Evidence for a unique profile of phosphatidylcholine synthesis in late mitotic cells.

Evidence is presented that the structural rearrangements in late mitosis are accompanied by an alteration in membrane lipid synthesis. This evidence was derived from analyzing phospholipid classes after rapid-labeling, as well as from determining the intracellular site of incorporation of choline by HeLa S3 cells as they progressed from metaphase into early interphase (G1). Compared with postmitotic cell data, the recent mitotic cell data indicate a specific two- to threefold increase in the net synthesis of phosphatidylcholine (PC) species, which appeared to contain the more saturated fatty acids. Since this was observed with glycerol, choline, and orthophosphate labelings, and not with methyl labeling, it appears that the CDP-choline plus diacylglycerol pathway rather than the phosphatidylethanolamine to PC pathway was augmented. Electron microscope autoradiography of anaphase, telophase, and early G1 cells demonstrated that the reformed nuclear envelope was the incorporation site of a significant proportion of the newly synthesized PC. This incorporation occurred by early telophase prior to chromosome decondensation. The potential significance of PC metabolism with regard to membrane rearrangements, such as nuclear envelope reformation, is discussed.

Persistence of messenger RNA through mitosis in HeLa cells.

The decrease in protein synthesis which occurs in mammalian cells during cell division is associated with significant disaggregation of polyribosomes. For determining whether messenger RNA survives this disaggregation, the reformation of polyribosomes was investigated in synchronized HeLa cells as they progressed from metaphase into interphase in the presence of 2 microg/ml Actinomycin D. The persistence of messenger during cell division was evidenced by: (1) a progressive increase in the rate of protein synthesis in both treated and untreated cells for 45 min after metaphase; (2) reformation of polyribosomes, as determined by both sucrose gradients and electron microscopy, within 30 min after the addition of Actinomycin D to metaphase cells; (3) the persistence of approximately 50% of the rapidly labeled nonribosomal RNA which had associated with polyribosomes just before metaphase; (4) the resumption of synthesis, following cell division, of 6 selected peptides in Actinomycin-treated cells.

Nuclear envelope-associated resumption of RNA synthesis in late mitosis of HeLa cells.

The restitution of RNA synthesis in cultures progressing from metaphase into interphase (G(1)) has been investigated in synchronized HeLa S(3) cells by using inhibitors of macro-molecular synthesis and the technique of electron microscope autoradiography. The rate of incorporation of radioactive uridine into RNA approached interphase levels in the absence of renewed protein synthesis. In contrast, maintenance of this rate in G(1) was dependent upon renewed protein synthesis. Restoration of synthesis of heterogeneous nuclear RNA occurred under conditions that inhibited production of ribosomal precursor RNA. In autoradiographs of individual cells exposed to radioactive uridine, silver grains were first detected after nuclear envelope reformation at the periphery of the chromosome mass but before chromosomal decondensation. These data are consistent with the following interpretation. Multiple RNA polymerase activities persist through mitosis and are involved in the initiation of RNA synthesis in early telophase at sites on the nuclear envelope.

Nuclear matrix of HeLa S3 cells. Polypeptide composition during adenovirus infection and in phases of the cell cycle.

A subnuclear fraction has been isolated from HeLa S3 nuclei after treatment with high salt buffer, deoxyribonuclease, and dithiothreitol. This fraction retains the approximate size and shape of nuclei and resembles the nuclear matrix recently isolated from rat liver nuclei. Ultrastructural and biochemical analyses indicate that this structure consists of nonmembranous elements as well as some membranous elements. Its chemical composition is 87% protein, 12% phospholipid, 1% DNA, and 0.1% RNA by weight. The protein constituents are resolved in SDS-polyacrylamide slab gels into 30-35 distinguishable bands in the apparent molecular weight range of 14,000 - 200,000 with major peptides at 14,000 - 18,000 and 45,000 - 75,000. Analysis of newly synthesized polypeptides by cylindrical gel electrophoresis reveals another cluster in the 90,000-130,000 molecular weight range. Infection with adenovirus results in an altered polypeptide profile. Additional polypeptides with apparent molecular weights of 21,000, 23,000, and 92,000 become major components by 22 h after infection. Concomitantly, some peptides in the 45,000-75,000 mol wt range become less prominent. In synchronized cells the relative staining capacity of the six bands in the 45,000-75,000 mol wt range changes during the cell cycle. Synthesis of at least some matrix polypeptides occures in all phases of the cell cycle, although there is decreased synthesis in late S/G2. In the absence of protein synthesis after cell division, at least some polypeptides in the 45,000-75,000 mol wt range survive nuclear dispersal and subsequent reformation during mitosis. The possible significance of this subnuclear structure with regard to structure-function relationships within the nucleus during virus replication and during the life cycle of the cell is discussed.

Prevalence of zoonotic agents in dogs visiting hospitalized people in Ontario: implications for infection control.

Visitation of hospitalized people by dogs is becoming commonplace, but little is known about the potential health risks of introducing dogs to healthcare settings. This cross-sectional study evaluated the prevalence of zoonotic agents in a group of 102 visitation dogs from a variety of sources across Ontario. Between May and July 2004, owners were interviewed by a standardized questionnaire while dogs underwent a standardized physical examination. One specimen of faeces, hair-coat brushings and one rectal, aural, nasal, oral and pharyngeal swab were collected from each dog and tested for 18 specific pathogens. All dogs were judged to be in good health. Zoonotic agents were isolated from 80 out of 102 (80%) dogs. The primary pathogen was Clostridium difficile, which was isolated from 58 (58%) faecal specimens. Seventy-one percent (41/58) of these isolates were toxigenic. Extended-spectrum beta-lactamase Escherichia coli was isolated from one (1%) dog, extended-spectrum cephalosporinase E. coli was isolated from three (3%) dogs, and organisms of the genus Salmonella were isolated from three (3%) dogs. Pasteurella multocida or Pasteurella canis was isolated from 29 (29%) oral swabs, and Malassezia pachydermatis was isolated from eight (8%) aural swabs. Giardia antigen was present in the faeces of seven (7%) dogs, while Toxocara canis and Ancylostoma caninum were detected in two (2%) dogs and one (1%) dog, respectively. Methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Campylobacter spp., Microsporum canis, group A streptococci, Pseudomonas aeruginosa and Cryptosporidium spp. were not detected. Further information is needed before the full implications of these findings for infection control can be assessed properly.

Whole-exome analysis reveals novel somatic genomic alterations associated with outcome in immunochemotherapy-treated diffuse large B-cell lymphoma.

Lack of remission or early relapse remains a major clinical issue in diffuse large B-cell lymphoma (DLBCL), with 30% of patients failing standard of care. Although clinical factors and molecular signatures can partially predict DLBCL outcome, additional information is needed to identify high-risk patients, particularly biologic factors that might ultimately be amenable to intervention. Using whole-exome sequencing data from 51 newly diagnosed and immunochemotherapy-treated DLBCL patients, we evaluated the association of somatic genomic alterations with patient outcome, defined as failure to achieve event-free survival at 24 months after diagnosis (EFS24). We identified 16 genes with mutations, 374 with copy number gains and 151 with copy number losses that were associated with failure to achieve EFS24 (P<0.05). Except for FOXO1 and CIITA, known driver mutations did not correlate with EFS24. Gene losses were localized to 6q21-6q24.2, and gains to 3q13.12-3q29, 11q23.1-11q23.3 and 19q13.12-19q13.43. Globally, the number of gains was highly associated with poor outcome (P=7.4 × 10(-12)) and when combined with FOXO1 mutations identified 77% of cases that failed to achieve EFS24. One gene (SLC22A16) at 6q21, a doxorubicin transporter, was lost in 54% of EFS24 failures and our findings suggest it functions as a doxorubicin transporter in DLBCL cells.

Biosynthesis of 3 beta-hydroxy-5,7-pregnadien-20-one by the horse fetal gonad.

The production of equilin and the other ring B-unsaturated estrogens by the pregnant mare is anomalous in that they are biosynthesised by a cholesterol-independent pathway. Fetal horse gonads were incubated with tritiated sodium acetate and radiochemically pure 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were isolated. A fetal gonad--placental system is proposed for equilin production, 3 beta-hydroxy-5,7-pregnadien-20-one being a precursor for 3 beta-hydroxy-5,7-androstadien-17-one in the fetal gonad and the latter being the precursor of equilin in the placenta. The nature of the possible precursor of 3 beta-hydroxy-5,7-pregnadien-20-one is discussed.

The biosynthesis of 3 beta-hydroxy-5,7-androstadien-17-one by the horse fetal gonad.

Horse fetal gonadal tissue was incubated with 3 beta-hydroxy-5,7-pregnadien-20-one and 5,7-cholestadien-3 beta-ol and it was shown that both substrates were converted to 3 beta-hydroxy-5,7-androstadien-17-one. These findings support the proposal that in this tissue there is a 5,7-diene pathway producing 3 beta-hydroxy-5,7-androstadien-17-one, the putative precursor of equilin in the placenta.

An electronic medical record system with direct data-entry and research capabilities.

The transfer of medical records from a paper system to a computer-based system is inevitable. However, the widespread acceptance of electronic medical records has been delayed by problems such as high cost, inefficiency, data entry errors and poor physician acceptance. We have developed a database system that has overcome these difficulties and now serves as an electronic medical record. Our system has been in use for a year and a half, and currently contains information on over two thousand patients. The database provides an electronic radiation oncology chart containing patients' demographic information, technical treatment data and dictated reports. All dictated notes are captured, including consultation notes, treatment summaries, on-treatment visits, letters and follow up reports. The system provides data validation upon entry, required few additional software or hardware purchases, and allows for efficient retrieval of data. Unlike other database systems which require the hiring of data entry clerks to input the data, ours combines transcription and data entry. The database runs on a local area network of computers and uses a commercially available relational database package. It makes extensive use of mouse interface features such as pull-down menus, pop-up lists, buttons, multi-page forms, and scrolling fields, making the system easy to use with minimal training. Many custom features are built in, such as help screens, control functions, audit trails, and a system that keeps track of each patient's referring and other relevant physicians. For research purposes, the system has the capability to perform survival analyses on arbitrary user-defined subsets of patients. Data may also be exported transparently to statistical packages for other types of analyses.

Psoriasis: current concepts in management.

Psoriasis is a chronic, relapsing dermatosis characterised by scaling, erythema, and less commonly pustulation. Although the clinical spectrum is broad and the nature of the underlying defect responsible for the production of psoriasis uncertain, all currently available effective remedies are capable of inhibiting epidermal mitosis and this remains the most widely accepted concept in the management of psoriasis.