EZH2 promotes neoplastic transformation through VAV interaction-dependent extranuclear mechanisms.

Recently, we reported that the histone methyltransferase, EZH2, controls leukocyte migration through interaction with the cytoskeleton remodeling effector, VAV, and direct methylation of the cytoskeletal regulatory protein, Talin. However, it is unclear whether this extranuclear, epigenetic-independent function of EZH2 has a profound impact on the initiation of cellular transformation and metastasis. Here, we show that EZH2 increases Talin1 methylation and cleavage, thereby enhancing adhesion turnover and promoting accelerated tumorigenesis. This transforming capacity is abolished by targeted disruption of EZH2 interaction with VAV. Furthermore, our studies demonstrate that EZH2 in the cytoplasm is closely associated with cancer stem cell properties, and that overexpression of EZH2, a mutant EZH2 lacking its nuclear localization signal (EZH2ΔNLS), or a methyl-mimicking Talin1 mutant substantially promotes JAK2-dependent STAT3 activation and cellular transformation. Taken together, our results suggest a critical role for the VAV interaction-dependent, extranuclear action of EZH2 in neoplastic transformation.

Quality of life in patients with peritoneal surface malignancies after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: An increasing number of patients are presenting with peritoneal carcinomatosis and more centers are performing cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). While morbidity and mortality are shown to be acceptable, quality of life after surgery should be assessed. METHODS: 63 patients who had CRS and HIPEC from 2001 to 2012 and who were still alive and on follow up were included. The EORTC-QLQ-C30 was administered to the patients. RESULTS: Median age was 53 years (14-71). 44% had ovarian primaries, 21% had appendicael primaries and 19% had colorectal primaries. Median follow-up was 1.08 years (0.06-9.8). The median time from surgery to the questionnaire was 1.3 years (0.24-10.18). There was no statistical difference in scores when comparing by age, gender, recurrence, gender, PCI score, presence of a complication and type of primary cancer. Scores were highest less than 6 months after surgery, dropped subsequently but rose again after 2 years. Our patients had better scores compared to a control group of outpatient cancer patients at our institution as well as the reference EORTC group. CONCLUSIONS: In keeping with previous quality of life studies done for CRS and HIPEC patients, we have shown that our patients can achieve a good quality of life after CRS and HIPEC even with recurrent disease.

Reovirus decreases azoxymethane-induced aberrant crypt foci and colon cancer in a rodent model.

Reovirus type 3 Dearing has demonstrated oncolytic efficacy in vitro and in vivo against a variety of cancer cell lines, tumor xenografts and syngeneic cancer models. In this study, we investigated the effectiveness of reovirus against aberrant crypt foci (ACF) and colon cancer induced by the carcinogen azoxymethane (AOM) in an immunocompetent rat model. Sprague-Dawley rats received 15 mg/kg AOM intraperitoneally once per week for 4 weeks and reovirus was administered rectally once a week for 5 weeks starting 20 weeks after the last dose of AOM. Two weeks after completion of reovirus therapy, animals were examined for tumor burden in the colon and other tissues. Reovirus-treated animals showed a decrease in total ACF numbers (P=0.014), in large ACFs (P=0.0069) and in tumor number (P=0.03) compared to vehicle-treated animals. Fewer obstructing tumors in the colon (P=0.07) and duodenum (P=0.03) and reduced hepatic metastases were also noted. In addition, a tumor cell line derived from hepatic metastases was found to be susceptible to reovirus in vitro. Our results show that repeated rectal reovirus administration had some efficacy in the treatment and prevention of AOM-induced ACFs, colon cancers and metastases.

Aquaporin expression is downregulated in a murine model of colitis and in patients with ulcerative colitis, Crohn's disease and infectious colitis.

Colitis is associated with alterations in electrolyte and water transport. These changes give rise to some of the symptoms experienced by patients with colitis. Alterations in fluid flux may also contribute to increased susceptibility to mucosal injury. Recently, endogenous water channel proteins (aquaporins; AQPs), have been identified in colonic tissue. The expression of AQP4, AQP7 and AQP8 was examined, via reverse transcription/polymerase chain reaction, Western blotting and immunohistochemistry, in a murine model of colitis and in patients with inflammatory bowel disease or infectious colitis. Colitis was induced in C57BL/6 mice by the addition of 2.5% dextran sodium sulphate (DSS) to their drinking water. AQP expression in these mice was assessed following 12 h to 7 days of DSS exposure and during the recovery phase from 1 to 15 days following cessation of DSS exposure. Colonic water transport was measured after 1 and 3 days of DSS and following 7 days of recovery. The expression of AQP4 and AQP8 mRNA was significantly decreased after 12-24 h of DSS exposure and remained depressed throughout the treatment period. Expression of AQP7 was more variable. Protein expression followed a similar pattern to that observed for AQP mRNA. Significant alteration in colonic fluid secretion was correlated with reduced expression of AQP isoforms. Significantly, patients with active ulcerative colonic, Crohn's colitis or infectious colitis had similar dramatic reductions in AQP expression that appeared to be correlated with disease activity. Thus, colonic injury in both mouse and man is associated with a downregulation in AQP expression.

Chemotherapy- and radiotherapy-induced intestinal damage is regulated by intestinal trefoil factor.

BACKGROUND & AIMS: Injury to the intestinal mucosa is frequently a dose-limiting complication of radiotherapy and chemotherapy. Approaches to limit the damage to the intestine during radiation and chemotherapy have been largely ineffective. Trefoil factors are produced throughout the gastrointestinal tract and regulate cell migration, restitution, and repair. Studies were undertaken to define the role of intestinal trefoil factor in modulating the intestinal response to chemotherapy and radiation. METHODS: The effect of intestinal trefoil factor on migration and cell survival in intestinal epithelial monolayer exposed to methotrexate was studied in vitro. Chemotherapy and radiation damage was assessed in wild-type and intestinal trefoil factor-null mice in the presence or absence of supplemental intestinal trefoil factor administered in drinking water. RESULTS: Radiation and chemotherapy induced a marked reduction in goblet cell number and intestinal trefoil factor messenger RNA, as well as intestinal trefoil factor promoter activity. Intestinal trefoil factor improved intestinal epithelial cell viability and wound repair after chemotherapy exposure in vitro. Intestinal trefoil factor-deficient mice (intestinal trefoil factor(-/-)) were more susceptible to chemotherapy- and radiation-induced mucositis. Oral recombinant intestinal trefoil factor reduced the severity of both chemotherapy-induced and chemotherapy/radiotherapy-induced intestinal mucositis. CONCLUSIONS: These studies suggest that intestinal trefoil factor is involved in protection against and recovery from intestinal mucositis induced by radiation and chemotherapy.

Molecular characterization and expression analysis of a highly conserved rice mago nashil homolog.

Mago Nashi, a protein initially shown to be essential in the development of the Drosophila oocyte, is highly conserved among species and shows no homology to any other known cellular proteins. Here we report the nucleotide sequence of a cDNA and a partial gene that encode rice Mago Nashi protein homologs. In addition, we present the tissue-specific expression pattern of mago nashi at the level of RNA and protein. The rice Mago Nashi protein shares at least 73% amino acid identity with all known animal homologs. Genomic DNA gel blot analysis indicates that two copies of the mago nashi gene exist in the rice genome, one of which has identical intron positions to those found in an Arabidopsis homolog. mago nashi is expressed in root, leaf and developing seed tissue as determined by RNA and protein gel blot analysis. Evidence from Drosophila, Caenorhabditis elegans and human studies of Mago Nashi suggests that a major function of this protein is its involvement in RNA localization. The highly conserved amino acid sequence of all Mago Nashi protein homologs across kingdoms suggests that the plant version of this protein may similarly be involved in RNA localization.

Marcus Gunn jaw-winking phenomenon: a new supplemental test in the preoperative evaluation.

PURPOSE: To introduce a new method for the evaluation of Marcus Gunn jaw-winking ptosis that more precisely defines the severity of blepharoptosis. METHODS: A retrospective review of 16 consecutive patients with Marcus Gunn jaw-winking ptosis presenting to our institution between 1993 to 1999 was performed. The position of the affected eyelid was observed after applying a technique of jaw immobilization and disruption of fusion with temporary occlusion of the ipsilateral side. RESULTS: In patients presenting with mild to moderate Marcus Gunn jaw-winking, the majority (62.5%) demonstrated a positive test, uncovering complete or near complete ptosis. Test results were partially positive in 3 patients (18.8%) with increased but not complete ptosis and negative in 3 patients (18.8%) with no change in eyelid position. CONCLUSIONS: Blepharoptosis associated with Marcus Gunn jaw-winking phenomenon is often more severe than found by conventional clinical evaluation. This finding may explain the frequent undercorrection and unpredictable results following levator resection. In patients exhibiting a positive jaw-winking ptosis test, disappointing outcomes with levator resection may be avoided by instead proceeding with a frontalis suspension with levator disinsertion as recommended for ptosis with severe jaw winking.

Porous polyethylene implant fibrovascularization rate is affected by tissue wrapping, agarose coating, and insertion site.

PURPOSE: Often used in facial and ocular reconstruction, biointegratable materials, such as hydroxyapatite and high density porous polyethylene, can be associated with migration, exposure, and infection. Complications are less likely after implants become fibrovascularly integrated. A model was sought to study the influence of multiple factors on the rate of fibrovascular ingrowth into porous implants. METHODS: High density porous polyethylene cubes were implanted into paraspinous skeletal muscles in rabbits. The cubes were explanted at weekly intervals using survival surgery. The number of fibroblasts at the center of each cube was counted, generating a time-dependent standard curve of cell accumulation. Porous polyethylene cubes uncoated, coated with agarose (a plant-derived carbohydrate), or coated with nonperforated sclera (human or rabbit) were implanted into suprascapular adipose and paraspinous skeletal muscle in other rabbits. RESULTS: Fibrovascular ingrowth occurred more rapidly with cube implantation into skeletal muscle versus adipose, with increased surface area contact between implants and muscle, and with removal of muscle capsules. While the rate of fibroblast accumulation decreased in cubes coated with sclera, coating the cubes with agarose increased the fibrous capsule formation without altering the rate of biointegration. CONCLUSIONS: This study provides a novel approach for the study of fibrovascular ingrowth into implants treated under a variety of conditions. Modification of current surgical techniques may increase the rate of porous polyethylene implant biointegration.

Epidermal and fibroblast growth factors enhance fibrovascular integration of porous polyethylene implants.

PURPOSE: Porous implants used in functional and aesthetic reconstruction of the orbit, face, and cranium are less likely to develop complications after they become biointegrated. We investigated whether the administration of exogenous growth factors could increase the rate of implant integration. METHODS: High-density porous polyethylene cubes were placed in dorsal paraspinal muscles of rabbits, and daily transcutaneous injections of saline, epidermal growth factor, or basic fibroblast growth factor were administered directly over the cubes for 10 days. At serial time points up to 10 weeks, cubes were explanted and the fibroblasts present at the center of the cubes were counted. RESULTS: Injections of epidermal growth factor and basic fibroblast growth factor increased the rate at which fibroblasts accumulated in porous polyethylene implants and decreased the time required to achieve a maximal rate of cellular accumulation within the cubes. At 4 weeks, when all cell populations had attained a linear rate of accumulation, cubes previously injected with saline, epidermal growth factor, or basic fibroblast growth factor contained an average of 10, 40, and 80 cells per 0.0156 mm2, at their centers, respectively. CONCLUSIONS: Enhancement of the rate of biointegration of porous polyethylene cubes in rabbits is achievable by repeated, transcutaneous administration of exogenous growth factors.

Growth factors embedded in an agarose matrix enhance the rate of porous polyethylene implant biointegration.

PURPOSE: Repeated injections of epidermal and basic fibroblastic growth factors have been shown to enhance the biointegration rate of implanted porous polyethylene. A study was done to determine whether agarose, introduced at the time of implant placement, might serve as an adequate "single dose" delivery system for endogenous and exogenous growth factors. METHODS: Polyethylene cubes coated with agarose-containing growth factors were implanted into fat and muscle in rabbits. Factors studied included autogenous whole blood, autogenous serum, ascorbic acid, epidermal growth factor, basic fibroblast growth factor, transforming growth factor alpha, and transforming growth factor beta. The rate and character of the fibrovascular ingrowth into implants and surrounding capsule thickness were assessed. RESULTS: Fibroblast infiltration enhanced two- to sixfold with the use of autogenous or allogenic factors introduced in an agarose matrix at the time of cube implantation. CONCLUSIONS: Growth factors studied altered the thickness of the capsule surrounding implants as well as both the vascularity and stromal density within implants.

A multidisciplinary approach to atypical lacrimal obstruction in childhood.

PURPOSE: This study explores the diagnosis and management of unusual anomalies involving the canaliculi, nasolacrimal duct, nasal cavity, and sinuses in childhood. METHODS: A case series of eight children with lacrimal outflow anomalies ranging from distal nasolacrimal duct cyst formation to persistent dacryocystitis following failed probing or silicone intubation were reviewed retrospectively. Diagnostic studies including intranasal endoscopy and preoperative or intraoperative dacryocystography (DCG) were of value. RESULTS: Treatment modalities included endoscopically guided resection of lacrimal cyst mucosa, endoscopic dacryocystorhinostomy (DCR), and monocanalicular or bicanalicular intubation of the lacrimal outflow system. In our series, endoscopic surgery was well tolerated by all patients with improvement in symptoms. CONCLUSIONS: This initial experience suggests that endoscopic techniques may be useful in the management of atypical lacrimal outflow obstruction in childhood.

Incidence and risk factors for delirium and other adverse outcomes in older adults after coronary artery bypass graft surgery.

OBJECTIVE: To determine the incidence and risk factors for delirium after coronary artery bypass graft (CABG) surgery. DESIGN: Prospective cohort. SETTING: Cardiac surgery units of a tertiary care hospital. PARTICIPANTS: Consecutive patients over age 65 years undergoing elective CABG surgery. Exclusion criteria included preoperative sensory or language barriers. INTERVENTIONS: Each patient was assessed within 24 h before surgery for baseline demographic, medical and functional data. Incident delirium (within four postoperative days) was diagnosed by a study physician. Nine potential risk factors for delirium were subjected to univariate and multivariate analysis. MAIN RESULTS: Of 75 consenting patients, three died during or soon after surgery and one was still comatose at follow-up. Of the remaining 71 participants, 23 (32%) experienced delirium. Those with delirium were more likely than those without delirium to have a history of a stroke (21% versus 4%, respectively, P=0.032) and to have had a longer duration of cardiopulmonary bypass (CPB) (113 mins versus 95 mins, respectively, P=0.025). A tendency to have experienced low cardiac output (83% versus 58%, respectively, P=0.061) postoperatively was also noted. Multivariate analysis confirmed past stroke and duration of cardiopulmonary bypass as risk factors. CONCLUSIONS: Delirium in the elderly after CABG surgery is common. Its occurrence may be predisposed by a history of a stroke and precipitated by a longer duration of CPB.

Pharmacology of insect GABA receptors.

A GABA-operated Cl- channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl- channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABAA receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl- channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active at t-butylbicyclophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.

The complete nucleotide sequence of the Xenopus laevis mitochondrial genome.

The complete sequence of the 17,553-nucleotide Xenopus laevis mitochondrial genome has been determined. A comparison of this amphibian mitochondrial genomic sequence with those of the mammalian mitochondrial genomes reveals a similar gene order and compact genomic organization. The encoded genes for 22 tRNAs, two ribosomal RNAs, and 13 proteins (COI, COII, COIII, ATPase 6, cytochrome b, and eight additional unidentified reading frames) in the amphibian mitochondria are highly homologous to their mammalian counterparts. Although the amphibian mitochondrial genome contains a significantly larger displacement loop region than the mammalian mitochondrial genomes, there are several regions of sequence homology near the putative sites for heavy and light strand transcription initiation and heavy strand replication. The unique mitochondrial genetic code observed in the mammalian mitochondrial systems is similar to that of the X. laevis mitochondrial genome because of the presence of only 22 encoded tRNAs and the high degree of homology between the predicted protein sequences. However, the amphibian system exclusively utilizes AUG as the start codon in all 13 open reading frames and shows a preference for codons ending in U rather than ending in C. In addition, the X. laevis mitochondrial genome employs the encoded AGA stop codon once and the UAA stop codon three times and requires polyadenylation to provide the nine other UAA stop codons. These observations suggest that the mechanisms of replication, transcription, processing, and translation in mitochondria are highly conserved throughout higher vertebrates.

A new class of endogenous human retroviral genomes.

Human DNA contains multiple copies of a novel class of endogenous retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2) containing one such proviral genome revealed that it is a mosaic of retroviral-related sequences with the organization and length of known endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized with the long terminal repeat of the squirrel monkey virus, a type D retrovirus. The HLM-2 gag and pol genes share extensive nucleotide sequence homology with those of the M432 retrovirus (a type A-related retrovirus), mouse mammary tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a type C retrovirus). Nucleotide sequence analysis revealed regions in the HLM-2 pol gene that were as much as 70 percent identical to the mouse mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridized with the corresponding region of the M432 viral genome.

Molecular cloning and sequencing of pheU, a gene for Escherichia coli tRNAPhe.

A recombinant plasmid (designated pID2) carrying the E. coli gene for tRNAPhe has been isolated from a plasmid bank constructed by the ligation of a total EcoRI digest of E. coli K12 DNA into the EcoRI site of pACYC184 DNA. The plasmid was selected by virtue of its ability to complement a temperature-sensitive lesion in the gene (PheS) for the alpha-subunit of phenylalanyl-tRNA synthetase. Crude tRNA isolated from such transformants exhibited elevated levels of phenylalanine acceptor activity. The tRNAPhe gene has been localized within the first 300 base pairs of a 3.6 kb SalI fragment of pID2. The sequence of the gene and its flanking regions is presented.

DNA sequence of the Xenopus laevis mitochondrial heavy and light strand replication origins and flanking tRNA genes.

We have determined the primary structure of the two regions of the Xenopus laevis mitochondrial genome which encompass the origins of heavy (H) and light (L) strand replication. The first segment, which consists of 2398 nucleotides, contains the displacement loop (D-loop), the tRNA genes for threonine, proline and phenylalanine, the origin of H-strand replication, and the promoters of H- and L-strand transcription. The second segment, which consists of 447 nucleotides, contains the L-strand replication origin flanked by the tRNA genes for tryptophan, alanine, asparagine, cysteine, and tyrosine. A comparison of the sequences of the Xenopus laevis mitochondrial L-strand replication origin region and the eight tRNA genes with their counterparts from the mammalian mitochondrial genomes reveals that these regions are quite homologous, while its D-loop region shows only slight homology with those of the mammalian mitochondrial genomes.

A mammalian mitochondrial serine transfer RNA lacking the "dihydrouridine" loop and stem.

A unique transfer RNA has been identified in human and bovine mitochondria that lacks the "dihydrouridine" loop and stem structure. This tRNA is mitochondrially coded as shown by DNA sequence analysis of the human and bovine mitochondrial DNA. Sequence analysis of the RNA shows that it is post-transcriptionally modified by the addition of CCA at the 3' terminus and that at least one base is modified. As predicted by its anticodon (GCU, corresponding to the serine codons AGU/C) this tRNA can be aminoacylated with serine when purified mitochondria are incubated in a medium containing 3H-serine.

Cellular immune response to Yersinia pestis modulated by product(s) from thymus-derived lymphocytes.

Resistance to infection with Yersinia pestis was found to depend on whether the macrophage can inactivate and withstand the cytotoxic effects of phagocytized Y. pestis. Serum from mice immunized with antigens of Y. pestis enhanced the resistance of monolayers of normal cultures to the cytotoxic effects of Y. pestis and increased the capacity of peritoneal exudate cells from immune mice to inactivate these bacteria. The enhancing component of the serum was not removed by absorption with heat-killed Y. pestis. Similar enhancement was provided by supernatant fluids of spleen cultures from immunized mice but not by those from unimmunized mice. Pretreatment of spleen cells with rabbit hyperimmune antiserum to mouse brain theta-antigen plus complement caused a reduction in the enhancing capacity of the spleen cell culture fluids. Removal of the glass-adherent cell population from suspensions of primed spleen cells prior to in vitro antigenic stimulation resulted in a loss of activity from the subsequently harvested culture fluids. Thus, the enhancing component of the serum appears to be a product of thymus-derived lymphocytes (T-cells). Furthermore, splenic macrophages seem to be required for the interaction of primed T-cells with heat-killed Y. pestis.