The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis.

Mycorrhizal symbioses--the union of roots and soil fungi--are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants. Boreal, temperate and montane forests all depend on ectomycorrhizae. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains approximately 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Simultaneous flow cytometric analysis of human T cell activation antigen expression and DNA content.

Cell-surface antigens that are induced to appear on T cells activated by the lectin phytohemagglutinin-P (PHA) can be classified both on the basis of the kinetics of their appearance and on their growth-association properties. Seven distinct T cell activation antigens, defined by monoclonal antibodies, were classified as early, intermediate, or late antigens based on their temporal appearance relative to DNA synthesis. Four antigens, the transferrin receptor, the T cell activation antigen Tac, the 4F2 antigen, and the 49.9 antigen were early antigens, whereas the OKT10 antigen appeared at intermediate times and both HLA-DR and antigen 19.2 appeared late. The use of a dye, Hoechst 33342, which stains DNA stoichiometrically, allowed the simultaneous analysis of immunofluorescence and cell cycle position of individual cells. This analysis unexpectedly revealed that essentially all cells in the proliferative phase of the cell cycle expressed each of the four early-activation antigens. The correlation between expression of the four early-activation antigens and T cell proliferation suggests that these molecules are important for the growth of all T cells. The relationship of two of these activation antigens, known to be the receptors for transferrin and interleukin 2, a T cell growth factor, is discussed with special reference to the roles of their ligands in supporting the growth of T cells.

The Canadian Assisted Reproductive Technologies Register (CARTR) Plus database: a validation study.

STUDY QUESTION: Are data accurately documented in the Canadian Assisted Reproductive Technologies Register (CARTR) Plus database? SUMMARY ANSWER: Measures of validity were strong for the majority of variables evaluated while those with moderate agreement were FSH levels, oocyte origin and elective single embryo transfer. WHAT IS KNOWN ALREADY: Health databases and registries are excellent sources of data. However, as these databases are typically not established for the primary purpose of performing research, they should be evaluated prior to utilization for research both to inform the study design and to determine the extent to which key study variables, such as patient characteristics or therapies provided, are accurately documented in the database. CARTR Plus is Canada's national register for collecting extensive information on IVF and corresponding pregnancy outcomes, and it has yet to be validated. STUDY DESIGN SIZE DURATION: This study evaluating the data translation CARTR Plus database examined IVF cycles performed in 2015 using data directly from patient charts. Six clinics across Canada were recruited to participate, using a purposive sampling strategy. Fixed random sampling was employed to select 146 patient cycles at each clinic, representing unique patients. Only a single treatment cycle record from a unique patient at each clinic was considered during chart selection. PARTICIPANTS/MATERIALS SETTING METHODS: Twenty-five data elements (patient characteristics, treatments and outcomes) were reabstracted from patient charts, which were declared the reference standard. Data were reabstracted by two independent auditors with relevant clinical knowledge after confirming inter-rater reliability. These data elements from the chart were then compared to those in CARTR Plus. To determine the validity of these variables, we calculated kappa coefficients, sensitivity, specificity, positive predictive value and negative predictive value with 95% CI for categorical variables and calculated median differences and intraclass correlation coefficients (ICC) for continuous variables. MAIN RESULTS AND THE ROLE OF CHANCE: Six clinics agreed to participate in this study representing five Canadian provinces. The mean age of patients was 35.5 years, which was similar between the two data sources, resulting in a near perfect level of agreement (ICC = 0.99; 95% CI: 0.99, 0.99). The agreement for FSH was moderate, ICC = 0.68 (95% CI: 0.64, 0.72). There was nearly perfect agreement for cycle type, kappa = 0.99 (95% CI: 0.98, 1.00). Over 90% of the cycles in the reabstracted charts used autologous oocytes; however, data on oocyte source were missing for 13% of cycles in CARTR Plus, resulting in a moderate degree of agreement, kappa = 0.45 (95% CI, 0.37, 0.52). Embryo transfer and number of embryos transferred had nearly perfect agreement, with kappa coefficients greater than 0.90, whereas that for elective single or double embryo transfer was much lower (kappa = 0.55; 95% CI: 0.49, 0.61). Agreement was nearly perfect for pregnancy type, and number of fetal sacs and fetal hearts on ultrasound, all with kappa coefficients greater than 0.90. LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: CARTR Plus contains over 200 variables, of which only 25 were assessed in this study. This foundational validation work should be extended to other CARTR Plus database variables in future studies. WIDER IMPLICATIONS OF THE FINDINGS: This study provides the first assessment of the quality of the data translation process of the CARTR Plus database, and we found very high quality for the majority of the variables that were analyzed. We identified key data points that are either too often lacking or inconsistent with chart data, indicating that changes in the data entry process may be required. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Canadian Institutes of Health Research (CIHR) (Grant Number FDN-148438) and by the Canadian Fertility and Andrology Society Research Seed Grant (Grant Number: N/A). The authors report no conflict of interest. TRIAL REGISTRATION NUMBER: Not applicable.

Tetraspanin-induced death of myeloma cell lines is autophagic and involves increased UPR signalling.

BACKGROUND: Multiple myeloma (MM) therapy is hindered by the interaction of the heterogeneous malignant plasma cells with their microenvironment and evolving drug resistance. We have previously shown that the membranal tetraspanins, CD81 and CD82, are under-expressed in MM cells and that their reintroduction causes massive non-apoptotic death. In this study, we aimed to characterise the tetraspanin-induced MM death. METHODS: Multiple myeloma cell lines were transiently transfected with eGFP-CD81N1/CD82N1 fusion proteins and assessed for death mode by flow cytometry (propidium iodide, ZVAD-fmk, 3MA), activation of unfolded protein response (UPR), and autophagy (immunoblot, RT-PCR). RESULTS: Cell death induced by CD81N1 and CD82N1 in MM cell lines was autophagic and involved endoplasmic reticulum (ER)-stress manifested by activation of UPR pathways, PERK (protein kinase-like ER kinase) and IRE1 (inositol-requiring 1). We also established the relative X-box binding protein 1 baseline expression levels in a panel of MM cell lines and their general dependence on autophagy for survival. Timeline of UPR cascades and cell fate supported our results. INTERPRETATION: This is the first publication implicating tetraspanins in UPR signalling pathways, autophagy, and autophagic death. Integration of our findings with published data highlights the unifying dependence of MM cells on ER-Golgi homoeostasis, and underscores the potential of tetraspanin complexes and ER-stress as leverage for MM therapy.

Isolation and culture of a tetraploid subpopulation of smooth muscle cells from the normal rat aorta.

Smooth muscle cells with 4C (double diploid) DNA content have been found in major arteries. The proportion of 4C cells increases with normal aging and with hypertension. These cells may represent a state of arrest at the G2 phase of the cell cycle or may be examples of true tetraploidy. Flow cytometric cell sorting was used to isolate 4C smooth muscle cells from the rat aorta, and the cells were cultured. Flow cytometry, Feulgen microdensitometry, and karyotyping of the progeny of the 4C cells established the presence of true tetraploid cells. These findings demonstrate the presence of reproductively viable tetraploid cells in a normal mammalian tissue.

Subthalamic deep brain stimulation and impulse control in Parkinson's disease.

BACKGROUND AND PURPOSE: Experimental studies suggest that deep brain stimulation (DBS) of the subthalamic nucleus (STN) induces impulsivity in patients with Parkinson's disease (PD). The purpose of this study was to assess various measures of impulse control in PD patients with STN DBS in comparison to patients receiving medical therapy. METHODS: In a cross-sectional evaluation, 53 consecutively eligible patients were assessed for impulsivity with the Barratt Impulsiveness Scale, for impulse control disorders (ICDs) using the Minnesota Impulsive Disorders Interview, and for obsessive-compulsive symptoms using the Maudsley Obsessional-Compulsive Inventory. RESULTS: Independent samples t-tests revealed that compulsivity scores were not different between DBS patients and patients without DBS. However, impulsivity scores were significantly higher in DBS patients. Additionally, ICDs were observed in 3 of 16 (19%) DBS patients and in 3 of 37 (8%) medically treated patients. No association was found between the use of dopamine agonists and impulsivity in DBS patients. CONCLUSIONS: Our data suggest that screening for impulsivity and ICDs should be performed prior to DBS, and that patients should be monitored for these problems during follow-up. Prospective trials are needed to confirm the findings of this exploratory study and to elucidate the reasons of a possible induction of impulsivity by STN DBS.

A systematic review of database validation studies among fertility populations.

STUDY QUESTION: Are routinely collected data from fertility populations adequately validated? SUMMARY ANSWER: Of the 19 studies included, only one validated a national fertility registry and none reported their results in accordance with recommended reporting guidelines for validation studies. WHAT IS KNOWN ALREADY: Routinely collected data, including administrative databases and registries, are excellent sources of data, particularly for reporting, quality assurance, and research. However, these data are subject to misclassification bias due to misdiagnosis or errors in data entry and therefore need to be validated prior to using for clinical or research purposes. STUDY DESIGN SIZE DURATION: We conducted a systematic review by searching Medline, Embase, and CINAHL from inception to 6 October 2016 to identify validation studies of databases that contain routinely collected data in an ART setting. Webpages of international ART centers were also searched. PARTICIPANTS/MATERIALS SETTING METHODS: We included studies that compared at least two data sources to validate ART population data. Key words and MeSH terms were adapted from previous systematic reviews investigating routinely collected data (e.g. administrative databases and registries), measures of validity (including sensitivity, specificity, and predictive value), and ART (including infertility, IVF, advanced reproductive age, and diminished ovarian reserve). Only full-text studies in English were considered. Results were synthesized qualitatively. The electronic search yielded 1074 citations, of which 19 met the inclusion criteria. MAIN RESULTS AND THE ROLE OF CHANCE: Two studies validated a fertility database using medical records; seven studies used an IVF registry to validate vital records or maternal questionnaires, and two studies failed to adequately describe their reference standard. Four studies investigated the validity of mode of conception from birth registries; two studies validated diagnoses or treatments in a fertility database; four studies validated a linkage algorithm between a fertility registry and another administrative database; one study created an algorithm in a single database to identify a patient population. Sensitivity was the most commonly reported measure of validity (12 studies), followed by specificity (9 studies). Only three studies reported four or more measures of validation, and five studies presented CIs for their estimates. The prevalence of the variable in the target population (pre-test prevalence) was reported in seven studies; however, only four of the studies had prevalence estimates from the study population (post-test prevalence) within a 2% range of the pre-test estimate. The post-test estimate was largely discrepant from the pre-test value in two studies. LIMITATIONS REASONS FOR CAUTION: The search strategy was limited to the studies and reports published in English, which may not capture validation studies from countries that do not speak English. Furthermore, only three specific fertility-based diagnostic variables (advanced reproductive age, diminished ovarian reserve, and chorionicity) were searched in Medline, Embase, and CINAHL. Consequently, published studies with other diagnoses or conditions relevant to infertility may not have been captured in our review. WIDER IMPLICATIONS OF THE FINDINGS: There is a paucity of literature on validation of routinely collected data from a fertility population. Furthermore, the prevalence of the markers that have been validated are not being presented, which can lead to biased estimates. Stakeholders rely on these data for monitoring outcomes of treatments and adverse events; therefore, it is essential to ascertain the accuracy of these databases and make the reports publicly available. STUDY FUNDING/COMPETING INTERESTS: This study was supported by Canadian Institutes of Health Research (CIHR) (FDN-148438). There are no competing interests for any of the authors. REGISTRATION NUMBER: International Prospective Register of Systematic Reviews ID: CRD42016048466.

Studies on the control of sodium excretion in experimental uremia.

A study of the mechanisms governing the high rate of sodium excretion per nephron characteristic of patients with chronic renal disease has been made in dogs. A "remnant kidney" was produced by 85% infarction of the left kidney while the right kidney was left intact. A bladder-splitting procedure allowed simultaneous measurement of glomerular filtration rate and the rate of sodium excretion by each kidney. The animals were fed a constant known amount of sodium chloride and 0.1 mg of 9 alpha-fluorohydrocortisone twice daily throughout the study. In a group of dogs fed 3 or 5 g of salt per day, sodium excretion by the remnant kidney averaged 6.5 muEq/min while the intact kidney was present and 53.7 muEq/min when the animals became uremic after the intact kidney was removed. The increased sodium excretion per nephron by the remnant organ often occurred within 18 hr after contralateral nephrectomy and persisted despite experimentally induced acute reductions in the glomerular filtration rate to below prenephrectomy levels. A second group of animals studied in the same manner but receiving 1 g of salt per day or less failed to develop a natriuresis after contralateral nephrectomy despite high grade uremia. Thus an increased impermeable solute load per nephron was not a regulatory factor in the production of the natriuresis. The increased rate of sodium excretion per nephron in uremia resembles that after saline loading in that it may occur without an increase in glomerular filtration rate or a reduction in mineralocorticoid stimulation. It follows that an additional factor or factors must be involved in the genesis of the natriuresis. In contrast to the natriuresis that is seen in normal animals subjected to saline loading, these uremic animals were found not to have a detectable increase in extracellular fluid volume or blood volume in the presence of high fractional sodium excretion rates. Sodium excretion in response to a small salt load by the remnant organ in uremia was 30% greater than the response of both kidneys in the preuremic state despite a markedly reduced total GFR. These data are consistent with the view that the volume control mechanism becomes more responsive in uremia.

On the adaptation in potassium excretion associated with nephron reduction in the dog.

An effort to examine certain aspects of the adaptation in potassium excretion associated with nephron reduction was made in dogs with unilateral remnant kidneys. A constant intake of potassium was maintained by tube feeding and studies were performed before and after removal of the intact control kidney. The removal of the intact kidney created the need for the remaining nephrons of the remnant kidney to increase their rate of potassium excretion markedly. Sodium intake was held constant either at a normal or a low level. Mineralocorticoid hormone activity was maintained either at a high level by the administration of 0.2 mg 9-alpha-fluorohydrocortisone daily or at a low level by performing bilateral adrenalectomy and administering a minimal maintenance dose of deoxycorticosterone acetate (DOCA) and cortisol. Potassium excretion per nephron increased strikingly within 18 hr of contralateral nephrectomy and by 7 days, excretion rates were 600% of control values for the remnant kidney. More potassium was excreted in the first 5 hr after administration of a test dose of potassium by the remnant kidney alone in the postnephrectomy state than by both the remnant and intact kidneys in the prenephrectomy state. 24 hr excretion of potassium by the remnant kidney postnephrectomy averaged 92% of the administered load of potassium. The adaptation in potassium excretion was independent of the concurrent rate of sodium excretion and of mineralocorticoid hormone activity and persisted during constriction of the renal artery, a stimulus which presumably decreased distal delivery of sodium. The adaptation and the continued modulation of potassium excretion could not be explained adequately by an increase in impermeant anion excretion per nephron. Finally, known changes in hydrogen ion excretion per nephron associated with nephron reduction are in a direction opposite to those which would explain the acquired kaliuresis per nephron.

Differences in primary cellular factors influencing the metabolism and distribution of 3,5,3'-L-triiodothyronine and L-thyroxine.

Administration of phenobarbital, which acts exclusively on cellular sites, results in an augmentation of the liver/plasma concentration ratio of L-thyroxine (T4) in rats but no change in the liver/plasma concentration ratio of L-triiodothyronine (T3). Whereas phenobarbital stimulates the fecal clearance rate both of T3 and T4, it increases the deiodinative clearance rate of T4 only. These findings suggest basic differences in the cellular metabolism of T3 and T4. Further evidence pointing to cellular differences was obtained from a comparison of the distribution and metabolism of these hormones with appropriate corrections for the effect of differential plasma binding. The percentage of total exchangeable cellular T4 within the liver (28.5) is significantly greater than the corresponding percentage of exchangeable cellular T3 within this organ (12.3). Extrahepatic tissues bind T3 twice as firmly as T4. The cellular metabolic clearance rate (= free hormone clearance rate) of T3 exceeds that of T4 by a factor 1.8 in the rat. The corresponding ratio in man, 2.4, was determined by noncompartmental analysis of turnover studies in four individuals after the simultaneous injection of T4-(125)I and T3-(131)I. The greater cellular metabolic clearance rate of T3 both in rat and man may be related to the higher specific hormonal potency of this iodothyronine.

Decreased expression of human class II antigens on monocytes from patients with acquired immune deficiency syndrome. Increased expression with interferon-gamma.

The expression of HLA-DR (a class II histocompatibility antigen) on monocytes isolated from the peripheral blood of normal individuals and patients with acquired immune deficiency syndrome (AIDS) was investigated by the use of dual fluorescent staining and cytofluorometry. In animal models the absence of class II positive monocytes is linked to a failure of T cells to respond to antigens. We now report that patients with AIDS have a paucity of HLA-DR+ monocytes. The percentage of HLA-DR+ monocytes among eight normal individuals ranged from 49.3 to 95.0%+, and only one individual had less than 50% HLA-DR+ monocytes. HLA-DR expression on monocytes from homosexual male patients with lymphadenopathy was similar to that of normal subjects (range, 58.0 to 97.4%+). In contrast, seven of nine patients with AIDS had less than 50% HLA-DR+ monocytes (range, 13.4 to 78.8%+). The in vitro incubation of monocytes from AIDS patients with cloned human interferon-gamma resulted in an increase of the expression of HLA-DR to near normal levels.

Growth suppression mediated by transfection of p53 in Hut292DM human lung cancer cells expressing endogenous wild-type p53 protein.

This study was undertaken to analyze the effect of wild-type p53 transfection on the growth potential of a human lung cancer cell line Hut292DM expressing endogenous wild-type p53. Transfection efficiencies obtained with either the wild-type or a mutant p53 complementary DNA revealed a significant decrease in the number of colonies obtained with the wild-type p53 as compared to the mutant p53 complementary DNA (27%) or control vector DNA only (20%), suggesting that wild-type p53 inhibited the growth of Hut292DM cells. A series of wild-type and mutant p53 transfection clones were then analyzed for the presence and expression of the exogenous p53 gene. Polymerase chain reaction amplification revealed that 98% of mutant p53 transfection clones analyzed contained the exogenous p53 gene as opposed to 47% for wild-type p53 clones. The majority of mutant p53 clones expressed high levels of exogenous p53 mRNA and protein as analyzed by Northern and Western blots, respectively. In contrast, all wild-type p53 clones analyzed failed to express exogenous p53 mRNA transcript or protein of a normal size. Aberrant-size p53 mRNA was detected in two wild-type p53 clones (X833.W2 and W18), and Western blot analysis revealed that these clones expressed truncated p53 proteins (M(r) 45,000 and 33,000 respectively). No difference in proliferation rates in vitro or in tumorigenic potential in nude mice were observed between mutant p53 clones or control cell lines. In contrast, a wild-type p53 clone (X833.W2) exhibited a significantly reduced tumorigenic potential in nude mice, whereas its in vitro proliferation rate was comparable to parental Hut292DM cells. The data indicate that exogenous expression of wild-type p53 is incompatible with Hut292DM lung cancer cell proliferation in vitro and suggest that p53-mediated growth control in vitro and in vivo may be dissociated and exerted by separate domains of the p53 protein.

Biological behavior of human breast carcinoma-associated antigens expressed during cellular proliferation.

The cell surface-binding properties of two murine monoclonal antibodies reactive with human mammary tumor cells are described. Fluorescence-activated cell sorter analyses demonstrate that both monoclonal antibodies, B6.2 and B38.1, are reactive with the surface of the majority of human breast tumor cell lines tested but are unreactive with a variety of normal human cell lines, melanomas, sarcomas, and lymphoid tumors. Antibody B6.2 was also reactive with selective carcinomas, while antibody B38.1 showed even broader reactivity. The two monoclonal antibodies were unreactive with the surface of a variety of normal human tissues obtained at biopsy, including lymph nodes, bone marrow, and spleen, but were reactive with mammary tumor cells obtained from four of six pleural effusions. Surface binding to mammary tumor cells by both monoclonal antibodies was shown to decrease during density-dependent arrest; further cell cycle analysis demonstrated differential antibody surface binding during S phase. Prolonged exposure of mammary tumor cells to antibody showed no evidence of antigen capping or internalization. Both monoclonal antibodies were shown to lyse mammary tumor cells in antibody-dependent cell-mediated cytotoxicity.

Escherichia coli mut T1. II. Consequences of modification on the association of DNA with the cell membrane.

1. Two isogenic strains of Escherichia coli, K-12 which differ by mutator gene character (mut T1) have been studied. This characteristic caused introduction of a high frequency of undirectional transversions, A-T leads to -CG, into the DNA of the strain harboring it. 2. It had been previously shown that the presence of this gene is accompanied by an alteration of a cell membrane component. Now, the nuclease susceptibility of DNA associated with membrane/DNA/DNA polymerase complexes is reported. DNA of mut T1 membranes is more sensitive towards exogenous nuclease than DNA of membrane complexes from the wild type mut+ strain. 3. Auto-digestion of this DNA by endogenous nuclease associated with the membrane complex is, also, more severe in preparations derived from mut T1 than from the wild-type strain, mut+, but to a lesser extent than observed with exogenous nucleases. 4. Nuclease susceptibility of mut+ membrane bound DNA is markedly influenced by the growth state of the cell. The nuclease susceptibility of membrane bound DNA from mut T1 cells, however, shows no differences between stationary and log states. 5. These differential sensitivities may be due to conformational changes in the membrane introduced as a pleiotrophic consequence of an altered membrane protein. A pertinent role of this protein in a modified replication/repair complex is an attractive suggestion, especially in the context of the mutator character of this strain.

Consequences of modification on the association of DNA polymerase with the cell membrane.

1. This is a report of a possible causal relation between the structure of the membrane-bound DNA polymerase and the mutator characteristic of Exherichia coli, mut T1. The characteristics of the membrane-bound polymerase are compatible with its identification as DNA polymerase II, and enzyme which has been determined to be genetically closely linked to the mut T1 gene. Several genes concerned with membrane structure are also known to be linked to the mutator locus. 2. Differences exist between membrane-bound polymerase activity of a wild-type E. coli K-12 and of an isogenic strain harboring the mutator gene. (a) The cell membranes of the mutator strain retain 5--6 times as much activity as do membrane complexes from wild-type cells. (b) The DNA polymerase activity of the membranes from the mut T1 strain is less sensitive to inhibition by the sulfhydryl-binding reagent, N-ethylmaleimide. (c) The membrane-DNA polymerase complex of mut T1 cells uses endogenous, membrane-bound DNA for replication-repair in preference to exogenous DNA. 3. The differences described are specific to structural differences in the membrane complex. When DNA polymerase activity is solubilized from the complexes, the enzymes of the two strains exhibit similar characteristics. These results are consistent with the thesis that an alteration in membrane structure is the basis of mut T1 activity. The results do not support any hypothesis that mut T1 phenotype is a reflection of mutations in the structural gene for DNA replicase (polymerase) or its components.

Maintenance of DNA integrity during murine erythroleukemia cell differentiation induced by ethionine and other hypomethylation agents.

The extent of single-strand nicks in DNA from murine erythroleukemia cells induced to differentiate to hemoglobin synthesis in the presence of the hypomethylating agent ethionine was estimated and compared to those levels in uninduced cells and from cells induced to differentiate upon exposure to dimethylsulfoxide or butyrate ion. Although ethionine has been shown to cause more extensive hypomethylation in the DNA of induced cells than that caused by dimethylsulfoxide or butyrate ion, the frequency of detected single-strand breaks in the DNA of uninduced, control cells was not significantly different from that of cells exposed to any of these inducing chemicals. This data indicates that no correlation exists between DNA hypomethylation and DNA single-strand breaks and that unmethylated CpG loci likely do not operate as specific endonuclease recognition sites or as potential origins of transcription in these mammalian cells.

Increased expression of neutrophil and monocyte adhesion molecules LFA-1 and Mac-1 and their ligand ICAM-1 and VLA-4 throughout the acute phase of myocardial infarction: possible implications for leukocyte aggregation and microvascular plugging.

OBJECTIVES: This study sought to evaluate expression of adhesion molecules on neutrophils and monocytes throughout the acute phase of myocardial infarction. BACKGROUND: Neutrophil and monocyte counts increase within days from onset of acute myocardial infarction. Because leukocytes are recruited to the involved myocardial region, we postulated that these activated cells would display an increased expression of adhesion molecules necessary for effective endothelial transmigration. METHODS: We measured the expression of neutrophil and monocyte lymphocyte function associated antigen-1 (LFA-1), Mac-1, very late after activation antigen-4 (VLA-4) and intercellular adhesion molecule-1 (ICAM-1) by flow cytometry throughout the acute phase of acute myocardial infarction in 25 patients and 10 age-matched control subjects. RESULTS: Expression of Mac-1 on neutrophils increased significantly, whereas no expression of VLA-4 and ICAM-1 was detected. The expression of LFA-1, Mac-1, VLA-4 and ICAM-1 on the monocyte cell membrane in patients with an acute myocardial infarction was increased compared with that in control subjects by 22% (on day 7), 67%, 13% and 44% (all on day 4), respectively (all p < 0.001). Elevated density of monocyte-specific CD14 in the AMI versus the control group was also shown (30%, p < 0.001). CONCLUSIONS: Increased expression of neutrophil and monocyte adhesion molecules may contribute to their adhesion to endothelium in the ischemic territory. This adhesion could feasibly precipitate vasoconstriction or add a local thrombotic effect due to tissue factor expression secondary to Mac-1 engagement. In addition, the manifestation of increased density of LFA-1 and Mac-1 by activated leukocytes with monocytes also expressing ICAM-1 suggests that leukocytes may form microaggregates that could cause microvascular plugging. This mechanism may facilitate the occurrence of the "no-reflow" phenomenon or slow coronary filling after acute myocardial infarction.

Rapid ventricular filling in left ventricular hypertrophy: I. Physiologic hypertrophy.

The effects of endurance training on the diastolic properties of the left ventricle were examined by comparing left ventricular filling rates in 11 male distance runners and 12 age-matched nonathletic control subjects selected to have nearly similar heart rates at rest. Maximal oxygen consumption was 69 +/- 11 ml/kg-min for the athletes and 48 +/- 8 ml/kg X min for the control subjects (p less than 0.001). Left ventricular end-diastolic dimension, posterior wall thickness and mass were determined by echocardiography, and average left ventricular filling rate was determined with a nonimaging scintillation probe. Electrocardiographic voltage was significantly greater in the athlete group than in the control group (sums of the voltages of the S wave in lead V1 and the R wave in lead V5 were 40 +/- 10 and 26 +/- 7 mV, respectively) (p less than 0.001), whereas ejection fraction was similar in the two groups. Despite a modest degree of left ventricular hypertrophy in the athlete group compared with the control group (left ventricular mass index 127 +/- 30 and 82 +/- 13 g/m2, respectively) (p less than 0.001), the average left ventricular filling rate was similar in the two groups (2.53 +/- 0.34 versus 2.38 +/- 0.29 end-diastolic counts/s, p = NS). There was no trend for the athletes with a higher left ventricular mass to exhibit a slower filling rate. These findings demonstrate that unlike pathologic hypertrophy associated with chronic hemodynamic over-loading, physiologic left ventricular hypertrophy is not accompanied by slowed left ventricular diastolic filling.

Detecting antibodies with similar reactivity patterns in the HLDA8 blind panel of flow cytometry data.

The blind panel collected for the 8th Human Leucocyte Differentiation Antigens Workshop (HLDA8; ) included 49 antibodies of known CD specificities and 76 antibodies of unknown specificity. We have identified groups of antibodies showing similar patterns of reactivity that need to be investigated by biochemical methods to evaluate whether the antibodies within these groups are reacting with the same molecule. Our approach to data analysis was based on the work of Salganik et al. (in press) [Salganik, M.P., Milford E.L., Hardie D.L., Shaw, S., Wand, M.P., in press. Classifying antibodies using flow cytometry data: class prediction and class discovery. Biometrical Journal].