Polycomb contraction differentially regulates terminal human hematopoietic differentiation programs.

BACKGROUND: Lifelong production of the many types of mature blood cells from less differentiated progenitors is a hierarchically ordered process that spans multiple cell divisions. The nature and timing of the molecular events required to integrate the environmental signals, transcription factor activity, epigenetic modifications, and changes in gene expression involved are thus complex and still poorly understood. To address this gap, we generated comprehensive reference epigenomes of 8 phenotypically defined subsets of normal human cord blood. RESULTS: We describe a striking contraction of H3K27me3 density in differentiated myelo-erythroid cells that resembles a punctate pattern previously ascribed to pluripotent embryonic stem cells. Phenotypically distinct progenitor cell types display a nearly identical repressive H3K27me3 signature characterized by large organized chromatin K27-modification domains that are retained by mature lymphoid cells but lost in terminally differentiated monocytes and erythroblasts. We demonstrate that inhibition of polycomb group members predicted to control large organized chromatin K27-modification domains influences lymphoid and myeloid fate decisions of primary neonatal hematopoietic progenitors in vitro. We further show that a majority of active enhancers appear in early progenitors, a subset of which are DNA hypermethylated and become hypomethylated and induced during terminal differentiation. CONCLUSION: Primitive human hematopoietic cells display a unique repressive H3K27me3 signature that is retained by mature lymphoid cells but is lost in monocytes and erythroblasts. Intervention data implicate that control of this chromatin state change is a requisite part of the process whereby normal human hematopoietic progenitor cells make lymphoid and myeloid fate decisions.

Fungal Planet description sheets: 868-950.

Novel species of fungi described in this study include those from various countries as follows: Australia, Chaetomella pseudocircinoseta and Coniella pseudodiospyri on Eucalyptus microcorys leaves, Cladophialophora eucalypti, Teratosphaeria dunnii and Vermiculariopsiella dunnii on Eucalyptus dunnii leaves, Cylindrium grande and Hypsotheca eucalyptorum on Eucalyptus grandis leaves, Elsinoe salignae on Eucalyptus saligna leaves, Marasmius lebeliae on litter of regenerating subtropical rainforest, Phialoseptomonium eucalypti (incl. Phialoseptomonium gen. nov.) on Eucalyptus grandis × camaldulensis leaves, Phlogicylindrium pawpawense on Eucalyptus tereticornis leaves, Phyllosticta longicauda as an endophyte from healthy Eustrephus latifolius leaves, Pseudosydowia eucalyptorum on Eucalyptus sp. leaves, Saitozyma wallum on Banksia aemula leaves, Teratosphaeria henryi on Corymbia henryi leaves. Brazil, Aspergillus bezerrae, Backusella azygospora, Mariannaea terricola and Talaromyces pernambucoensis from soil, Calonectria matogrossensis on Eucalyptus urophylla leaves, Calvatia brasiliensis on soil, Carcinomyces nordestinensis on Bromelia antiacantha leaves, Dendryphiella stromaticola on small branches of an unidentified plant, Nigrospora brasiliensis on Nopalea cochenillifera leaves, Penicillium alagoense as a leaf endophyte on a Miconia sp., Podosordaria nigrobrunnea on dung, Spegazzinia bromeliacearum as a leaf endophyte on Tilandsia catimbauensis, Xylobolus brasiliensis on decaying wood. Bulgaria, Kazachstania molopis from the gut of the beetle Molops piceus. Croatia, Mollisia endocrystallina from a fallen decorticated Picea abies tree trunk. Ecuador, Hygrocybe rodomaculata on soil. Hungary, Alfoldia vorosii (incl. Alfoldia gen. nov.) from Juniperus communis roots, Kiskunsagia ubrizsyi (incl. Kiskunsagia gen. nov.) from Fumana procumbens roots. India, Aureobasidium tremulum as laboratory contaminant, Leucosporidium himalayensis and Naganishia indica from windblown dust on glaciers. Italy, Neodevriesia cycadicola on Cycas sp. leaves, Pseudocercospora pseudomyrticola on Myrtus communis leaves, Ramularia pistaciae on Pistacia lentiscus leaves, Neognomoniopsis quercina (incl. Neognomoniopsis gen. nov.) on Quercus ilex leaves. Japan, Diaporthe fructicola on Passiflora edulis × P. edulis f. flavicarpa fruit, Entoloma nipponicum on leaf litter in a mixed Cryptomeria japonica and Acer spp. forest. Macedonia, Astraeus macedonicus on soil. Malaysia, Fusicladium eucalyptigenum on Eucalyptus sp. twigs, Neoacrodontiella eucalypti (incl. Neoacrodontiella gen. nov.) on Eucalyptus urophylla leaves. Mozambique, Meliola gorongosensis on dead Philenoptera violacea leaflets. Nepal, Coniochaeta dendrobiicola from Dendriobium lognicornu roots. New Zealand, Neodevriesia sexualis and Thozetella neonivea on Archontophoenix cunninghamiana leaves. Norway, Calophoma sandfjordenica from a piece of board on a rocky shoreline, Clavaria parvispora on soil, Didymella finnmarkica from a piece of Pinus sylvestris driftwood. Poland, Sugiyamaella trypani from soil. Portugal, Colletotrichum feijoicola from Acca sellowiana. Russia, Crepidotus tobolensis on Populus tremula debris, Entoloma ekaterinae, Entoloma erhardii and Suillus gastroflavus on soil, Nakazawaea ambrosiae from the galleries of Ips typographus under the bark of Picea abies. Slovenia, Pluteus ludwigii on twigs of broadleaved trees. South Africa, Anungitiomyces stellenboschiensis (incl. Anungitiomyces gen. nov.) and Niesslia stellenboschiana on Eucalyptus sp. leaves, Beltraniella pseudoportoricensis on Podocarpus falcatus leaf litter, Corynespora encephalarti on Encephalartos sp. leaves, Cytospora pavettae on Pavetta revoluta leaves, Helminthosporium erythrinicola on Erythrina humeana leaves, Helminthosporium syzygii on a Syzygium sp. bark canker, Libertasomyces aloeticus on Aloe sp. leaves, Penicillium lunae from Musa sp. fruit, Phyllosticta lauridiae on Lauridia tetragona leaves, Pseudotruncatella bolusanthi (incl. Pseudotruncatellaceae fam. nov.) and Dactylella bolusanthi on Bolusanthus speciosus leaves. Spain, Apenidiella foetida on submerged plant debris, Inocybe grammatoides on Quercus ilex subsp. ilex forest humus, Ossicaulis salomii on soil, Phialemonium guarroi from soil. Thailand, Pantospora chromolaenae on Chromolaena odorata leaves. Ukraine, Cadophora helianthi from Helianthus annuus stems. USA, Boletus pseudopinophilus on soil under slash pine, Botryotrichum foricae, Penicillium americanum and Penicillium minnesotense from air. Vietnam, Lycoperdon vietnamense on soil. Morphological and culture characteristics are supported by DNA barcodes.

Skeletal Metastasis of Canine Urothelial Carcinoma: Pathologic and Computed Tomographic Features.

Invasive urothelial (transitional cell) carcinoma (UC) is the most common cancer in the canine urinary tract. Prolonged survival of dogs with UC due to better management of the primary tumor and prevention of urethral obstruction might have contributed to an apparent increase in distant metastasis. Metastasis to bone is particularly concerning because the ensuing pain often leads to euthanasia; however, little is known of the frequency, site, or nature of UC skeletal metastasis. In a retrospective analysis, 17 (9%) of 188 canine UC cases had histologically confirmed skeletal metastasis, mainly to the vertebrae. In a prospective analysis of 21 dogs with UC that underwent total body computed tomography (CT) at euthanasia followed by a standardized pathologic examination, skeletal lesions detected on CT were suspected to be metastatic in 4 dogs and were confirmed as metastatic UC histologically in 3 (14%) dogs. In all 3 cases, skeletal metastasis had been suspected based on history and physical examination; however, 1 dog had additional CT-detected skeletal metastases in a clinically unsuspected location, and 2 dogs had histologically confirmed skeletal metastases that corresponded to nonspecific osseous lesions on CT. These findings suggest that total body CT could be helpful in detecting skeletal metastasis as a cause of bone pain in dogs with UC as well as in identifying clinically "silent" sites of skeletal metastasis.

Canine urothelial carcinoma: genomically aberrant and comparatively relevant.

Urothelial carcinoma (UC), also referred to as transitional cell carcinoma (TCC), is the most common bladder malignancy in both human and canine populations. In human UC, numerous studies have demonstrated the prevalence of chromosomal imbalances. Although the histopathology of the disease is similar in both species, studies evaluating the genomic profile of canine UC are lacking, limiting the discovery of key comparative molecular markers associated with driving UC pathogenesis. In the present study, we evaluated 31 primary canine UC biopsies by oligonucleotide array comparative genomic hybridization (oaCGH). Results highlighted the presence of three highly recurrent numerical aberrations: gain of dog chromosome (CFA) 13 and 36 and loss of CFA 19. Regional gains of CFA 13 and 36 were present in 97 % and 84 % of cases, respectively, and losses on CFA 19 were present in 77 % of cases. Fluorescence in situ hybridization (FISH), using targeted bacterial artificial chromosome (BAC) clones and custom Agilent SureFISH probes, was performed to detect and quantify these regions in paraffin-embedded biopsy sections and urine-derived urothelial cells. The data indicate that these three aberrations are potentially diagnostic of UC. Comparison of our canine oaCGH data with that of 285 human cases identified a series of shared copy number aberrations. Using an informatics approach to interrogate the frequency of copy number aberrations across both species, we identified those that had the highest joint probability of association with UC. The most significant joint region contained the gene PABPC1, which should be considered further for its role in UC progression. In addition, cross-species filtering of genome-wide copy number data highlighted several genes as high-profile candidates for further analysis, including CDKN2A, S100A8/9, and LRP1B. We propose that these common aberrations are indicative of an evolutionarily conserved mechanism of pathogenesis and harbor genes key to urothelial neoplasia, warranting investigation for diagnostic, prognostic, and therapeutic applications.

Dark septate endophytic pleosporalean genera from semiarid areas.

Dark septate endophytes (DSE) are distributed worldwide as root-colonising fungi, and frequent in environments with strong abiotic stress. DSE is not a taxon, but constitutes numerous fungal taxa belonging to several orders of Ascomycota. In this study we investigate three unidentified DSE lineages belonging to Pleosporales that were found previously in semiarid sandy grasslands. For molecular phylogenetic studies seven loci (ITS, partial 18S nrRNA, 28S nrRNA, actin, calmodulin, transcription-elongation factor 1- α and ß -tubulin genes) were amplified and sequenced. Based on morphology and the resulting molecular phylogeny these isolates were found to represent three novel genera within the Pleosporales, namely Aquilomyces, Flavomyces and Darksidea, with eight novel species. Molecular data revealed that monotypic Aquilomyces belongs to Morosphaeriaceae, monotypic Flavomyces represents an incertae sedis lineage related to Massarinaceae, and Darksidea, with six new species, is allied to the Lentitheciaceae. During this study we tested numerous conditions to induce sporulation, and managed for the first time to induce several DSE to form their sexual morphs.

Association between cigarette smoke exposure and urinary bladder cancer in Scottish terriers in a cohort study.

Canine urothelial carcinoma (UC) initially responds favorably to treatment, but is ultimately lethal in most cases. Research to identify modifiable risk factors to prevent the cancer is essential. The high breed-associated risk for UC, e.g. 20-fold higher in Scottish terriers, can facilitate this research. The objective was to identify environmental and host factors associated with UC in a cohort of Scottish terriers. Information was obtained through dog owner questionnaires for 120 Scottish terriers ≥ 6 years old participating in a bladder cancer screening study, with comparisons made between dogs that did or did not develop UC during the 3 years of screening. Univariable models were constructed, and variables with P < 0.20 were included when building the multivariable model, and then removed using a backward stepwise procedure. P < 0.05 was considered statistically significant. Urine cotinine concentrations were measured by liquid chromatography-mass spectrometry to further investigate potential cigarette smoke exposure. Biopsy-confirmed UC which was found in 32 of 120 dogs, was significantly associated with the dogs living in a household with cigarette smokers (odds ratio [OR], 6.34; 95 % confidence intervals [CI], 1.16-34.69; P = 0.033), living within a mile of a marsh or wetland (OR, 21.23; 95 % CI, 3.64-123.69; P = 0.001), and history of previous bladder infections (OR, 3.87; 95 % CI, 1.0-14.98; P = 0.050). UC was diagnosed in 18 of 51 dogs (35.3 %) with quantifiable cotinine concentrations, and six of 40 dogs (15.0 %) without quantifiable cotinine concentrations in their urine (P = 0.0165). In conclusion, the main modifiable risk factor for UC in this cohort of dogs was exposure to second-hand tobacco smoke.

Cutaneous metastasis of transitional cell carcinoma in 12 dogs.

In humans, cutaneous metastasis of transitional cell carcinoma (TCC) has been attributed to direct extension, lymphatic or hematogenous dissemination, or surgical implantation. The purpose of this study was to characterize the clinical and histologic features of cutaneous TCC metastasis, confirmed by uroplakin-III immunohistochemistry, in dogs. The 12 cases were 9 spayed female and 3 neutered male dogs, 6 to 14 years old (mean, 11 years). Four dogs had a history of urinary incontinence. Three had undergone abdominal surgery for TCC diagnosis or treatment. The primary neoplasms were 7 papillary infiltrating and 5 nonpapillary infiltrating TCC. Cutaneous lesions were detected at a mean of 123 days (median, 38 days) after diagnosis of the primary TCC and appeared as plaques, papules, or nodules in, with 1 exception, perineal, inguinal, or ventral abdominal dermis or subcutis. Of 8 dogs with dermal TCC, 5 had epidermal erosion or ulceration. In 10 dogs, TCC was detected in cutaneous lymphatic vessels, identified by endothelial immunoreactivity for Prox1. Metastases were also detected in lymph nodes in all dogs and at distant noncutaneous sites, usually the lungs, in 10 dogs. Mean survival after diagnosis was 162 days (median, 90 days). Despite medical treatment of 10 dogs after the development of cutaneous metastasis, remission was not achieved; 4 dogs had stable disease. Although TCC could have spread to skin by direct extension or lymphatic or vascular dissemination, the proximity of most cutaneous metastases to the vulva or prepuce raises the additional possibility of transepidermal spread through urine-scalded skin.

Phase I clinical trial and pharmacokinetics of intravesical mitomycin C in dogs with localized transitional cell carcinoma of the urinary bladder.

BACKGROUND: Transitional cell carcinoma (TCC) is the most common cancer of the urinary tract in dogs. The most frequent cause of death is urinary obstruction from the primary tumor. Standard medical therapy for TCC is only partially effective. HYPOTHESIS/OBJECTIVES: Intravesical administration of mitomycin C (MMC) in dogs with invasive TCC will result in antitumor activity against the primary tumor and minimal systemic drug absorption. ANIMALS: Thirteen privately owned dogs with naturally occurring, histopathologically diagnosed TCC of the urinary bladder. METHODS: A prospective phase I trial was performed. MMC was given intravesically (600 µg/mL initial concentration) for 1 h/d for 2 consecutive days each month. The MMC concentration was escalated to a maximum of 800 µg/mL in groups of 3 dogs until the maximum tolerated dose (MTD) was determined. Serum assays for MMC were performed to determine the extent of systemic absorption of the MMC. RESULTS: The MTD of MMC based on local toxicoses was 700 µg/mL (1-h dwell time, 2 consecutive days). In addition, 2 dogs had severe myelosuppression and appeared to have systemic absorption of MMC. Five dogs had partial remission, and 7 dogs had stable disease. CONCLUSIONS: Intravesical MMC has antitumor activity in dogs with invasive TCC. Further study is needed to determine the cause of the myelosuppression associated with MMC administration, and to develop strategies to minimize this risk.

Design, fabrication and test of a microfluidic nebulizer chip for desorption electrospray ionization mass spectrometry.

This paper presents design, microfabrication, and test of a microfluidic nebulizer chip for desorption electrospray ionization mass spectrometry (DESI-MS) in proteomic analysis. The microfluidic chip is fabricated using cyclic olefin copolymer (COC) substrates. The fluidic channels are thermally embossed onto a base substrate using a nickel master and then a top substrate is thermally bonded to seal the channels. Carbon ink embossed into the top COC substrate is used to established electrical connection between the external power supply and the liquid in the channel. The microfluidic chip to external capillary connection is fabricated using Nanoport() interconnection system. Preliminary leakage test was performed to demonstrate the interconnection system is leak-free and pressure test was performed to evaluate the burst pressure. Finally, the nebulizer chip was used to perform DESI-MS for analyzing peptides (BSA and bradykinin) and reserpine on the nanoporous alumina surface. DESI-MS performance of the microfluidic nebulizer chip is compared with that obtained using a conventional DESI nebulizer.

Cognitive evaluation in children and adolescents with Möbius sequence.

BACKGROUND: Möbius sequence is a rare condition usually defined as uni- or bilateral congenital facial weakness with impairment of ocular abduction. Mental retardation is estimated to occur in 10-15% of cases, but at present there have been no studies focusing on the intellectual capacities of children and adolescents with Möbius sequence. METHODS: Twenty-three children and adolescents aged 6-16 years could be recruited following a request of the German Möbius foundation. The primary caregivers of all subjects filled out a special questionnaire to compile personal, somatic and psychosocial history of the probands. All subjects had a physical examination. To assess intellectual capacities, the German version of the Wechsler Intelligence Test-III (WISC-III) was administered. In case of a severe mental retardation, the Vineland Adaptive Behavior Rating Form was used as an alternative. RESULTS: Twenty-two subjects [12 males, 10 females; mean age: 11.3 (6-16) years] could be included; 21 could be examined with the WISC-III. Compared with the normative sample, Full Scale IQ (mean: 92.05; standard deviation: 14.84) was significantly lower (P = 0.023) which was the consequence of a very low Performance IQ (mean: 80.48; standard deviation: 15.84). Compared with the normative sample, the results of all performance subtests were significantly lower (P = 0.033-0.000), whereas verbal subtest scores did not differ or were even higher ['Similarities' (P = 0.026) and 'Vocabulary' (P = 0.019)]. Verbal IQ (mean: 106.24; standard deviation: 15.31) was not significantly different from the normative sample. Two boys met ICD-10 criteria for mental retardation. Full Scale IQ was not predictive for academic success. CONCLUSIONS: The WISC-III is not an adequate predictor for academic success in Möbius patients; intelligence tests which are less dependant on time constraints should be preferred for subjects with Möbius sequence.

Evaluation of two-dimensional ultrasonography and computed tomography in the mapping and measuring of canine urinary bladder tumors.

Determining the dimensions of transitional cell carcinomas (TCCs) of the urinary bladder in dogs is important in assessing tumor progression and the response to treatment. The primary aim of this study was to evaluate the reliability of a standardized two-dimensional (2-D) ultrasound (US) protocol performed by a single experienced operator. Secondary aims were to compare World Health Organization (WHO) and Response Evaluation Criteria in Solid Tumors (RECIST) guidelines, and to compare measurements by two operators following these guidelines. These were evaluated by inter-operator and intra-operator reliability using the concordance correlation coefficient (CCC) and Cohen's κ statistics, which demonstrated substantial to better agreement by an experienced operator using either set of guidelines. It was demonstrated that 2-D US provides a reliable means to determine the dimensions of urinary bladder TCC when an experienced operator used a standardized protocol. In a subset of dogs, urinary bladder distension was varied, which resulted in differences in measurement with 2-D US and computed tomography.

The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1.

DNA replication in budding yeast cells depends on the activation of the Cdc28 kinase (Cdk1 of Saccharomyces cerevisiae) associated with B-type cyclins Clb1 to Clb6. Activation of the kinase depends on proteolysis of the Cdk inhibitor p40SIC1 in late G1, which is mediated by the ubiquitin-conjugating enzyme Cdc34 and two other proteins, Cdc4 and Cdc53. Inactivation of any one of these three proteins prevents p40SIC1 degradation and causes cells to arrest in G1 with active Cln kinases but no Clb-associated Cdc28 kinase activity. Deletion of SIC1 allows these mutants to replicate. p40SIC1 disappears at the G1/S transition and reappears only after nuclear division. Cell cycle-regulated proteolysis seems largely responsible for this pattern, but transcriptional control could also contribute; SIC1 RNA accumulates to high levels as cells exit M phase. To identify additional factors necessary for the inhibition of the Cdk1/Cdc28 kinase in G1, we isolated mutants that can replicate DNA in the absence of Cdc4 function. Mutations in three loci (SIC1, SWI5, and RIC3) were identified. We have shown that high SIC1 transcript levels at late M phase depend on Swi5. Swi5 accumulates in the cytoplasm during S, G2, and M phases of the cell cycle but enters the nuclei at late anaphase. Our data suggest that cell cycle-regulated nuclear accumulation of Swi5 is responsible for the burst of SIC1 transcription at the end of anaphase. This transcriptional control may be important for inactivation of the Clb/Cdk1 kinase in G2/M transition and during the subsequent G1 period.

The fluoride cleavable 2-(cyanoethoxy)methyl (CEM) group as reversible 3'-O-terminator for DNA sequencing-by-synthesis - synthesis, incorporation, and cleavage.

A new and promising sequencing technology called sequencing-by-synthesis (SBS) enables fast determination of DNA sequences. 2'-Deoxynucleotides containing the (2-cyanoethoxy)methyl (CEM) group at the 3'-O-position are potential reversible terminators for the SBS technology. Herein we describe the synthesis, the incorporation by several polymerases, and the cleavage of this 3'-O-blocking group using 3'-O-CEM-thymidinyl-5'-O-triphosphate 7 as an example.

Airborne laser-guided imaging spectroscopy to map forest trait diversity and guide conservation.

Functional biogeography may bridge a gap between field-based biodiversity information and satellite-based Earth system studies, thereby supporting conservation plans to protect more species and their contributions to ecosystem functioning. We used airborne laser-guided imaging spectroscopy with environmental modeling to derive large-scale, multivariate forest canopy functional trait maps of the Peruvian Andes-to-Amazon biodiversity hotspot. Seven mapped canopy traits revealed functional variation in a geospatial pattern explained by geology, topography, hydrology, and climate. Clustering of canopy traits yielded a map of forest beta functional diversity for land-use analysis. Up to 53% of each mapped, functionally distinct forest presents an opportunity for new conservation action. Mapping functional diversity advances our understanding of the biosphere to conserve more biodiversity in the face of land use and climate change.

Pharmacokinetics and toxicity of the novel oral demethylating agent zebularine in laboratory and tumor bearing dogs.

The purpose of this study was to determine the plasma pharmacokinetics (PK) and toxicity of zebularine, an oral cytidine analog with demethylating activity, in dogs. Plasma zebularine concentrations were determined by HPLC-MS/MS following an oral zebularine dose of 8 or 4 mg kg-1 . Plasma zebularine clearance was constant. Mean maximum concentration (Cmax ) was 23 ± 4.8 and 8.6 ± 1.4 µM following 8 and 4 mg kg-1 , respectively. Mean half-life was 5.7 ± 0.84 and 7.1 ± 2.1 following 8 and 4 mg kg-1 , respectively. A single 8 mg kg-1 dose was well tolerated. Daily 4 mg kg-1 treatment in three laboratory dogs resulted in grade 4 neutropenia (n = 3), grade 1 anorexia (n = 2) and grade 1 or 2 dermatologic changes (n = 2). All adverse events resolved with supportive care. A 4 mg kg-1 dose every 21 days was well tolerated. A follow-up dose escalation study is in progress with a lower starting dose.

Involvement of protein kinase A in ethanol-induced locomotor activity and sensitization.

RATIONALE: Mutant mice lacking the RIIbeta subunit of protein kinase A (regulatory subunit II beta(-/-)) show increased ethanol preference. Recent evidence suggests a relationship between heightened ethanol preference and susceptibility to ethanol-induced locomotor sensitization. It is currently unknown if protein kinase A signaling modulates the stimulant effects and/or behavioral sensitization caused by ethanol administration. To address this question, we examined the effects of repeated ethanol administration on locomotor activity RIIbeta(-/-) and littermate wild-type (RIIbeta(+/+)) mice on multiple genetic backgrounds. METHODS: Over three consecutive days, mice were given single i.p. saline injections and immediately placed in a locomotor activity apparatus to establish a composite baseline for locomotor activity. Next, mice maintained on a hybrid 129/SvEvxC57BL/6J or pure C57BL/6J genetic background were given 10 i.p. ethanol injections before being placed in the activity apparatus. Each ethanol injection was separated by 3-4 days. To determine if changes in behavior were specific to ethanol injection, naïve mice were tested following repeated daily saline injections. The effects of ethanol injection on locomotor behavior were also assessed using an alternate paradigm in which mice were given repeated ethanol injections in their home cage environment. RESULTS: Relative to RIIbeta(+/+) mice, RIIbeta(-/-) mice, regardless of genetic background, consistently showed significantly greater ethanol-induced locomotor activation. RIIbeta(-/-) mice also showed increased sensitivity to ethanol-induced locomotor sensitization resulting from repeated administration, an effect that was dependent on genetic background and testing paradigm. Increased locomotor activity by RIIbeta(-/-) mice was specific to ethanol injections, and was not related to altered blood ethanol levels. CONCLUSIONS: These data provide novel evidence implicating an influence of protein kinase A signaling on ethanol-induced locomotor activity and behavioral sensitization. The observation that RIIbeta(-/-) mice are more sensitive to the effects of repeated ethanol administration suggests that normal protein kinase A signaling limits, or is protective against, the stimulant effects of ethanol and the plastic alterations that underlie behavioral sensitization.

Crystal structure of the DNA-binding domain of Mbp1, a transcription factor important in cell-cycle control of DNA synthesis.

BACKGROUND: During the cell cycle, cells progress through four distinct phases, G1, S, G2 and M; transcriptional controls play an important role at the transition between these phases. MCB-binding factor (MBF), a transcription factor from budding yeast, binds to the so-called MCB (MluI cell-cycle box) elements found in the promoters of many DNA synthesis genes, and activates the transcription of those at the G1-->S phase transition. MBF is comprised of two proteins, Mbp1 and Swi6. RESULTS: The three-dimensional structure of the N-terminal DNA-binding domain of Mbp1 has been determined by multiwavelength anomalous diffraction from crystals of the selenomethionyl variant of the protein. The structure is composed of a six-stranded beta sheet interspersed with two pairs of alpha helices. The most conserved core region among Mbp1-related transcription factors folds into a central helix-turn-helix motif with a short N-terminal beta strand and a C-terminal beta hairpin. CONCLUSIONS: Despite little sequence similarity, the structure within the core region of the Mbp1 N-terminal domain exhibits a similar fold to that of the DNA-binding domains of other proteins, such as hepatocyte nuclear factor-3gamma and histone H5 from eukaryotes, and the prokaryotic catabolite gene activator. However, the structure outside the core region defines Mbp1 as a larger entity with substructures that stabilize and display the helix-turn-helix motif.

Immunohistochemical staining for markers of future neoplastic progression in the larynx.

We performed a retrospective, longitudinal study to determine whether abnormalities in immunohistochemical staining for the epidermal growth factor receptor (EGFR), p53, or cyclin D1 occur before the development of laryngeal carcinoma. Staining was performed on 63 paraffin-embedded biopsies from 18 patients who subsequently developed carcinoma in situ (CIS) or invasive carcinoma of the larynx. These were compared to 71 biopsies from 20 patients who did not develop CIS/cancer (minimum follow-up period, 4 years). Also studied were the 34 biopsies containing CIS and/or carcinoma from those patients who progressed and 22 biopsies obtained concurrently. The two patient groups did not differ significantly in terms of tobacco and alcohol use. Distinct patterns of staining correlated with malignant progression. These included EGFR staining of two thirds or more of the epithelium thickness, a linear basal p53 staining pattern, and strong (3+) staining for cyclin D1 (P < 0.01 for each). These staining patterns also correlated with increasing atypia. In our study population, linear basal staining for p53 and strong staining for cyclin D1 had higher specificity for progression than did EGFR overexpression, which was also seen in association with inflammation and chronic irritation. Marked site-to-site variation was seen in the immunohistochemical staining and in the degree of atypia, suggesting that multiple biopsies are necessary to properly assess risk. These immunohistochemical staining patterns may be clinically useful to predict patients at risk for neoplastic progression.

Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder.

Cyclooxygenase (COX)-inhibiting drugs have antitumor activity in canine and rodent models of urinary bladder cancer. Two isoenzymes of COX have been identified, COX-1 and COX-2. The purpose of this study was to characterize COX-1 and COX-2 expression in human invasive transitional cell carcinoma of the urinary bladder by immunohistochemistry and Western blot analysis. COX-2 was not expressed in normal urinary bladder samples but was detected in 25 of 29 (86%) invasive transitional cell carcinomas of the urinary bladder and in 6 of 8 (75%) cases of carcinoma in situ. These results indicate that COX-2 may play a role in bladder cancer in humans and support further study of COX-2 inhibitors as potential antitumor agents in human bladder cancer.

Human lung carcinomas express Fas ligand.

To reach a clinically detectable size, neoplasms must be able to suppress or evade a host immune response. Activated T cells may enter apoptosis in the presence of Fas ligand (FasL) (1), and tissue expression of FasL has been shown to contribute to immune privilege in the eye and testis (2, 3). We have demonstrated that all human lung carcinoma cell lines tested (16 of 16) express a Mr 38,000 protein consistent with FasL by immunoblotting, whereas the majority of resected tumors (23 of 28) show positive staining for FasL by immunohistochemistry. DNA sequencing of reverse transcription-PCR products from lung cancer cells and resected lung tumors confirms the presence of human FasL mRNA in these neoplastic tissues. Furthermore, lung carcinoma cells are capable of killing a Fas-sensitive human T cell line (Jurkat) in coculture experiments; this killing was inhibited by a recombinant form of the soluble portion of the Fas receptor (FasFc). FasL expression by neoplastic cells represents a potential mechanism for peripheral deletion of tumor-reactive T-cell clones.