High temperature aggravates the effects of pesticides in goldfish.

In human-altered rivers, fish are often conjointly exposed to an increase in water temperature due to global warming and to a contamination by organic pollutants such as pesticides, but their combined effects are still elusive. Thermal and chemical stressors could potentially interact because high temperature increases metabolism and toxicant uptake, and can alter the ability of organisms to set up adequate stress responses and to maintain homeostasis. These combined stressors could thus potentially result in higher level of molecular and cellular damage, and stronger effects on behavior and physiology, but experimental evidence across biological levels is still scarce. In this study, goldfish Carassius auratus were experimentally exposed to an environmentally realistic cocktail of pesticides (S-metolachlor, isoproturon, linuron, atrazine-desethyl, aclonifen, pendimethalin and tebuconazol) commonly found in rivers of South-West of France at low or high dose in two different thermal conditions: a common summer temperature (22 °C) or a high temperature recorded during heat waves (32 °C). Results showed that high temperature alone caused behavioral and physiological changes (increased swimming activity, increased hepatosomatic index, decreased reproductive index) but limited cellular damage. However, high temperature aggravated the effects of pesticides at the molecular and cellular level. Indeed, pesticide exposure resulted in higher genotoxic effects (micronuclei rate) and irreversible cellular damage of the gills and liver (apoptosis, inflammation, necrosis) at 32 °C compared to 22 °C. This suggests potential synergistic effects of climate change and pollution, and highlights the need for multiple stress approaches to better predict the impacts of human activities on aquatic wildlife.

A case of bilateral frontal tumors without "frontal syndrome".

We report the longitudinal case study of a right-handed patient harboring two frontal tumors that benefited from bilateral simultaneous surgery. The tumors were WHO Grade II gliomas located in the left inferior frontal area (including the cingulate gyrus) and the right anterior superior frontal gyrus. The double tumor resection was guided by direct electrical stimulation of brain areas while the patient was awake. Neuropsychological assessments were administered before and after the surgery to analyse how the brain functions in the presence of two frontal gliomas that affect both hemispheres and reacts to a bilateral resection, which can brutally compromise the neuronal connectivity, progressively established during the infiltrating process. We showed that both the tumor infiltration and their bilateral resection did not lead to a "frontal syndrome" or a "dysexecutive syndrome" predicted by the localization models. However, a subtle fragility was observed in fine-grain language, memory and emotional skills. This case study reveals the significance of brain plasticity in the reorganization of cognitive networks, even in cases of bilateral tumors. It also confirms the clinical relevance of hodotopical brain models, which considers the brain to be organized in parallel-distributed networks around cortical centers and epicenters.

Genetic evidence for the involvement of DNA ligase IV in the DNA-PK-dependent pathway of non-homologous end joining in mammalian cells.

Cells of vertebrates remove DNA double-strand breaks (DSBs) from their genome predominantly utilizing a fast, DNA-PKcs-dependent form of non-homologous end joining (D-NHEJ). Mutants with inactive DNA-PKcs remove the majority of DNA DSBs utilizing a slow, DNA-PKcs-independent pathway that does not utilize genes of the RAD52 epistasis group, is error-prone and can therefore be classified as a form of NHEJ (termed basic or B-NHEJ). We studied the role of DNA ligase IV in these pathways of NHEJ. Although biochemical studies show physical and functional interactions between the DNA-PKcs/Ku and the DNA ligase IV/Xrcc4 complexes suggesting operation within the same pathway, genetic evidence to support this notion is lacking in mammalian cells. Primary human fibroblasts (180BR) with an inactivating mutation in DNA ligase IV, rejoined DNA DSBs predominantly with slow kinetics similar to those observed in cells deficient in DNA-PKcs, or in wild-type cells treated with wortmannin to inactivate DNA-PK. Treatment of 180BR cells with wortmannin had only a small effect on DNA DSB rejoining and no effect on cell radiosensitivity to killing although it sensitized control cells to 180BR levels. This is consistent with DNA ligase IV functioning as a component of the D-NHEJ, and demonstrates the unperturbed operation of the DNA-PKcs-independent pathway (B-NHEJ) at significantly reduced levels of DNA ligase IV. In vitro, extracts of 180BR cells supported end joining of restriction endonuclease-digested plasmid to the same degree as extracts of control cells when tested at 10 mM Mg(2+). At 0.5 mM Mg(2+), where only DNA ligase IV is expected to retain activity, low levels of end joining ( approximately 10% of 10 mM) were seen in the control but there was no detectable activity in 180BR cells. Antibodies raised against DNA ligase IV did not measurably inhibit end joining at 10 mM Mg(2+) in either cell line. Thus, in contrast to the situation in vivo, end joining in vitro is dominated by pathways with properties similar to B-NHEJ that do not display a strong dependence on DNA ligase IV, with D-NHEJ retaining only a limited contribution. The implications of these observations to studies of NHEJ in vivo and in vitro are discussed.

Down-regulation of DNA replication in extracts of camptothecin-treated cells: activation of an S-phase checkpoint?

Extracts prepared from camptothecin (CPT)-treated cells have a reduced ability to support SV40 DNA replication in vitro. This reduction derives mainly from a reduction in the frequency of initiation events because DNA chain elongation remains practically unchanged. Mixing of extract from nontreated cells with small amounts of extract of CPT-treated cells indicates that the reduction in DNA replication is due to the synthesis/activation of a dominant inhibitor. The observed reduction in DNA replication activity cannot be attributed to inactivation of Topo I, the molecular target of camptothecin, because levels and activity of this protein remain unchanged in extracts of CPT-treated cells and addition of purified Topo I does not restore replication activity. Although replication protein A (RP-A) is phosphorylated in CPT-treated cells, reduced replication may not be caused by RP-A inactivation, because neither loss of phosphorylation nor the addition of recombinant RP-A restore replication activity. We interpret these observations as biochemical evidence for the activation of a checkpoint in S phase and discuss the ramifications of this activation on the mechanism of CPT-induced cytotoxicity.

Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase.

Replication protein A (RPA, also known as human single-stranded DNA-binding protein) is a trimeric, multifunctional protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other DNA-damaging agents, which implicates the modified protein in the regulation of DNA replication after DNA damage or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these kinases and completely abolished in wild-type, ATM, or DNA-PK-deficient cells after treatment with wortmannin at a concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the protein. In an in vitro assay for DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the protein from DNA replication to DNA repair.

The protein composition of reconstituted 30S ribosomal subunits: the effects of single protein omission.

Using reverse phase HPLC, we have been able to quantify the protein compositions of reconstituted 30S ribosomal subunits, formed either with the full complement of 30S proteins in the reconstitution mix or with a single protein omitted. We denote particles formed in the latter case as SPORE (single protein omission reconstitution) particles. An important goal in 30S reconstitution studies is the formation of reconstituted subunits having uniform protein composition, preferably corresponding to one copy of each protein per reconstituted particle. Here we describe procedures involving variation of the protein:rRNA ratio that approach this goal. In SPORE particles the omission of one protein often results in the partial loss in uptake of other proteins. We also describe procedures to increase the uptake of such proteins into SPORE particles, thus enhancing the utility of the SPORE approach in defining the role of specific proteins in 30S structure and function. The losses of proteins other than the omitted protein provide a measure of protein:protein interaction within the 30S subunit. Most of these losses are predictable on the basis of other such measures. However, we do find evidence for several long-range protein:protein interactions (S6:S3, S6:S12, S10:S16, and S6:S4) that have not been described previously.

Mechanisms of DNA double strand break repair and chromosome aberration formation.

It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10-30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2-10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for exchanges through the joining of incorrect ends and cause the formation of chromosome aberrations in wild type and D-NHEJ mutant cells.

Replication protein A as a potential regulator of DNA replication in cells exposed to hyperthermia.

It is well known that exposure of cells to heat leads to a drastic inhibition of DNA synthesis as assayed in vivo by the incorporation of radioactive precursors into acid-insoluble material. Here we introduce an SV40 in vitro DNA replication assay and show that this inhibition may be partly due to the activation of a checkpoint in S phase that stalls the initiation of DNA replication by inactivating replication protein A (RPA), an essential factor for replication. The results implicate trans-acting processes in the regulation of DNA replication after heat exposure and suggest that such processes may be an integral part of the normal response to heat insult. The observations extend and complement previous studies that have implicated heat-induced chromatin damage acting in cis as a cause for the observed inhibition of DNA synthesis in cells exposed to hyperthermia. A model is proposed postulating that the presence of single-stranded DNA, or heat-induced damage to chromatin structures directly, albeit passively, inhibits the elongation stages of ongoing DNA replication. It is hypothesized that arrested replication forks subsequently act as signals to activate the S-phase checkpoint that actively inhibits the initiation of new replicons. The ultimate purpose of this response will be the minimization of the toxic consequences of heat-induced damage, as it may delay DNA replication until chromatin conformation has been restored. DNA replication in the presence of chromatin damage has been implicated in the formation of lethal chromosome aberrations observed in cells heated during S phase. The operation of active processes in the regulation of DNA replication in cells exposed to hyperthermia offers new targets for intervention and sensitization of cells to heat.

In vitro rejoining of DNA double strand breaks: a comparison of genomic-DNA with plasmid-DNA-based assays.

PURPOSE: To evaluate potential similarities between the enzymatic activities required to rejoin DNA double strand breaks (dsb) in an in vitro assay based on genomic DNA and an in vitro assay based on plasmid DNA. Because the latter assay is simpler and faster, it should be preferred for the characterization of repair factors if both assays are found to probe for the same activities. If, however, the enzymatic requirements for dsb rejoining are different between the two assays, both should be used as they are likely to play complementary roles in the characterization of repair factors. MATERIALS AND METHODS: A cell-free assay has been used, developed to study rejoining DNA dsb induced by radiation in 'naked' DNA prepared from agarose embedded cells using an extract of HeLa cells as a source of enzymes. Also employed was an in vitro assay using the ligation of linearized plasmid DNA to model dsb rejoining. RESULTS: Evidence is presented that, under the conditions employed, different sets of activities are involved in the ligation of linearized plasmid DNA and in the rejoining of dsb in 'naked' genomic DNA. Optimal rejoining of dsb induced in genomic DNA is observed with cytoplasmic cell extract at 37 degrees C, whereas optimal ligation of plasmid DNA is observed with nuclear extract at 25 degrees C. Rejoining of dsb in genomic DNA comes to a near halt at 14 degrees C, but plasmid DNA ligation proceeds at significant rates at this temperature. Furthermore, the activities required for the rejoining of dsb induced in genomic DNA are partly stable to heating at 50 degrees C for 1 h, whereas activities required for the ligation of plasmid DNA are completely inactivated by a similar treatment. Both reactions require ATP for optimal performance, and in both, DNA joining is inhibited at high ATP concentrations. CONCLUSIONS: These observations indicate that the plasmid-and the genomic-DNA-based assays probe for, at least partly, different sets of activities and therefore are expected to play complementary roles in the purification and characterization of activities involved in dsb rejoining.

DNA-PK-independent rejoining of DNA double-strand breaks in human cell extracts in vitro.

PURPOSE: To investigate the role of DNA-dependent protein kinase (DNA-PK) in the rejoining of ionizing radiation-induced DNA double-strand breaks (dsb). MATERIALS AND METHODS: This study employed previously described in vitro assays that utilize nuclei or 'naked' DNA prepared from agarose-embedded cells as a substrate and S-HeLa cell extracts as a source of enzymes. Rejoining of dsb in these assays is absolutely dependent on cell extract and it proceeds, under optimal reaction conditions, to an extent similar to that observed in intact cells. Results were confirmed in a plasmid-based assay for in vitro rejoining of dsb. RESULTS: It is shown that concentrations of wortmannin completely inhibiting DNA-PK activity profoundly affect the rejoining of dsb in vivo, but have no effect on dsb rejoining in vitro. Furthermore, fractionation of cell extracts using ammonium sulphate precipitation, generates protein fractions that are able to support dsb rejoining, despite the fact that they do not contain detectable amounts of either DNA-PKcs or Ku80. Efficient rejoining of dsb in vitro is also observed with extracts of MO59J cells that lack DNA-PK activity. Finally, rejoining of dsb remains unaffected by wortmannin in a plasmid-based assay, and is also detectable with extracts of MO59J cells. CONCLUSIONS: These findings are in contrast with genetic studies demonstrating a requirement for DNA-PK activity for efficient rejoining of dsb in vivo. The difference between in vitro and in vivo results may not be attributed to chromatin structure since wortmannin was without an effect when using nuclei as a substrate. It is speculated that the differences between in vivo and in vitro results can be explained either by assuming the operation of multiple pathways in dsb rejoining, some of which do not require DNA-PK, or by postulating a purely regulatory/damage-sensing role for DNA-PK in intact cells but no direct involvement in dsb rejoining.

Review of outcome measurement instruments in Alzheimer's disease drug trials: introduction.

This article introduces a four-part series on outcome scales used in Alzheimer's disease drug trials. First, it discusses the division of scales into four domains: cognition, functional ability/quality of life, behavior/mood, and global. Within each domain, the shortcomings of existing literature reviews are outlined, and the need for a more coherent view of the psychometric properties of the scales is emphasized. Second, the key concepts of reliability, validity, and responsiveness to change are defined and explained. This explanation also provides an overview of the statistical techniques used to assess measurement properties. Finally, the methods used to select the scales for review in the subsequent articles are explained, and each article is briefly introduced.

Review of outcome measurement instruments in Alzheimer's disease drug trials: psychometric properties of global scales.

The use of global outcome measures with strong psychometric properties in Alzheimer's disease (AD) drug trials is encouraged. This article focuses on Clinician Global Impression of Change scales, the Clinical Dementia Rating, and the Global Deterioration Scale to provide (1) a review of psychometric properties, (2) a critique of how these properties are assessed in the literature, and (3) a basis for evaluating, from the standpoint of psychometric properties, the appropriateness of using a given global scale in a drug trial. Reported reliability and validity estimates for the aforementioned scales range from fair to very good, but small sample sizes and/or inappropriate measures of correlation weaken the quality of the evidence. There is also a dearth of published information on responsiveness to change. Researchers planning AD drug trials should consider these issues, along with the interval between test administrations for test-retest reliability, to help select appropriate global outcome measurement instruments.

Review of outcome measurement instruments in Alzheimer's disease drug trials: psychometric properties of functional and quality of life scales.

The psychometric properties of functional and quality of life outcome measures that were used for the purpose of showing changes in antidementia drug trials for Alzheimer's disease are described and critiqued. The seven functional scales reviewed for reliability, validity, and responsiveness to change included the Geriatric Evaluation by Relative's Rating Instrument, the Physical Self-Maintenance Scale, the Instrumental Activities of Daily Living, the Blessed Dementia Scale, Part 1 and its revised version, the Interview for Deterioration in Daily Living with Dementia, the Unified Activities of Daily Living, and the Dependence Scale. The Progressive Deterioration Scale and Quality of Life Assessment were classified as quality of life scales. The majority of the scales were found to exhibit serious limitations, such as incomplete reliability and validity assessment for the intended uses. The most pervasive problem was a lack of data on responsiveness to change. It is recommended that further research be conducted to develop new tools or enhance existing measures for the assessment of both quality of life and functional ability.

Review of outcome measurement instruments in Alzheimer's disease drug trials: psychometric properties of behavior and mood scales.

This article reviews the reliability and validity of eight scales for behavior and mood problems that were identified in a comparative analysis of Alzheimer's disease (AD) drug trials. The scales are the Brief Psychiatric Rating Scale, the Alzheimer's Disease Assessment Scale-noncognitive, the Relative's Assessment of Global Symptomatology, the Consortium to Establish a Registry for Alzheimer's Disease-Behavior Rating Scale for Dementia, the Dementia Behavior Disturbance scale, the Neuropsychiatric Inventory, and two scales for depressive symptoms, the Cornell Scale for Depression in Dementia and the Dementia Mood Assessment Scale. This article also examines methodological limitations in the way the published literature has assessed the psychometric properties of these scales. The aim is to help clinicians and potential trial investigators select appropriate measurement instruments with which to assess behavior and mood problems in AD and to assist AD researchers in the evaluation of the psychometric properties of such scales.

Reliable individual change scores on the 3MS in older persons with dementia: results from the Canadian Study of Health and Aging.

We examined the degree of interrater agreement on the Modified Mini-Mental State Examination (3MS), administered both in the home and at the clinical examination, to determine the boundaries of reliable individual changes for 257 community-dwelling older persons who received a diagnosis of dementia at CSHA-1. Individual score differences were approximately normally distributed (mean of differences 0.2; SD 8.0; 95% confidence interval -16 to 16). The intraclass correlation coefficient was 0.85. Except for the language of testing, there was no relationship between score differences and the determinants investigated (i.e., age, education, type and severity of dementia). This study provides evidence that, in a time frame compatible with no change in cognition, the discrepancy between repeat 3MS scores can be as large as +/- 16. These limits represent the range of variability consistent with no change and should be considered when interpreting individual change scores.

Molecular analysis of the mitochondrial genome of Helianthus annuus in relation to cytoplasmic male sterility and phylogeny.

A circular supercoiled mitochondrial DNA plasmid P1 (1.45 kb) is shown in both normal fertile plants of Helianthus annuus, and some cytoplasmic male sterile lines (CMS A and CMS P). In contrast, no plasmid is found in some other types of CMS C, I, B and K. A circular supercoiled DNA (P2) of higher molecular weight (1.8 kb) is observed in CMS F. The mitochondrial plasmid P1 was cloned, nick-translated and hybridized with native mitochondrial DNA from different lines of male fertile, CMS or wild Helianthus. No sequence homology has been detected between plasmid DNA P1 and high molecular weight mitochondrial DNA in any line examined. A slight hybridization occurs between plasmids P1 and P2. Thus, there is no apparent relationship between mitochondrial plasmid DNA and CMS or Helianthus species. On the contrary, each Helianthus CMS and male fertile strain can be characterized by digestion fragment patterns (Sal I and Bgl I). Analysis of mitochondrial DNA from wild Helianthus strains indicated a relation between some CMS and the strain from which they were maternally derived, as for example CMS I and H. annuus ssp lenticularis and CMS F and H. petiolaris fallax. On the basis of restriction endonuclease patterns, a CMS phylogenic tree is proposed which illustrates a molecular polymorphism in the mitochondrial genome of Helianthus.

Properties of spermatozoa in relation to their elimination after vasectomy.

Sperm disposal and the formation of sperm granulomas are two related critical aspects of vasectomy which have been studied in man and several animal models. Since mammalian spermatozoa present a keratinoid quality, a comparison was made of the resistance of human and rat spermatozoa. When exposed to dithiothreitol and sodium dodecyl sulfate, human sperm decondense readily while rat sperm resist decondensation for long periods of time. A cooperative effect in the rate of decondensation was observed for human sperm but not for rat sperm. Oxidation of sulfhydryl groups renders human spermatozoa as resistant as rat spermatozoa, indicating that disulfide crosslinks are involved in this resistance. Human spermatozoa constitute a heterogeneous population with respect to sulfhydryl group content, suggesting variations in the state of maturation and/or elimination. The sulfhydryl group content of rat epididymal spermatozoa was similar from one cell to another, suggesting little sperm reabsorption in the epididymis. Human and rat spermatozoa differ in size, resistance to decondensing agents, cooperative effect during decondensation, and content and localization of sulfhydryl groups; these differences can explain why vasectomized rats invariably develop huge granulomas while this side effect is not severe in vasectomized men.

PIP: This study compares rat spermatozoa with human spermatozoa in respect to disulfide cross links; sulfhydryl group content; and resistance of the structures to chemical agents. The spermatozoa were decondensed, and whole sperm; sperm heads; and tails were counted in a Neubauer chamber and photographed under phase contrast microscopy. The spermatozoa were incubated; centrifuged; and resuspended in PBS for oxidation of sulfhydryl groups. For autoradiography, the spermatozoa were washed in PBS and exposed to a mixture of a maleimide compound and nonradioactive N-ethylmaleimide. Smears were prepared and the slides were developed with Kodak D19 developer. The autoradiograms were then analyzed for distribution and number of grains over the spermatozoa. Upon exposure to dithiothreitol and sodium dodecyl sulfate, human sperm decondensed readily while the rat sperm resisted decondensation for long periods of time. The human sperm exhibited a cooperative effect in the rate of decondensation but not the rat sperm. Oxidation of sulfhydryl groups rendered human sperm as resistant as rat spermatozoa, suggesting the involvement of the disulfide crosslinks in this resistance. Human spermatozoa are a heterogenous population with respect to sulfhydryl group content, indicating variations in the state of maturation and/or elimination. The differences between human and rat spermatozoa as to size; resistance to decondensing agents; cooperative effect during decondensation; and content and localization of sulfhydryl groups explain why vasectomized rats invariably develop huge granulomas while a small percentage of vasectomized men develop small granulomas.

Cytoplasmic male sterility in sunflower: comparison of molecular biology and genetic studies.

The genetics of male fertility restoration and the RFLP of mitochondrial DNA were studied for 16 sunflower cytoplasms (15 male-sterile and a male-fertile). Male fertility restoration/male sterility maintenance patterns distinguished 12 cytotypes. Four cytoplasms were completely unrestored so they were not distinguished genetically. The sunflower lines, tested for their restorer/maintenance reaction, showed that there was a continuous range between 0% and 100% of restorer genotypes according to the CMS considered. Restoration/maintenance patterns indicated that at least some restorer genes are specific to certain CMS. RFLP of mitochondrial DNA revealed specific differences between the cytotypes studied. Three restriction enzymes and 12 probes permitted distinction of 13 cytotypes. No relationship exists between CMS cytotypes and the species from which they originated. For genetical and mitochondrial RFLP studies, phenograms were constructed according to the similarity indexes between cytotypes. Most of the CMS defined by restoration patterns correspond with a restriction fragment pattern of mitochondrial DNA.

Interrater reliability of the modified Jadad quality scale for systematic reviews of Alzheimer's disease drug trials.

Drug therapies for Alzheimer's disease (AD) have been evaluated in clinical trials over the past 2 decades. Systematic reviews of AD drug trials can shed more light on the efficacy of pharmaceutical interventions. The modified Jadad scale can be used to assess the quality of trial reports that are candidates for inclusion in these systematic reviews. The interrater reliability of the modified Jadad scale was examined during such a review. Three blinded reviewers rated the quality of 42 AD drug trial reports: the intraclass correlation coefficient was 0.90. The modified Jadad scale appears to be a useful tool for AD research because of the very good interrater reliability. Also, it is composed of items that are well suited to the specific disease characteristics of AD. Further research should focus on the validity of this instrument.

Incorporation of dinitrophenyl protein L23 into totally reconstituted Escherichia coli 50 S ribosomal subunits and its localization at two sites by immune electron microscopy.

Escherichia coli ribosomal protein L23 was derivatized with [3H]2, 4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.