flanking stem-loop III.
PMCID: PMC178564  PMID: 8955285

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