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1.  Bicalutamide-induced hypoxia potentiates RUNX2-mediated Bcl-2 expression resulting in apoptosis resistance 
British Journal of Cancer  2012;107(10):1714-1721.
Background:
We have previously shown that hypoxia selects for more invasive, apoptosis-resistant LNCaP prostate cancer cells, with upregulation of the osteogenic transcription factor RUNX2 and the anti-apoptotic factor Bcl-2 detected in the hypoxia-selected cells. Following this observation, we questioned through what biological mechanism this occurs.
Methods:
We examined the effect of hypoxia on RUNX2 expression and the role of RUNX2 in the regulation of Bcl-2 and apoptosis resistance in prostate cancer.
Results:
Hypoxia increased RUNX2 expression in vitro, and bicalutamide-treated LNCaP tumours in mice (previously shown to have increased tumour hypoxia) exhibited increased RUNX2 expression. In addition, RUNX2-overexpressing LNCaP cells showed increased cell viability, following bicalutamide and docetaxel treatment, which was inhibited by RUNX2 siRNA; a range of assays demonstrated that this was due to resistance to apoptosis. RUNX2 expression was associated with increased Bcl-2 levels, and regulation of Bcl-2 by RUNX2 was confirmed through chromatin immunoprecipitation (ChIP) binding and reporter assays. Moreover, a Q-PCR array identified other apoptosis-associated genes upregulated in the RUNX2-overexpressing LNCaP cells.
Conclusion:
This study establishes a contributing mechanism for progression of prostate cancer cells to a more apoptosis-resistant and thus malignant phenotype, whereby increased expression of RUNX2 modulates the expression of apoptosis-associated factors, specifically Bcl-2.
doi:10.1038/bjc.2012.455
PMCID: PMC3493869  PMID: 23073173
hypoxia; RUNX2; Bcl-2; bicalutamide; prostate cancer; LNCaP
2.  Targeted inhibition of mitochondrial Hsp90 suppresses localised and metastatic prostate cancer growth in a genetic mouse model of disease 
British Journal of Cancer  2011;104(4):629-634.
Background:
The molecular chaperone heat shock protein-90 (Hsp90) is a promising cancer drug target, but current Hsp90-based therapy has so far shown limited activity in the clinic.
Methods:
We tested the efficacy of a novel mitochondrial-targeted, small-molecule Hsp90 inhibitor, Gamitrinib (GA mitochondrial matrix inhibitor), in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model. The TRAMP mice receiving 3-week or 5-week systemic treatment with Gamitrinib were evaluated for localised or metastatic prostate cancer, prostatic intraepithelial neoplasia (PIN) or localised inflammation using magnetic resonance imaging, histology and immunohistochemistry. Treatment safety was assessed histologically in organs collected at the end of treatment. The effect of Gamitrinib on mitochondrial dysfunction was studied in RM1 cells isolated from TRAMP tumours.
Results:
Systemic administration of Gamitrinib to TRAMP mice inhibited the formation of localised prostate tumours of neuroendocrine or adenocarcinoma origin, as well as metastatic prostate cancer to abdominal lymph nodes and liver. The Gamitrinib treatment had no effect on PIN or prostatic inflammation, and caused no significant animal weight loss or organ toxicity. Mechanistically, Gamitrinib triggered acute mitochondrial dysfunction in RM1 cells, with loss of organelle inner membrane potential and release of cytochrome-c in the cytosol.
Conclusions:
The Gamitrinib has pre-clinical activity and favourable tolerability in a genetic model of localised and metastatic prostate cancer in immunocompetent mice. Selective targeting of mitochondrial Hsp90 could provide novel molecular therapy for patients with advanced prostate cancer.
doi:10.1038/bjc.2011.9
PMCID: PMC3049604  PMID: 21285984
prostate cancer; metastasis; mitochondria; Hsp90; TRAMP
3.  The Role of RUNX2 in Osteosarcoma Oncogenesis 
Sarcoma  2010;2011:282745.
Osteosarcoma is an aggressive but ill-understood cancer of bone that predominantly affects adolescents. Its rarity and biological heterogeneity have limited studies of its molecular basis. In recent years, an important role has emerged for the RUNX2 “platform protein” in osteosarcoma oncogenesis. RUNX proteins are DNA-binding transcription factors that regulate the expression of multiple genes involved in cellular differentiation and cell-cycle progression. RUNX2 is genetically essential for developing bone and osteoblast maturation. Studies of osteosarcoma tumours have revealed that the RUNX2 DNA copy number together with RNA and protein levels are highly elevated in osteosarcoma tumors. The protein is also important for metastatic bone disease of prostate and breast cancers, while RUNX2 may have both tumor suppressive and oncogenic roles in bone morphogenesis. This paper provides a synopsis of the current understanding of the functions of RUNX2 and its potential role in osteosarcoma and suggests directions for future study.
doi:10.1155/2011/282745
PMCID: PMC3005824  PMID: 21197465
4.  Systemically Transplanted Bone Marrow Stromal Cells Contributing to Bone Tissue Regeneration 
Journal of cellular physiology  2008;215(1):204-209.
Bone marrow stromal cells (BMSCs) are a rich source of osteogenic progenitor cells. A fundamental question is whether systemically transplanted BMSCs participate in bone regeneration. Luciferase and GFP double-labeled BMSCs were transplanted into irradiated mice. Five weeks after transplantation, artificial bone wounds were created in the mandibles and calvaria of the recipients. Animals were sacrificed at weeks 2, 4, and 6 after surgery and the expressions of luciferase and GFP were determined using Xenogen IVIS Imaging System, immunohistochemical staining and RT-PCR. The results demonstrated that transplanted BMSCs can be detected in wound sites as early as 2 weeks and lasted the whole experimental period. Luciferase expression peaked at 2 weeks after surgery and decreased thereafter, exhibiting a similar expression pattern as that of BSP, while GFP expression was relatively stable during the experimental period. In conclusion, BMSCs can migrate to bone wound sites and participate in bone regeneration in orocraniofacial region.
doi:10.1002/jcp.21302
PMCID: PMC2828813  PMID: 17960569
5.  Advanced glycation end product modification of bone proteins and bone remodelling: hypothesis and preliminary immunohistochemical findings 
Annals of the Rheumatic Diseases  2006;65(1):101-104.
Background
The process of bone remodelling is disturbed in the development of osteoporosis.
Objective
To investigate if proteins in osteoporotic bone are modified by advanced glycation end products (AGEs), and whether these alterations are related to measures of bone remodelling based on histomorphometric findings.
Methods
Bone specimens taken from the iliac crest by bone biopsy of eight osteoporotic patients were investigated by histomorphometry and by immunohistochemical staining with the AGEs imidazolone and Nε‐carboxymethyllysine.
Results
Both AGEs were found in all bone specimens. The intensity of staining correlated with patient age. The percentage of bone surface covered with osteoblasts showed a significantly negative correlation with the staining intensity of both AGEs.
Conclusions
It is known that AGEs can regulate proliferation and differentiation of osteoblastic cells and that AGE‐specific binding sites are present in cultured osteoblast‐like cells. Moreover, AGE induced biological effects in these cells might be mediated by RAGE (receptor of AGE) or by other AGE receptors in different stages of osteoblast development. The inverse relation between AGE staining intensity and the percentage of bone surface covered with osteoblasts in the trabecular bone may provide evidence that AGE modification of bone proteins disturbs bone remodelling.
doi:10.1136/ard.2004.034348
PMCID: PMC1797982  PMID: 16344492
glycation end products; bone remodelling; histomorphometry; immunohistochenical staining; osteoporosis
6.  Molecular detection (k-ras) of exfoliated tumour cells in the pelvis is a prognostic factor after resection of rectal cancer? 
BMC Cancer  2008;8:213.
Background
After total mesorectal excision (TME) for rectal cancer around 10% of patients develops local recurrences within the pelvis. One reason for recurrence might be spillage of cancer cells during surgery. This pilot study was conducted to investigate the incidence of remnant cancer cells in pelvic lavage after resection of rectal cancer. DNA from cells obtained by lavage, were analysed by denaturing capillary electrophoresis with respect to mutations in hotspots of the k-ras gene, which are frequently mutated in colorectal cancer.
Results
Of the 237 rectal cancer patients analyzed, 19 had positive lavage fluid. There was a significant survival difference (p = 0.006) between patients with k-ras positive and negative lavage fluid.
Conclusion
Patients with k-ras mutated cells in the lavage immediately after surgery have a reduced life expectation. Detection of exfoliated cells in the abdominal cavity may be a useful diagnostic tool to improve the staging and eventually characterize patients who may benefit from aggressive multimodal treatment of rectal cancer.
doi:10.1186/1471-2407-8-213
PMCID: PMC2525659  PMID: 18655729
7.  Pseudomyxoma peritonei – two novel orthotopic mouse models portray the PMCA-I histopathologic subtype 
BMC Cancer  2007;7:116.
Background
Pseudomyxoma peritonei (PMP) is a rare malignant disease, most commonly originating from appendiceal lesions and characterized by accumulation of mucinous tumor tissue in the peritoneal cavity. Since the disease is infrequent, the task of carrying out studies of treatment efficacy and disease biology in the clinical setting is challenging, warranting the development of relevant in vitro and in vivo PMP models.
Methods
Human tumor tissue was implanted in the peritoneal cavity of nude mice to establish two orthotopic models exhibiting noninvasive intraperitoneal growth without metastasis development.
Results
Xenograft tissues have retained essential properties of the original human tumors, such as macro- and microscopic growth patterns, mucin production as well as expression of carcinoembryonal antigen, cytokeratins 20 and 7 and the proliferation marker pKi67. Upon microscopic examination, the human tumors were categorized as the PMCA-I (peritoneal mucinous carcinomatosis of intermediate features) subtype, which was conserved through 14 examined passages in mice, for the first time modeling this particular histopathologic category.
Conclusion
In conclusion, two novel orthotopic models of human PMP have been established that consistently portray a distinct histopathologic subtype and reflect essential human tumor properties. Xenografts can easily and reproducibly be transferred to new generations of mice with acceptable passage periods, rendering the models as attractive tools for further studies of PMP biology and treatment.
doi:10.1186/1471-2407-7-116
PMCID: PMC1920528  PMID: 17603904
8.  P183-T Analysis of Glycoproteins in Human Serum by Means of Glyco-Specific Magnetic Bead Separation and LC-MALDI with Automated Glycopeptide Detection 
Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins and automated analysis procedures for the detection, identification, and structural characterization of the corresponding peptide modification.
Selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task.
Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids). Isolated proteins were released from the beads under acidic conditions, dried, and subsequently re-dissolved and digested with trypsin. The resulting complex mixture of peptides was subjected to LC-MALDI analysis. The respective glycoproteins were identified by direct MS/MS analysis and subsequent database searching. Intact glyco-peptides enriched by a second magnetic-bead purification on peptide level were directly subjected to LC-MALDI analysis to get structural information about the glycan and peptide parts. A precondition to this novel approach was the discovery of certain consensus peak patterns in the MALDI-MS/MS spectra, allowing the automatic determination of the peptide part and the glycosidic information of the glycopeptides supported by bioinformatics tools.
Applying this fast and simple approach, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from experiments using magnetic ConA, LCA, WGA, jacalin, and boronic acid beads, respectively. The use of jacalin and boronic acid beads facilitates the enrichment of O-glycosidically modified proteins. In contrast, ConA, WGA, and LCA specifically bind N-glycosylated peptides and proteins. Few non-glycosylated proteins were identified, probably due to co-precipitation with glycosylated proteins.
PMCID: PMC2291947
9.  P41-M Automated LC-MALDI Analysis of Glycopeptides from Glycoprotein Digests Using DHB as Matrix 
2,5-Dihydroxybenzoic acid (DHB) is the matrix of choice for carbohydrate and glycopeptide analysis, but due to the inhomogeneous surface morphology of samples prepared with DHB, it is typically incompatible with automated measurements.
We describe a simple and rapid method for the analysis of glycoproteins, which combines (a) reducing the complexity of the digest mixtures with glyco-specific enrichment and (b) subsequent LC-MALDI-TOF MS/MS analysis with DHB as MALDI matrix. All samples were prepared on hydrophobic sample plates with hydrophilic anchors 400 or 600 μm in diameter confining the sample dimensions.
In a first step, the matrix was applied to the 384 sample spots (“anchors”) of a microtiter plate–shaped MALDI target. The LC eluate from CAP-RP-HPLC subsequently dissolved the DHB matrix confined to the hydrophobic boundaries of the anchors. Co-crystallization of glycopeptides in DHB suitable for the automated analysis was achieved.
This method was applied to recombinant human inte-grin alpha and beta; glycosylation sites were identified and described.
The MALDI-MSMS spectra of glycopeptides (N-linked type) include information about the structure of peptide moiety as well as the glycan part of the molecules. MALDI-TOF/TOF spectra permitted (a) the detection of N-linked glycopeptides by a neutral loss analysis across the entire LC-MALDI-MS/MS dataset, (b) the determination of the molecular weight of the pure peptide chain by typical fragmentation patterns, (c) the identification of the peptide part of the fragmented glycopeptide by means of simple database searching, and (d) initial information about the glycan composition and the attachment site.
LC-MALDI-TOF/TOF on DHB matrix preparation is a powerful approach for the detailed characterization of glycoproteins.
PMCID: PMC2291954
10.  Pupillary evaluation for differential diagnosis of coma 
Postgraduate Medical Journal  2003;79(927):49-51.
Objectives: To determine the usefulness of bedside evaluation of pupils in determining the aetiology of coma by adopting a probabilistic approach.
Patients and methods: One hundred and fifteen consecutive patients presenting with coma were enrolled in this prospective cohort during the 12 month study period in the emergency room of a community teaching hospital. Patients underwent structured clinical examinations and laboratory and imaging tests. Assignment of aetiology of coma was based on strict adherence to predetermined criteria and achieved by consensus of the two physician investigators. One year follow up was obtained in all patients.
Results: Aetiology of coma was determined in 98% of the patients. It was metabolic in 69 patients (60%) and structural in 46 patients (40%). Metabolic causes included drug overdose, acute alcohol intoxication, hypoglycaemia, sepsis, and pneumonia. Structural causes included intracerebral haemorrhage, subarachnoid haemorrhage, cerebral infarction, subdural haematoma, and epidural haematoma. Multivariate logistic regression analysis showed light reflex loss (likelihood ratio for positive test result 3.59) and anisocoria (likelihood ratio for positive test result 9.0) as independent predictors of structural origin.
Conclusions: In this prospective study of patients presenting to the emergency room of a community based teaching hospital with coma, in about 60% the coma is of metabolic origins and in about 40% of structural origins. Light reflex loss and anisocoria suggest a structural aetiology.
doi:10.1136/pmj.79.927.49
PMCID: PMC1742582  PMID: 12566553
11.  Identification of the advanced glycation end products N -carboxymethyllysine in the synovial tissue of patients with rheumatoid arthritis 
Annals of the Rheumatic Diseases  2002;61(6):488-492.
Background: Generation of advanced glycation end products (AGEs) is an inevitable process in vivo and can be accelerated under pathological conditions such as oxidative stress. In serum and synovial fluid of patients with rheumatoid arthritis (RA) raised AGE levels have been found.
Objective: To determine the presence of N -carboxymethyllysine (CML; marker of oxidative stress) in RA synovial tissue by immunohistology.
Methods: Frozen synovial tissue samples from 10 patients with RA and eight controls (four patients without joint disease and four patients with osteoarthritis (OA)) were treated with rabbit-anti-CML-IgG and goat-antirabbit-IgG. Immunostaining was visualised by streptavidine-alkaline phosphatase (chromogen fuchsin). Cell differentiation was performed with antibodies against CD68, CD45RO, and CD20.
Results: CML was detected in the synovial lining, sublining, and endothelium in 10/10 RA and 4/4 OA synovial specimens. In RA some macrophages (CD68+) and T cells (CD45RO+) showed positive immunostaining for CML, whereas B cells were negative. Staining in OA synovial sublining was weak compared with RA.
Conclusions: CML was detected for the first time in RA and OA synovial tissue. Different patterns of immunostaining in RA and OA and the presence of CML on macrophages and T cells, suggest a role for CML in the pathogenesis of RA. This might be due to presentation of new epitopes which can maintain or even trigger an autoimmune response.
doi:10.1136/ard.61.6.488
PMCID: PMC1754129  PMID: 12006318
12.  Standardization of Broth Microdilution and Disk Diffusion Susceptibility Tests for Actinobacillus pleuropneumoniae and Haemophilus somnus: Quality Control Standards for Ceftiofur, Enrofloxacin, Florfenicol, Gentamicin, Penicillin, Tetracycline, Tilmicosin, and Trimethoprim-Sulfamethoxazole 
Journal of Clinical Microbiology  2001;39(12):4283-4287.
Quality control (QC) standards for the in vitro antimicrobial susceptibility testing of two fastidious veterinary pathogens, Actinobacillus pleuropneumoniae and Haemophilus somnus, were developed in a multilaboratory study according to procedures established by the National Committee for Clinical Laboratory Standards for broth microdilution and disk diffusion testing. The medium recommended for the broth microdilution testing is cation-adjusted Mueller-Hinton broth supplemented with 2% lysed horse blood, 2% yeast extract, and 2% supplement C. This medium has been designated veterinary fastidious medium. The medium recommended for the disk diffusion testing is chocolate Mueller-Hinton agar. The recommended QC organisms are A. pleuropneumoniae ATCC 27090 and H. somnus ATCC 700025. The QC MICs of ceftiofur, enrofloxacin, florfenicol, gentamicin, penicillin, tetracycline, tilmicosin, and trimethoprim-sulfamethoxazole were determined for each isolate, as were the zone size ranges. Of the results from the participating laboratories, 94.0% of the zone diameter results and 97.0% of the MIC results fell within the suggested QC ranges for all compounds. These QC guidelines should allow greater accuracy in interpreting results when testing these antimicrobial agents against fastidious pathogens.
doi:10.1128/JCM.39.12.4283-4287.2001
PMCID: PMC88537  PMID: 11724833
13.  Development of the osteoblast phenotype: molecular mechanisms mediating osteoblast growth and differentiation. 
The Iowa Orthopaedic Journal  1995;15:118-140.
The formation of bone tissue involves multiple activities of the osteoblast. The combined application of molecular, biochemical, histochemical and ultrastructural approaches has defined stages in the development of the osteoblast phenotype with each subpopulation of cells exhibiting unique morphologic and functional properties in relation to the ordered deposition of the mineralized bone extracellular matrix (ECM). Peak levels of expressed genes reflect a maturational sequence of osteoblast growth and differentiation characterized by three principal periods: proliferation, ECM maturation and mineralization. A plethora of new information in the past several years provides the basis for insight into molecular mechanisms regulating the development and activities of differentiating osteoblasts. These new concepts will be discussed within the context of understanding cellular responses of bone tissue. To be considered are the following: 1) maturational stages of the osteoblast reflected by the selective expression of transcription factors (e.g., oncogenes, cyclins, homeodomain proteins) and phenotypic genes that provide signals for differentiation through the osteoblast lineage; 2) role of the extracellular matrix in mediating osteoblast growth and differentiation; 3) osteoblast stage specific responses to physiologic mediators (e.g., growth factors and hormones); 4) the developmentally regulated expression and selective responses of osteoblast phenotypic genes are supported by cooperative, synergistic and/or antagonistic activities at multiple basal and enhancer or suppressor sequences in gene promoters; and 5) deregulation of these control mechanisms in transformed osteoblasts and osteosarcoma cells.
Images
PMCID: PMC2329080  PMID: 7634023
14.  Video thoracoscopy: routine application for recurrent spontaneous pneumothorax. 
Video thoracoscopy is a technique that allows a minimally invasive approach to common thoracic surgical problems. This article reports three patients with recurrent spontaneous pneumothorax managed by video thoracoscopic apical bleb resection and describes the surgical technique.
Images
PMCID: PMC2607596  PMID: 8064903
15.  Placebo-controlled trial of itraconazole for treatment of acute vaginal candidiasis. 
Itraconazole is a new orally active triazole antifungal agent with enhanced activity against Candida species. In the clinical trial described in this paper, we compared the efficacy and safety of itraconazole capsules with those of clotrimazole vaginal tablets and placebo oral capsules for women with acute vulvovaginal candidiasis. Ninety-five patients were randomized in a 2:1:1 fashion to receive itraconazole (200 mg/day), clotrimazole (200 mg/day), or placebo (two capsules per day) for 3 consecutive days. Clinical success rates (cure and improvement) were similar for women who received itraconazole (96%) and clotrimazole (100%) 1 week posttreatment. These response rates were statistically superior to those obtained with placebo treatment (77%, P < 0.05). Negative mycological cultures were found in 95, 73, and 32% of the patients treated with clotrimazole, itraconazole, and placebo, respectively (P < 0.005) [active treatments versus placebo]). By 4 weeks posttreatment, the clinical failure rate for itraconazole was less than that observed for clotrimazole (17 versus 30%), but this difference did not reach statistical significance (P > 0.05; beta = 0.81). Mycological response rates for itraconazole and clotrimazole were also similar. No patients enrolled in this study discontinued treatment because of an adverse event. Minor side effects were reported by 35, 4, and 41% of patients who received itraconazole, clotrimazole, and placebo, respectively. The most common side effects associated with itraconazole therapy were nausea and headache. In summary, itraconazole was found to be as effective and safe as clotrimazole in women with acute candida vaginitis. Moreover, oral therapy was highly favored over intravaginal treatment in our survey of patients.
PMCID: PMC187610  PMID: 8381643
16.  Pharmacokinetic evaluation of ceftiofur in serum, tissue chamber fluid and bronchial secretions from healthy beef-bred calves. 
Ceftiofur is a new broad spectrum cephalosporin marketed for the treatment of acute bovine respiratory disease. In this investigation ceftiofur was administered by intramuscular injection, at 24 h intervals, to healthy beef-bred calves for four days at dosages of 2.2 and 4.4 mg/kg of body weight, with 4 wk intervals between dosing regimens. Serum, tissue chamber fluid (TCF), and bronchial secretion (BS) concentrations of ceftiofur were measured by microbiological assay after the first and fourth dose of each dosing regimen. Peak serum concentrations (Cmax) of 8.8 micrograms/mL and 17.3 micrograms/mL were obtained approximately 2 h (Tmax), the time of mean peak concentration) after single injections of 2.2 mg/kg and 4.4 mg/kg, respectively. The Cmax was increased approximately twofold following multiple doses of 2.2 mg/kg (Cmax = 13.1 micrograms/mL) and 4.4 mg/kg (Cmax = 24.1 micrograms/mL). Ceftiofur accumulated slowly into TCF and peak concentrations were found to be approximately 14% of those observed in serum after the first dose and approximately 24% after multiple dosing. Concentrations of ceftiofur in BS were obtained rapidly with peak concentrations reaching 45% of the serum Cmax after the first dose. After multiple dosing the Cmax for BS was approximately 25% of the serum Cmax. This study found that both the 2.2 mg/kg and 4.4 mg/kg dosing regimens resulted in continuous serum, TCF and BS concentrations of ceftiofur that exceeded the minimal concentration required to inhibit the bacteria most frequently isolated from calves with acute bovine respiratory disease.
PMCID: PMC1263555  PMID: 1477795
17.  Overlapping and CpG methylation-sensitive protein-DNA interactions at the histone H4 transcriptional cell cycle domain: distinctions between two human H4 gene promoters. 
Molecular and Cellular Biology  1992;12(7):3273-3287.
Transcriptional regulation of vertebrate histone genes during the cell cycle is mediated by several factors interacting with a series of cis-acting elements located in the 5' regions of these genes. The arrangement of these promoter elements is different for each gene. However, most histone H4 gene promoters contain a highly conserved sequence immediately upstream of the TATA box (H4 subtype consensus sequence), and this region in the human H4 gene FO108 is involved in cell cycle control. The sequence-specific interaction of nuclear factor HiNF-D with this key proximal promoter element of the H4-FO108 gene is cell cycle regulated in normal diploid cells (J. Holthuis, T.A. Owen, A.J. van Wijnen, K.L. Wright, A. Ramsey-Ewing, M.B. Kennedy, R. Carter, S.C. Cosenza, K.J. Soprano, J.B. Lian, J.L. Stein, and G.S. Stein, Science, 247:1454-1457, 1990). Here, we show that this region of the H4-FO108 gene represents a composite protein-DNA interaction domain for several distinct sequence-specific DNA-binding activities, including HiNF-D, HiNF-M, and HiNF-P. Factor HiNF-P is similar to H4TF-2, a DNA-binding activity that is not cell cycle regulated and that interacts with the analogous region of the H4 gene H4.A (F. LaBella and N. Heintz, Mol. Cell. Biol. 11:5825-5831, 1991). The H4.A gene fails to interact with factors HiNF-M and HiNF-D owing to two independent sets of specific nucleotide variants, indicating differences in protein-DNA interactions between these H4 genes. Cytosine methylation of a highly conserved CpG dinucleotide interferes with binding of HiNF-P/H4TF-2 to both the H4-FO108 and H4.A promoters, but no effect is observed for either HiNF-M or HiNF-D binding to the H4-FO108 gene. Thus, strong evolutionary conservation of the H4 consensus sequence may be related to combinatorial interactions involving overlapping and interdigitated recognition nucleotides for several proteins, whose activities are regulated independently. Our results also suggest molecular complexity in the transcriptional regulation of distinct human H4 genes.
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PMCID: PMC364541  PMID: 1620129
19.  Changes in the stability of a human H3 histone mRNA during the HeLa cell cycle. 
Molecular and Cellular Biology  1991;11(1):544-553.
A major component of the regulation of histone protein synthesis during the cell cycle is the modulation of the half-life of histone mRNA. We have uncoupled transcriptional and posttranscriptional regulation by using a Drosophila hsp70-human H3 histone fusion gene that produces a marked human H3 histone mRNA upon heat induction. Transcription of this gene can be switched on and off by raising and lowering cell culture temperatures, respectively. HeLa cell lines containing stably integrated copies of the fusion gene were synchronized by double thymidine block. Distinct populations of H3 histone mRNA were produced by heat induction in early S-phase, late S-phase, or G2-phase cells, and the stability of the induced H3 histone mRNA was measured. The H3 histone mRNA induced during early S phase decayed with a half-life of 110 min, whereas the same transcript induced during late S phase had a half-life of 10 to 15 min. The H3 histone mRNA induced in non-S-phase cells is more stable than that induced in late S phase, with a half-life of 40 min. Thus, the stability of histone mRNA is actively regulated throughout the cell cycle. Our results are consistent with an autoregulatory model in which the stability of histone mRNA is determined by the level of free histone protein in the cytoplasm.
Images
PMCID: PMC359664  PMID: 1986245
20.  Abolition by omeprazole of aspirin induced gastric mucosal injury in man. 
Gut  1990;31(5):514-517.
This study investigates whether aspirin injury to the human gastric mucosa can be prevented by profound acid suppression with omeprazole, in a randomised, double blind, crossover design according to latin square. It was concluded that profound acid suppression can prevent aspirin induced gastric mucosal injury in normal subjects. This approach may prevent the development of peptic ulcers and their complications in patients taking aspirin and other non-steroidal anti-inflammatory drugs.
PMCID: PMC1378564  PMID: 2190865
21.  Piroxicam induced lithium toxicity. 
PMCID: PMC1001687  PMID: 4026412
22.  Randomized clinical trial of rifampin-trimethoprim and sulfamethoxazole-trimethoprim in the treatment of localized urinary tract infections. 
To investigate whether 10 days of rifampin-trimethoprim (RIF-TMP) or 6 weeks of sulfamethoxazole-trimethoprim (SMX-TMP) would decrease the relapse rate in patients with acute uncomplicated upper urinary tract infections in comparison with 10 days of SMX-TMP, we randomized 189 patients to receive RIF-TMP or SMX-TMP in a ratio of 1:2. After the site of infection was established by the antibody-coated bacterium (ACB) test, patients with upper-tract infections who received SMX-TMP were again randomized and received either a total of 6 weeks or 10 days of therapy. All patients who received RIF-TMP were treated for 10 days. Clinical and microbiological evaluations were repeated at 2 and 6 weeks posttreatment. Eighty-five patients (54 ACB positive) received 10 days of RIF-TMP, 71 patients (45 ACB positive) received 10 days of SMX-TMP, and 18 patients (18 ACB positive) received 6 weeks of SMX-TMP. The overall recurrence rates in patients who received 10 days of therapy were 32% for RIF-TMP and 23% for SMX-TMP (P = 0.13). There were 12 (14%) relapses in the RIF-TMP group compared with 2 (3%) relapses in the SMX-TMP group (P = 0.01). In patients with upper-tract infections, the relapse rates were not statistically significantly different (P = 0.13). There were two (11%) recurrences (one relapse and one reinfection) in the 6-week treatment group. This 6% relapse rate was not different from the 4% relapse rate observed in patients with upper-tract infections who received 10 days of SMX-TMP. The number of patients who discontinued treatment because of an adverse effect in the 6-week SMX-TMP treatment group was significantly greater than those in the 10-day SMX-TMP treatment group (P=0.003) and the RIF-TMP treatment group (P=0.05). Ten days of SMX-TMP was as effective as 6 weeks of SMP-TMP or 10 days of RIF-TMP in the treatment of uncomplicated upper urinary tract infections and caused the fewest untoward effects.
PMCID: PMC172286  PMID: 3046481
23.  Single-dose tioconazole compared with 3-day clotrimazole treatment in vulvovaginal candidiasis. 
A total of 80 patients were equally randomized to receive a single dose of 6.5% tioconazole ointment or a 3-day course of 100-mg clotrimazole vaginal tablets for the treatment of vulvovaginal candidiasis. Of the 32 evaluable patients treated with tioconazole, 27 (84%) remained asymptomatic 4 weeks posttreatment, compared with 28 of 33 patients (85%) treated with clotrimazole. A total of 34 patients in each group could be evaluated for mycological response based on culture results 1 and 4 weeks after treatment. Twenty patients (59%) who received tioconazole and twenty-one patients (62%) who received clotrimazole remained culture negative 4 weeks after therapy. Of 40 patients who received tioconazole, 12 (30%) experienced local irritation or itching, compared with 2 of 40 patients (5%) treated with clotrimazole (P less than 0.01). Single-dose tioconazole ointment was as effective as a 3-day course of clotrimazole tablets, but significantly more patients in the tioconazole-treated group experienced local side effects.
PMCID: PMC180485  PMID: 3524439
24.  The public health response to 2,3,7,8-TCDD environmental contamination in Missouri. 
Public Health Reports  1985;100(3):289-293.
In 1971, waste oil containing 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was sprayed for dust control on a number of residential, recreational, and work areas in Missouri. In several of them, the level and extent of environmental contamination were not known until late 1982 or 1983. Extrapolation from existing toxicological data indicated the potential for substantial adverse health effects in highly exposed populations. As a result, the Missouri Division of Health and the Centers for Disease Control initiated close collaboration with the Environmental Protection Agency (EPA) on review and evaluation of environmental data, the development of health advisories to EPA on the need for remedial or preventive actions at specific contaminated sites, a health education effort for the medical community and general public, establishment of a dermatological screening clinic, establishment of a central listing of potentially exposed persons through administration of a health effects survey questionnaire, and a pilot medical study of a "highest risk" cohort. Strategies for additional interventions will continue to be based on findings derived from this first phase of the investigation.
PMCID: PMC1424744  PMID: 3923536
25.  Effect of adenovirus infection on expression of human histone genes. 
Molecular and Cellular Biology  1984;4(7):1363-1371.
The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA synthesis, observed when DNA replication is inhibited by a variety of drugs, is not maintained after adenovirus infection.
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PMCID: PMC368919  PMID: 6095065

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