Doxorubicin-induced p53 interferes with mitophagy in cardiac fibroblasts.

Anthracyclines are the critical component in a majority of pediatric chemotherapy regimens due to their broad anticancer efficacy. Unfortunately, the vast majority of long-term childhood cancer survivors will develop a chronic health condition caused by their successful treatments and severe cardiac disease is a common life-threatening outcome that is unequivocally linked to previous anthracycline exposure. The intricacies of how anthracyclines such as doxorubicin, damage the heart and initiate a disease process that progresses over multiple decades is not fully understood. One area left largely unstudied is the role of the cardiac fibroblast, a key cell type in cardiac maturation and injury response. In this study, we demonstrate the effect of doxorubicin on cardiac fibroblast function in the presence and absence of the critical DNA damage response protein p53. In wildtype cardiac fibroblasts, doxorubicin-induced damage correlated with decreased proliferation and migration, cell cycle arrest, and a dilated cardiomyopathy gene expression profile. Interestingly, these doxorubicin-induced changes were completely or partially restored in p53-/- cardiac fibroblasts. Moreover, in wildtype cardiac fibroblasts, doxorubicin produced DNA damage and mitochondrial dysfunction, both of which are well-characterized cell stress responses induced by cytotoxic chemotherapy and varied forms of heart injury. A 3-fold increase in p53 (p = 0.004) prevented the completion of mitophagy (p = 0.032) through sequestration of Parkin. Interactions between p53 and Parkin increased in doxorubicin-treated cardiac fibroblasts (p = 0.0003). Finally, Parkin was unable to localize to the mitochondria in wildtype cardiac fibroblasts, but mitochondrial localization was restored in p53-/- cardiac fibroblasts. These findings strongly suggest that cardiac fibroblasts are an important myocardial cell type that merits further study in the context of doxorubicin treatment. A more robust knowledge of the role cardiac fibroblasts play in the development of doxorubicin-induced cardiotoxicity will lead to novel clinical strategies that will improve the quality of life of cancer survivors.

Oral toxicity of boric acid and other boron compounds to immature cat fleas (Siphonaptera: Pulicidae).

Oral toxicity was characterized in first-instar cat fleas, Ctenocephalides felis felis (Bouché), feeding on dried blood treated with boric acid. LC50 values ranged from 2.11% after 24 h to 0.21% after 7 d. In carpet tests with five different boron compounds and a number of different formulations, significant mortality for first instars was observed in all cases. In similar tests with prepupae and cocoons, there was no significant effect on mortality. The importance of these results is discussed in light of current application procedures for boron compounds, and suggestions are made for future research.

Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

Close association of a DNA replication origin and an ARS element on chromosome III of the yeast, Saccharomyces cerevisiae.

Two dimensional gel electrophoretic techniques were used to locate all functional DNA replication origins in a 22.5 kb stretch of yeast chromosome III. Only one origin was detected, and that origin is located within several hundred bp of an ARS element.

The in vivo replication origin of the yeast 2 microns plasmid.

We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.

Neonatal screening for sickle cell diseases in Camberwell: results and recommendations of a two year pilot study.

The sickle cell diseases are a major health problem for Afro-Caribbean peoples. Neonatal detection and prophylactic management can reduce mortality and morbidity in childhood. A study was therefore conducted analysing the results of the first two years of cord blood screening in the Camberwell health area. Thirteen cases of sickle cell disease and two of haemoglobin (Hb)C disease were identified among 2202 non-white infants screened. The carrier state, sickle cell trait (HbAS), was present in 11.9% and HbC trait (HbAC) in 4.1% of Afro-Caribbean infants. The incidence of disease and of carrier states was much higher in West Africans than in Caribbeans. The wider implications of screening and the need for a comprehensive plan of care are emphasised.

Normal blood cell values in the early mid-trimester fetus.

Samples of pure fetal blood from 116 fetuses of 15-21 weeks' gestation were obtained by direct vision fetoscopy. Ninety nine of these fetuses, presumed to be haematologically normal, were suitable for analysis. The data obtained show that the erythropoietic system is evolving rapidly in this gestational age range. The myeloid series shows no significant increase or decrease in numbers apart from eosinophils and basophils which increase significantly with gestational age whereas the platelet count remains constant. The growing application of fetoscopic blood sampling to the prenatal diagnosis and management of fetal blood disorders renders mandatory a knowledge of normal fetal blood values.

Experimental parvoviral infection in humans.

Healthy adult volunteers were inoculated intranasally with human parvovirus obtained from an asymptomatic blood donor. One week after inoculation, intense viremia was observed in seronegative volunteers, accompanied by a mild illness with pyrexia, malaise, myalgia, itching, and excretion of virus from the respiratory tract. In the following week hematologic studies revealed reticulocytopenia with an associated slight drop in hemoglobin concentration, lymphopenia, neutropenia, and a drop in platelet counts. At 17-18 days after inoculation a second-phase illness with rash and arthralgia lasting three to four days occurred in three of four infected volunteers. This study confirms the etiologic role of human parvovirus in erythematous rash illness, with the second-phase illness being consistent with adult cases of erythema infectiosum. Moreover, the hematologic changes associated with infection support the hypothesis that the same virus is responsible for the temporary arrest of erythropoiesis that leads to aplastic crisis in persons with chronic hemolytic anemia.

Occurrence of infection with a parvovirus-like agent in children with sickle cell anaemia during a two-year period.

The occurrence of infection with a parvovirus-like agent during the period April 1979-May 1981 in children attending a single sickle cell clinic in London was investigated. Virus was detected in serum by counter-current immunoelectrophoresis (CIE) and immunoelectron microscopy (IEM). Viral antibody was detected by CIE and specific IgM antibody by an IgM-antibody capture assay. Of the 68 children studied nine presented in aplastic crisis and evidence of infection with the parvovirus-like agent at the time of the crisis was found in all nine. Eighteen of the other children were antibody-positive at some time during the study. In 11 children there was no evidence of recent infection; however, two of these had a history of aplastic crisis in previous years. The other seven seroconverted during the course of the study but did not show any haematological effects. Five of these had a primary infection, one appeared to have reinfection and in the seventh there were insufficient data to distinguish between the two. Possible explanations for the difference between those presenting with aplastic crisis and those with asymptomatic seroconversion are discussed.

Both parental deoxyribonucleic acid strands at each replication fork of replicating simian virus 40 chromosomes are cut by a single-strand-specific endonuclease.

We have measured the relative accessibility to a single-strand-specific endonuclease of the single-stranded DNA on the leading and lagging sides of replication forks in replicating simian virus 40 (SV40) chromosomes. To do this we have digested replicating SV40 chromosomes with a single-strand-specific endonuclease (P1 nuclease) and then characterized the intermediate and final products of digestion by sucrose gradient sedimentation and agarose gel electrophoresis. P1 nuclease rapidly and specifically cleaves parental DNA strands at replication forks, yielding intermediate and final cleavage products which are consistent with an approximately equal rate of nuclease cleavage on both sides of the fork. Thus, single-stranded DNA is approximately as accessible to P1 nuclease on the leading side of the fork as on the lagging side; the simplest interpretation of this observation is that the stretch of single-stranded DNA on the leading side is as long as that on the lagging side.

Defective yeast opsonisation and functional deficiency of complement in sickle cell disease.

Opsonisation of heat-killed baker's yeast, functional activity of the total alternative pathway of complement, and factor B detected functionally and immunochemically were significantly reduced in 72 children with sickle cell disease compared with 40 age-matched black control children. There was significant correlation between functional activity of the total alternative pathway and functionally measured factor B, but not between factor B measured functionally and immunochemically. The opsonisation defect could be corrected in vitro by normal serum, and factor B-depleted serum, and was qualitatively similar to that seen in patients with primary yeast opsonisation deficiency. Serial studies showed that these serum defects were persistent. Reduction in the activity of components of the alternative pathway of complement and opsonisation was found in 4 patients who had recovered from pneumococcal meningitis and in one who developed osteomyelitis. Defects of yeast opsonisation and complement which are common in patients with sickle cell disease, may partly explain the children's increased susceptibility to infection, and might help to identify individuals especially at risk.

The development and use of an antibody capture radioimmunoassay for specific IgM to a human parvovirus-like agent.

An IgM-antibody capture radioimmunoassay (MACRIA) was developed for the detection of IgM antibody specific for the human parvovirus-like agent B19. Diagnosis of infection with this agent by either antigen detection or antibody seroconversion had been made by counter-current immunoelectrophoresis (CIE) in 18 cases of aplastic crisis occurring in children with homozygous sickle-cell disease. The MACRIA described here gave positive results in 17 of these 18 cases; in the remaining case only an acute specimen taken from the patient during viraemia and late convalescent specimens taken 184 and 247 days after onset of illness were available. The test was used to investigate 20 further cases of aplastic crisis in which neither viral antigen nor antibody seroconversion could be detected by CIE. Detection of virus-specific IgM permitted diagnosis of infection with this parvovirus-like agent in 17 of these cases. In the remaining three cases only single serum specimens taken late in convalescence, 82 days or more after the onset of symptoms, were available. In addition to these 34 cases of aplastic crisis in which primary infection with this agent was diagnosed by MACRIA, seven cases of apparent 'silent' infection detected by CIE were investigated. The test permitted the discrimination between primary infection and re-exposure to the virus in six of these patients. The use of this assay has added a considerable weight of evidence implicating primary infection with this parvovirus-like agent as an important cause of aplastic crisis in children with sickle-cell disease. Furthermore, MACRIA permits diagnosis of infection when only single serum specimens taken up to ten weeks after infection are available. Thus the use of this test will significantly facilitate the investigation of other clinical syndromes of presumptive infectious aetiology.

Mate-seeking behavior and reduced mating byEphestia cautella (Walker) in a sex pheromone-permeated atmosphere.

The mating rate ofEphestia cautella (Walker) was inversely related to the level of pheromone permeating the air. However, permeation did not prevent mating by altering the number of females calling or the frequency, duration, or pattern of male flight. Instead, permeation probably prevented mating by reducing the distance from which a male can respond to a female. The pheromone level necessary to achieve a particular reduction in mating increases with moth density.

Survey of sickle-cell disease in England and Wales.

The incidence and the clinical course of patients suffering from sickle-cell syndrome (Hb SS; Hb SC; Hb S thal) in England and Wales are not known. In 1979 an ad hoc committee was formed to investigate these problems. Initially, a questionnaire was sent to 227 haematologists in England and Wales to determine the number of cases in these countries. The replies have indicated that 1367 cases were seen in 1978 and 1979. Probably this may represent only half the total number of cases. From this survey it has been possible to draw up a composite map showing the location of patients, which has provided a basis to determine the clinical course of the disease, and for further studies into the complications and management of sickle-cell disease in England and Wales. From a second questionnaire preliminary data about the general management and mortality in England and Wales have been recorded.

Admissions to hospital of children with sickle-cell anaemia: a study in south London.

Admissions to hospital of 171 children with sickle-cell anaemia, genotype Hb SS, were reviewed over a 20-year period. Altogether 887 admissions occurred in 797 patient-years. The commonest cause of admission was painful vaso-occlusive crisis. Appreciable morbidity also resulted from pulmonary disease, infection, and anaemic episodes. The complications resulting in the most severe illness were acute splenic sequestration, pneumococcal meningitis, and some episodes of erythroid hypoplasia resulting in very low haemoglobin concentrations. Most deaths occurred in children aged under 5. Mortality and morbidity could be reduced by measures including prophylaxis of pneumococcal infections and more active treatment of seemingly minor illness in children with sickle-cell anaemia.

Copper chelate affinity chromatography of human fibroblast and leucocyte interferons.

Human fibroblast and human leucocyte interferons display a strong affinity for the copper chelate of bis-carboxymethyl amino agarose, binding tenaciously over a wide pH range (7.4 to 4.0). Their binding is apparently irreversible on a sorbent saturated with copper (24.8 mumol of Cu2+/ml of column bed). However, both interferons can be partially recovered from sorbents of lower copper content, prepared by leaching the columns with sodium citrate at pH 9.0. The recovery of fibroblast interferon from a leached sorbent (5.8 mumol of Cu2+/ml of column bed) is about 30% and that of leucocyte interferon about 60%. Moreover, the strength of binding of leucocyte interferon can be modulated by leaching copper chelate-agarose with citrate of varying concentration.