Efficient Repair Mechanisms Research Articles

The contribution of DNA repair and antioxidants in determining cell type-specific resistance to oxidative stress

The aims of this study were; (i) to elucidate the mechanisms involved in determining cell type-specific responses to oxidative stress and (ii) to test the hypothesis that cell types which are subjected to high oxidative burdens in vivo, have greater oxidative stress resistance. Cultures of the retinal pigment epithelium (RPE), corneal fibroblasts, alveolar type II epithelium and skin epidermal cells were studied. Cellular sensitivity to H2O2 was determined by the MTT assay. Cellular antioxidant status (CuZnSOD, MnSOD, GPX, CAT) was analyzed with enzymatic assays and the susceptibility and repair capacities of nuclear and mitochondrial genomes were assessed by QPCR. Cell type-specific responses to H2O2 were observed. The RPE had the greatest resistance to oxidative stress (P>0.05; compared to all other cell types) followed by the corneal fibroblasts (P < 0.05; compared to skin and lung cells). The oxidative tolerance of the RPE coincided with greater CuZnSOD, GPX and CAT enzymatic activity (P < 0.05; compared to other cells). The RPE and corneal fibroblasts both had up-regulated nDNA repair post-treatment (P < 0.05; compared to all other cells). In summary, variations in the synergistic interplay between enzymatic antioxidants and nDNA repair have important roles in influencing cell type-specific vulnerability to oxidative stress. Furthermore, cells located in highly oxidizing microenvironments appear to have more efficient oxidative defence and repair mechanisms.

Dynamics of photosystem II: a proteomic approach to thylakoid protein complexes

Oxygenic photosynthesis produces various radicals and active oxygen species with harmful effects on photosystem II (PSII). Such photodamage occurs at all light intensities. Damaged PSII centres, however, do not usually accumulate in the thylakoid membrane due to a rapid and efficient repair mechanism. The excellent design of PSII gives protection to most of the protein components and the damage is most often targeted only to the reaction centre D1 protein. Repair of PSII via turnover of the damaged protein subunits is a complex process involving (i) highly regulated reversible phosphorylation of several PSII core subunits, (ii) monomerization and migration of the PSII core from the grana to the stroma lamellae, (iii) partial disassembly of the PSII core monomer, (iv) highly specific proteolysis of the damaged proteins, and finally (v) a multi-step replacement of the damaged proteins with de novo synthesized copies followed by (vi) the reassembly, dimerization, and photoactivation of the PSII complexes. These processes will shortly be reviewed paying particular attention to the damage, turnover, and assembly of the PSII complex in grana and stroma thylakoids during the photoinhibition-repair cycle of PSII. Moreover, a two-dimensional Blue-native gel map of thylakoid membrane protein complexes, and their modification in the grana and stroma lamellae during a high-light treatment, is presented.

Coordinating stomatal and xylem functioning – an evolutionary perspective

Coordinating stomatal and xylem functioning – an evolutionary perspective

Radiation protection in humans: extending the concept of as low as reasonably achievable (ALARA) from dose to biological damage.

Ionizing radiation has proven to be a double-edged sword since its discovery by Dr Roentgen in 1895. Radiation is a potent mutagen and carcinogen; however, it is also used in the diagnosis and treatment of human diseases. Background radiation consists of cosmic radiation and radiation emitted from radioactive substances present in the ground or commercial sources. Thus, all living organisms have been exposed to background radiation since their appearance on Earth. The appearance of oxygen some 4.5 billion years ago, allowed the appearance of aerobic organisms that used oxygen for their survival. The usage of oxygen generated toxic chemical species, free radicals, as byproducts. The interaction of radiation with oxygen may have caused increased production of free radicals. In order to cope with these adverse conditions, aerobic organisms that survived have antioxidant defence systems to quench excessive levels of free radicals and repair systems that can correct mutagenic changes. Today, humans, well equipped with antioxidant systems (from dietary and endogenous sources) and efficient repair mechanisms, are constantly exposed to varying levels of background radiation, depending upon the region and altitude where they live. In the USA, the average annual effective dose equivalent from natural sources to a member in the population is about 3.0 mSv (2.0 mSv from radon and 1 mSv from cosmic, terrestrial and internal) [1]. Medical exposure, consumer products, occupational, fallout, nuclear fuel cycle and miscellaneous contribute to about 0.6 mSv [1]. Patients can receive medical radiation exposure of varying levels up to 100 mSv, generally about 20 mSv or less, for diagnostic purposes. The use of radiation in medicine has always been rationalized on the basis of risk vs benefit. Owing to the growing use of nuclear energy in the world, especially in developed countries for civilian and military purposes, the concept of risk was applied to all personnel involved with radiation. In order to further safeguard against potential radiation damage, the concept of maximum permissible dose (MPD) was developed for the two major population groups. The annual MPD value for the general population for stochasticeffects, where the probability of the biological effect is proportional to the dose (linear no-threshold effect) is about 50 times less (1 mSv) than that recommended for radiation workers (50 mSv). In the UK, the radiation worker standard is 20 mSv as effective dose [2]. Although the value of MPD for radiation workers for the above effect has remained fairly constant during the last several decades, this value for the US population was recommended to be reduced by a factor of 5 in 1991 [3] and made law in 1993. This is due to the emergence of new data on low doses of radiation on somatic and heritable mutations in mammals and in mammalian cells in culture [4–7], the synergistic effect of the interaction of radiation with other chemical and biological carcinogens and tumour promoters on cancer incidence and mutations, and epidemiological studies in humans. To address the growing concerns of radiation-induced somatic and heritable mutations, the concept of ALARA (as low as reasonably achievable) with respect to dose was recommended by national and international radiation protection agencies for radiation workers [2, 3]. Efforts to protect normal tissues were started soon after the discovery of X-rays. However, the observation by Dr Muller of Columbia University in 1927, that radiation causes gene mutations inDrosophila melanogaster (common fruit fly) provided new impetus to reduce exposures [5]. The initial concept of radiation protection involved three physical principles: (a) shielding (usually by lead) of unexposed areas, especially radiosensitive organs such as bone marrow, gonads and thyroid; (b) increased distance between the radiation source and radiation workers or patients; and (c) reduction of exposure time. Each of these factors has been very useful, but they have limitations. For example, during fluoroscopy, it may not be possible to protect the gastrointestinal tract (one of the most radiosensitive organs) against radiation damage by lead shielding. Increasing the distance between the radiation source and exposed individuals may not be practical for many radiation workers, patients, civilian or military personnel. Reducing exposure time may also not be pertinent to all populations, except those that are involved in taking care of patients who have received gammaemitting radioisotopes for medical purposes or who are responsible for radioactive decontamination as a result of accidents or attack. Nevertheless, radiation protection based on physical factors has served a useful purpose and has been successful in reducing the level of unnecessary medical exposure to patients and to radiation workers. In order to protect normal tissues from potential radiation Received 31 March 2003 and in final form 1 September 2003, accepted 22 September 2003. The British Journal of Radiology, 77 (2004), 97–99 E 2004 The British Institute of Radiology DOI: 10.1259/bjr/88081058

The Methanococcus jannaschiidCTP Deaminase Is a Bifunctional Deaminase and Diphosphatase

Most bacteria produce the dUMP precursor for thymine nucleotide biosynthesis using two enzymes: a dCTP deaminase catalyzes the formation of dUTP and a dUTP diphosphatase catalyzes pyrophosphate release. Although these two hydrolytic enzymes appear to catalyze very different reactions, they are encoded by homologous genes. The hyperthermophilic archaeon Methanococcus jannaschii has two members of this gene family. One gene, at locus MJ1102, encodes a dUTP diphosphatase, which can scavenge deoxyuridine nucleotides that inhibit archaeal DNA polymerases. The second gene, at locus MJ0430, encodes a novel dCTP deaminase that releases dUMP, ammonia, and pyrophosphate. Therefore this enzyme can singly catalyze both steps in dUMP biosynthesis, precluding the formation of free, mutagenic dUTP. Besides differing from the previously characterized Salmonella typhimurium dCTP deaminase in its reaction products, this archaeal enzyme has a higher affinity for dCTP and its steady-state turnover is faster than the bacterial enzyme. Kinetic studies suggest: 1) the archaeal enzyme specifically recognizes dCTP; 2) dCTP deamination and dUTP diphosphatase activities occur independently at the same active site, and 3) both activities depend on Mg(2+). The bifunctional activity of this M. jannaschii enzyme illustrates the evolution of a suprafamily of related enzymes that catalyze mechanistically distinct reactions.

Conditional Targeting of the DNA Repair Enzyme hOGG1 into Mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress.

Overexpression of enzymes that repair endogenous damage to DNA.

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.

SOS mutagenesis results from up-regulation of translesion synthesis

Irradiation of DNA with ultraviolet light generates a variety of photolesions. Among them, are cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts blocking lesions that interfere with DNA replication if left unrepaired. In addition to efficient pre-replicative excision repair mechanisms, cells have evolved damage tolerance pathways enabling them to replicate lesion-containing DNA molecules either by directly replicating through the damaged base (translesion synthesis, TLS) or by employing the locally undamaged complementary strand thus avoiding the lesion (damage avoidance pathways, DA). Using double-stranded vectors with a single T(6-4)T UV lesion and a strand segregation analysis (SSA), we have measured the relative utilization of the two tolerance pathways (TLS and DA) in Escherichia coli. During the SOS response the error-prone TLS pathway is strongly stimulated (≈20-fold) at the expense of the error-free DA pathways. Thus, up-regulation of TLS may turn out to be a general property of the SOS response; a similar conclusion was previously reached with the frameshift-inducing N-2-acetylaminofluorene adduct. Therefore, as far as its contribution to damaged DNA replication is concerned, the SOS response appears to be an induced mutator state rather than a survival strategy. Depending on the base inserted opposite the lesion, TLS can be error-free or mutagenic. In a wild-type strain, both forms of TLS are increased to a similar extent during the SOS response. In contrast, in a Δ umuDC strain induction of TLS is totally abolished, demonstrating that the UmuDC proteins usually thought to be specifically involved in mutagenesis facilitate the recovery of both error-free and mutagenic replication intermediates in vivo.

The adaptive response in radiobiology: evolving insights and implications.

The first of the regularly reproducible experiments to show that very low doses of ionizing radiation, like very low doses of chemical agents, could induce mechanisms whereby cells become better fit to cope with subsequent exposures to high doses were carried out on the induction of chromosome aberrations in cultures of human lymphocytes. If cells that had been exposed to a very low dose (1 cGy) of X rays were subsequently exposed to a relatively high dose (1 Gy), approximately half as many chromosome breaks were induced. Subsequent experiments showed that this adaptive response to low doses requires a certain minimal dose before it becomes active; occurs only within a relatively small window of dose; is dose-rate dependent; and depends on the genetic constitution of the people or animals exposed, with some being unresponsive. It was further shown that the response to the low-dose preexposure was not instantaneous but took approximately 4 to 6 hr to become fully active, and could be prevented if during this period protein synthesis was inhibited, i.e., a necessary protein (enzyme) was being induced. In fact, subsequent experiments with two-dimensional gel electrophoresis showed new proteins in cells irradiated with 1 to 2 cGy. The adaptation induced by low doses of radiation was therefore attributed to the induction of a novel efficient chromosome break repair mechanism that if active at the time of challenge with high doses would lead to less residual damage. This hypothesis was strengthened by a series of experiments in which it was found that inhibitors of poly(ADP-ribose)polymerase, an enzyme implicated in DNA strand break rejoining, could prevent the adaptive response. Although the phenomenon is well established in cellular systems, it is still problematical as to whether or not it will have any utility in establishing risks of ionizing radiation to humans. Newer experiments have now been carried out on the mechanisms underlying the effect and whether or not the effect can manifest itself as a decrease in the number of induced cancers and radiation-induced mortality. Experiments with restriction enzymes now indicate that double-strand breaks in DNA can be triggering events in adaptation. In addition, preliminary experiments on the survival of whole-body irradiated mice have shown that multiple exposures to low adapting doses can have profound effects on survival, and other experiments have shown that adaptation can affect the induction of thymic lymphoma in irradiated mice. It therefore appears that the initial experiments behind the adaptive response have led to a vigorous worldwide effort to understand the basic mechanisms behind it. This effort is stimulated both by a desire to understand the basic cell biology behind the response and a desire to see if indeed this phenomenon affects the estimation of risks of low-level radiation exposure.

Radioadaptive response: Efficient repair of radiation-induced DNA damage in adapted cells

To verify the hypothesis that the induction of a novel, efficient repair mechanism for chromosomal DNA breaks may be involved in the radioadaptive response, the repair kinetics of DNA damage has been studied in cultured Chinese hamster V79 cells with single-cell gel electrophoresis. The cells were adapted by priming exposure with 5 cGy of γ-rays and 4-h incubation at 37°C. There were no indication of any difference in the initial yields of DNA double-strand breaks induced by challenging doses from non-adapted cells and from adapted cells. The rejoining of DNA double-strand breaks was monitored over 120 min after the adapted cells were challenged with 5 or 1.5 Gy, doses at the same level to those used in the cytogenetical adaptive response. The rate of DNA damage repair in adapted cells was higher than that in non-adapted cells, and the residual damage was less in adapted cells than in non-adapted cells. These results indicate that the radioadaptive response may result from the induction of a novel, efficient DNA repair mechanism which leads to less residual damage, but not from the induction of protective functions that reduce the initial DNA damage.

Beta-cell defence and repair mechanisms in human pancreatic islets.

In vivo and in vitro data obtained in rodents indicate that beta-cells can trigger efficient repair mechanisms following non-lethal injury. Recent observations suggest that human pancreatic islets are more resistant than rodent islets to damage by alkylating agents, free oxygen radicals, nitric oxide and cytokines. This increased resistance to injury is associated with higher expression of heat shock protein 70, catalase and superoxide dismutase. These findings emphasise the potential relevance of beta-cell repair and/or defence mechanisms in the development of human IDDM.

A double-strand break in a herpesvirus genome stimulates targeted homologous recombination with exogenous, cloned viral sequences.

A method is described for the highly efficient recovery of recombinant pseudorabies virions; the approach should be applicable to other herpesviruses. Pseudorabies virus (PRV) strain PRV509 contains a unique EcoRI site in its genome, largely replacing the glycoprotein gC gene. By digesting PRV509 DNA with EcoRI prior to cotransfection with plasmid DNA that harbored a cloned copy of gC, we isolated recombinant viruses containing the cloned gC allele at a frequency exceeding 75%. This represented u to a 37-fold increase over the use of intact viral DNA in cotransfection experiments, and may eliminate the need for phenotypic screening of recombinants. Closer analysis of the recombinant viruses revealed that genetic markers up to 1 kilobase pair apart could be recombined into the genome using the EcoRI-digested DNA. Overall, the increased frequency of recombinant viruses can be explained if homologous recombination at sites of double-strand breakage is a more efficient repair mechanism than the re-annealing and ligation of the break itself.

Enhancement of the uvrA gene dosage reduces pyrimidine dimer excision in UV-irradiated Escherichia coli

E. coli possesses an efficient repair mechanism able to remove pyrimidine dimers from UV-irradiated DNA, which is catalyzed by UvrABC endonuclease. In E. coli B/r Hcr + cells transformed with a multicopy plasmid harboring a gene coding for UvrA, the excision capacity was greatly reduced. The course of thymine dimer excision was investigated using the enzymatic as well as the radiochromatographic method and the results are discussed in term of nonspecific interaction between the excess of UvrA protein and undamaged DNA duplex.

P-element-induced interallelic gene conversion of insertions and deletions in Drosophila melanogaster.

We studied the process by which whd, a P-element insertion allele of the Drosophila melanogaster white locus, is replaced by its homolog in the presence of transposase. These events are interpreted as the result of double-strand gap repair following excision of the P transposon in whd. We used a series of alleles derived from whd through P-element mobility as templates for this repair. One group of alleles, referred to collectively as whd-F, carried fragments of the P element that had lost some of the sequences needed in cis for mobility. The other group, whd-D, had lost all of the P insert and had some of the flanking DNA from white deleted. The average replacement frequencies were 43% for whd-F alleles and 7% for the whd-D alleles. Some of the former were converted at frequencies exceeding 50%. Our data suggest that the high conversion frequencies for the whd-F templates can be attributed at least in part to an elevated efficiency of repair of unexpanded gaps that is possibly caused by the closer match between whd-F sequences and the unexpanded gap endpoints. In addition, we found that the gene substitutions were almost exclusively in the direction of whd being replaced by the whd-F or whd-D allele rather than the reverse. The template alleles were usually unaltered in the process. This asymmetry implies that the conversion process is unidirectional and that the P fragments are not good substrates for P-element transposase. Our results help elucidate a highly efficient double-strand gap repair mechanism in D. melanogaster that can also be used for gene replacement procedures involving insertions and deletions. They also help explain the rapid spread of P elements in populations.

P-element-induced interallelic gene conversion of insertions and deletions in Drosophila melanogaster.

We studied the process by which whd, a P-element insertion allele of the Drosophila melanogaster white locus, is replaced by its homolog in the presence of transposase. These events are interpreted as the result of double-strand gap repair following excision of the P transposon in whd. We used a series of alleles derived from whd through P-element mobility as templates for this repair. One group of alleles, referred to collectively as whd-F, carried fragments of the P element that had lost some of the sequences needed in cis for mobility. The other group, whd-D, had lost all of the P insert and had some of the flanking DNA from white deleted. The average replacement frequencies were 43% for whd-F alleles and 7% for the whd-D alleles. Some of the former were converted at frequencies exceeding 50%. Our data suggest that the high conversion frequencies for the whd-F templates can be attributed at least in part to an elevated efficiency of repair of unexpanded gaps that is possibly caused by the closer match between whd-F sequences and the unexpanded gap endpoints. In addition, we found that the gene substitutions were almost exclusively in the direction of whd being replaced by the whd-F or whd-D allele rather than the reverse. The template alleles were usually unaltered in the process. This asymmetry implies that the conversion process is unidirectional and that the P fragments are not good substrates for P-element transposase. Our results help elucidate a highly efficient double-strand gap repair mechanism in D. melanogaster that can also be used for gene replacement procedures involving insertions and deletions. They also help explain the rapid spread of P elements in populations.

Organophosphate polyneuropathy in chicks

Young animals are resistant to organophosphate-induced delayed neuropathy (OPIDP), although biochemical changes on Neuropathy Target Esterase (NTE) caused by neuropathic organophosphorus esters (OP) are similar to those observed in the sensitive hen. We report here that the resistance of chicks to single doses of neuropathic OPs is not absolute because ataxia was produced in 40-day-old chicks by 2,2-dichlorovinyl dibutyl phosphate (DBDCVP, 5.0 or 10.0 mg/kg s.c.) and by diisopropyl phosphorofluoridate (DFP, 2.0 mg/kg s.c.). However, the clinical picture was different from that usually seen in hens; spasticity and complete recovery being the main features. α-Tolyl sulphonyl fluoride (PMSF, 300 mg/kg s.c.) promoted both DBDCVP neuropathy (5.0 or 10.0 mg/kg s.c.) and non-neuropathic doses of DFP (1.5 mg/kg s.c.) or DBDCVP (1.0 mg/kg s.c.). The lowest promoting dose of PMSF given 24 hr after 1.5 mg/kg of DFP was 30 mg/kg. Higher doses had a more severe effect but no further increase of OPIDP severity was obtained with doses ranging from 90 to 300 mg/kg. PMSF (30 mg/kg) protected 40-day-old chicks from subsequent doses of neuropathic OPs even when a promoting dose of PMSF followed. At 60 days of age, chicks' resistance to OPIDP decreased because lower doses of neuropathic OPs became effective and, similarly to hens, PMSF did not fully protect from subsequent promotion. In 40-day-old chicks the threshold of NTE inhibition for OPIDP development was 95–97% (DBDCVP 5.0 mg/kg). When promotion followed initiation, the minimal effective inhibition of NTE for initiation by neuropathic OPs was about 90%. In 36-day-old chicks, PMSF (300 mg/kg) promoted OPIDP when given up to 5 days after DFP (1.5 mg/kg) when residual NTE inhibition in brain and sciatic nerve was about 40%. We conclude that chicks' resistance to OPIDP might reflect either a less effective initiation by phosphorylated NTE or a more efficient repair mechanism or both, and also that promotion is likely to involve a target other than NTE.

Response to Commentary by D. Billen

In a recent Commentary (1), Billen, considering the deleterious effects of ionizing radiation on cells, equates damage produced by this agent on cellular DNA with that which occurs naturally by hydrolysis (2) and by oxidation resulting from metabolism (3). From these considerations Billen concludes that there is a negligible dose of ionizing radiation. This conclusion is not warranted when the quality of the damage induced by radiation is taken into account. There are fundamental differences between the nature of the damage produced by ionizing radiation and that caused by the endogenous processes. In previous publications we have pointed out these differences (4, 5). That work can be summarized: At equal levels of total amounts of DNA damage, hydrolysis (6) or oxidation products (7) are several orders of magnitude less biologically effective than ionizing radiation damage. We assign this difference in effectiveness to the double-strand breaks (DSBs) and locally multiply damaged sites (LMDS) which are induced in DNA by ionizing radiation (8). The damage introduced by hydrolysis (loss of purines, pyrimidines, and amino groups from bases) and oxidation (base alterations and introduction of single-strand breaks (SSBs)) is part of the cell's evolutionary experience. It is not surprising, therefore, that cells have developed several efficient and accurate repair mechanisms for handling these types of damage. DNA DSBs, on the other hand, as Billen points out (1), have not been detected as a result of endogenous processes so that repair of these lesions may not have evolved (an ancillary question here is: Is there a repair gene specific for DSB?). The DSBs produced by background radiation are formed infrequently: At a typical background dose rate of 0.1 cGy per year the average mammalian cell experiences a DNA DSB once every 25 years. It is questionable whether this frequency would be sufficient to educe a repair mechanism. In our comparisons of oxidative damage produced by hydrogen peroxide and ionizing radiation, the special nature of the DNA DSBs induced by the latter agent has been described. We pointed out (9) the ease with which singly damaged sites can be enzymatically repaired and contrasted this facility with the problems facing a cell in its attempts to repair DNA DSBs accurately and efficiently. From these arguments, we would challenge Billen's equating, on purely quantitative grounds, DNA damage produced by ionizing radiation with that caused by natural processes. The quality of the DNA damage is a major aspect which must be taken into account.

Age sensitivity to organophosphate-induced delayed polyneuropathy: Biochemical and toxicological studies in developing chicks

Young animals are resistant to organophosphate-induced delayed polyneuropathy (OPIDP). The putative target protein in the nervous system for initiation of OPIDP in the adult hen is an enzyme called Neuropathy Target Esterase (NTE), which is dissected by selective inhibitors among nervous tissue esterases hydrolysing phenyl valerate (PV). We report here that the pool of PV-esterases sensitive to paraoxon was different in peripheral nerves of chicks as compared to that of hens while that of brain and spinal cord was not. NTE activity decreased with age in brain, spinal cord and peripheral nerve, but its sensitivity to several inhibitors remained unchanged. In the adult hen more than 70% inhibition of peripheral nerve NTE by neuropathic OPs is followed by deficit of retrograde axonal transport, axonal degeneration and paralysis. Similar NTE inhibition in 40-day-old or younger chicks however is not followed by changes in retrograde axonal transport nor by OPIDP. Chicks aged 60 to 80 days are only marginally sensitive to a single dose of DFP otherwise clearly neuropathic to hens. In vitro and in vivo phosphorylation by DFP and subsequent aging of brain NTE is similar both in chicks and in hens. The recovery of NTE activity monitored in vivo after inhibition by DFP is faster (half-life of about 3 days) in chick peripheral nerves as compared to chick brain, hen brain and hen peripheral nerve (half-life of about 5 days). It is concluded that the reduced sensitivity to OPIDP in chicks is not due to differences in OP-NTE interactions. The resistance might be explained by a more efficient repair mechanism, as suggested by the faster recovery of peripheral nerve NTE activity.

3 Pathophysiology of aplastic anaemia

No single pathophysiological phenomenon--neither the intrinsic defect of haemopoiesis nor any of the described immune effects--explains aplastic anaemia. Since the intrinsic defect is compatible with near normal haemopoietic function, as seen in autologous bone marrow reconstitution, it cannot be the cause of severe pancytopenia. On the other hand, immune mechanisms cannot be the primary cause of the disease, otherwise haemopoietic function would recover to complete normality after immunosuppressive therapy. From these observations we deduce that the intrinsic defect, a premalignant haemopoietic disorder, can either be clinically quiescent by virtue of repair mechanisms, or induce auto-reactivity of the immune system against the abnormal haemopoietic tissue, drugs, chemicals and viruses acting as non-specific triggers or amplifiers. In this sense, aplastic anaemia could be interpreted as an attempt to 'self-cure' from a variant type of preleukaemia. This means that the original concept of aplastic anaemia being a hypoplastic variant of leukaemia may be true. The fact that aplastic anaemia can present either as acute severe bone marrow failure, as chronic mild pancytopenia or as a myelodysplasia-like syndrome does not imply that the underlying pathophysiological mechanisms are basically different. Variations of the clinical course and the response to immunosuppressive treatment could be explained by variations in the balance between the primary defect and the secondary immune reaction; the co-involvement of accessory cells in the primary disease; the relative time course of the two components and the efficiency of repair mechanisms. From repeated in vitro studies in a large group of aplastic anaemia patients at various stages of disease this concept can be applied to the majority of cases, including chloramphenicol- and virus-induced aplastic anaemia. In a small proportion of patients with pancytopenia occurring after exposure to certain drugs other than chloramphenicol, aplastic anaemia is rapidly and completely reversible after withdrawal of the drug. These patients probably have truly benign aplastic anaemia and thus differ from the majority of patients who are left with a permanently fragile bone marrow once they have acquired aplastic anaemia.

Repair replication in ultraviolet light-irradiated protoplasts of Daucus carota

Colony-forming ability and DNA replication in protoplasts isolated from suspension cultures of the wild carrot Daucus carota were found to be moderately resistant to ultraviolet irradiation. 37% survival was achieved after a dose of 100 J/m2, while 50% inhibition of replication occurred at a dose of 80 J/m2. Analysis of the thymine dimer content of DNA from irradiated protoplasts after dark incubation confirmed the existence of an efficient excision repair mechanism. A maximum rate of excision of 25 000 dimers/cell per h was achieved at a dose of 100 J/m2. A novel method for inhibiting DNA replication with the fungal metabolite compactin allowed the quantitation of repair replication in protoplasts isolated from dividing cells by chromatography of the DNA on BND-cellulose. Ultraviolet light-stimulated incorporation of [3H]thymidine was demonstrated and a patch size of 70 nucleotides calculated. The properties of the excision repair mechanism of Daucus protoplasts are therefore similar to those of mammalian systems. However, excision alone is insufficient to account for the removal of all dimers induced by natural sunlight.