The effect of Msh2 knockdown on toxicity induced by tert-butyl-hydroperoxide, potassium bromate, and hydrogen peroxide in base excision repair proficient and deficient cells.

The DNA mismatch repair (MMR) and base excision repair (BER) systems are important determinants of cellular toxicity following exposure to agents that cause oxidative DNA damage. To examine the interactions between these different repair systems, we examined whether toxicity, induced by t-BOOH and KBrO3, differs in BER proficient (Mpg (+/+), Nth1 (+/+)) and deficient (Mpg (-/-), Nth1 (-/-)) mouse embryonic fibroblasts (MEFs) following Msh2 knockdown of between 79 and 88% using an shRNA expression vector. Msh2 knockdown in Nth1 (+/+) cells had no effect on t-BOOH and KBrO3 induced toxicity as assessed by an MTT assay; knockdown in Nth1 (-/-) cells resulted in increased resistance to t-BOOH and KBrO3, a result consistent with Nth1 removing oxidised pyrimidines. Msh2 knockdown in Mpg (+/+) cells had no effect on t-BOOH toxicity but increased resistance to KBrO3; in Mpg (-/-) cells, Msh2 knockdown increased cellular sensitivity to KBrO3 but increased resistance to t-BOOH, suggesting a role for Mpg in removing DNA damage induced by these agents. MSH2 dependent and independent pathways then determine cellular toxicity induced by oxidising agents. A complex interaction between MMR and BER repair systems, that is, exposure dependent, also exists to determine cellular toxicity.

Phosphonoacetate biosynthesis: in vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of the marine Roseovarius nubinhibens ISM.

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.

Alkylation of sperm DNA is associated with male factor infertility and a reduction in the proportion of oocytes fertilised during assisted reproduction.

Approximately one-third of IVF cases in the UK are attributed to male factor infertility and in the majority of cases the origin of male infertility is unknown. The integrity of sperm DNA is important both for the success of assisted reproduction and the implications for the off-spring. One type of DNA damage that has not been investigated with respect to fertility outcomes is the adduct N7-methyldeoxyguanosine (N7-MedG), a biomarker for exposure to alkylating agents. A prospective cohort of couples attending for IVF had their N7-MedG levels in sperm measured using an immunoslot blot technique to examine whether sperm N7-MedG levels are associated with male factor infertility, semen quality measures or assisted reproduction outcomes. Sufficient DNA for analysis was obtained from 67/97 couples and N7-MedG was detected in 94% of sperm samples analysed. Men diagnosed with male factor infertility had significantly higher mean levels of N7-MedG in their sperm DNA (P=0.03). Logistic regression analysis showed that N7-MedG levels were significantly negatively associated with the proportion of oocytes successfully fertilised irrespective of the method of fertilisation used (IVF or intra-cytoplasmic sperm injection; ICSI, P<0.001). Therefore exposure to DNA alkylating agents is significantly associated with male infertility and the proportion of oocytes fertilised during assisted reproduction. Reducing such exposure may improve male fertility but further work is required to determine the relative importance of exogenous and endogenous sources of exposure.

The effect of Msh2 knockdown on methylating agent induced toxicity in DNA glycosylase deficient cells.

The DNA structure recognition protein MSH2 is an important protein in DNA mismatch repair due to its role in initiating the repair process. To examine the potential interactions between mismatch repair and base excision repair (BER) we have examined the effect of MSH2 knockdown on 6-thioguanine (6-TG), temozolomide (TMZ) and methylmethane sulphonate (MMS) induced toxicity in BER proficient and deficient cell lines. An shRNA expression vector containing Msh2 target sequences was designed and used to transfect mouse embryonic fibroblasts lacking either alkylpurine DNA N-glycosylase (Mpg) or endonuclease III homologue (Nth1). Significant knockdown of Msh2 gene expression was achieved with three different target sequences, with the highest level being shown by Msh2(283). Clonal selection resulted in differing levels of knockdown in Mpg(-/-) cells: (69.0+/-12.1% from 5 cell clones). Transfection of the Msh2(283) sequence in Mpg+/+, Nth1+/+ and Nth1(-/-) cells resulted in average knockdowns of 45.1+/-40.5% (3 clones), 58.0+/-21.4% (5 clones) and 74.9+/-14.8% (3 clones), respectively. Msh2 knockdown resulted in increased resistance to 6-TG in BER (MPG and NTH1) proficient and deficient cell lines with similar levels of knockdown (84+/-4%) but increased resistance to TMZ only in Mpg+/+ and Nth1(-/-) cell lines and not Mpg(-/-) or Nth1+/+ cells as assessed by an MTT assay. Msh2 knockdown had no effect on sensitivity to MMS induced toxicity. In a clonogenic assay, Msh2 silenced Mpg+/+, Mpg(-/-), Nth1+/+ and Nth1(-/-) cells were more resistant to TMZ. These results confirm previous studies showing that MSH2 is a key protein in influencing 6-TG and O(6)-methylguanine induced toxicity but also suggest that the effect of this protein depends upon the presence of other proteins in different DNA repair pathways.

Ecotypic differences in responses of Arabidopsis thaliana L. to elevated polychromatic UV-A and UV-B+A radiation in the natural environment: a positive correlation between UV-B+A inhibition and growth rate.

The effects of supplementary ultraviolet-A (UV-A) and ultraviolet-B+A (UV-B+A) in the natural environment on the growth and morphology of various ecotypes of Arabidopsis thaliana were investigated. The ecotypes investigated were Columbia (Col-4), Landsberg erecta (Ler-0), Cvi-0, Wassilewskija, Enkheim-D, Aa-0 and Di-1. The mutant hy-4 was also used. Results varied with the radiation treatment, ecotype and parameter measured. Plants subjected to elevated UV-A were both insensitive (all parameters Cvi-0 and Col-4) and sensitive. When responses to UV-A occurred they were mostly inhibitory (all significant responses of Di-1 and Enkheim-D, most parameters of Wassilewskija, and some parameters of hy-4), however, promotive affects were observed for some parameters of Aa-0 and Ler-0. Supplementary UV-B+A inhibited all parameters of Di-1 and Enkheim-D and most parameters of Col-4, Ler-0 and hy-4, but Wassilewskija, Aa-0 and Cvi-0 were mostly insensitive. The magnitude of the UV-B+A response varied with ecotype (compare Di-1 with Ler-0). Some ecotypes were sensitive to UV-A but not UV-B+A (Aa-0), whereas others (Ler-0, Col-4) show the opposite sensitivities. A linear relationship is reported between the degree of UV-B+A inhibition of each ecotype and growth rate. The higher the growth rate the more susceptible the ecotype is to UV-B+A inhibition. This relationship holds for the majority of growth parameters measured.

Growth and stomatal responses of temperate meadow species to enhanced levels of UV-A and UV-B+A radiation in the natural environment.

Some responses of various meadow species to enhanced UV-radiation of the natural daylight spectrum are described together with the experimental protocol employed. Growth responses to supplementary UV-B+A are mostly inhibitory when compared to the ambient daylight treatment for Bellis perennis, Cardamine pratensis, Cynosurus critatus and Ranunculus ficaria. However, the response of UV-A treatment compared to that of the UV-B+A varies in significance according to the species and parameter investigated. The pertinence of the ambient and UV-A treatment to data interpretation is discussed. Stomatal conductance of B. perennis was measured throughout the 24 h cycle. Although no significant difference could be found between the stomatal conductance of UV and ambient treated plants during the hours of daylight, at night it was found that the UV-B+A treated plants were unable to achieve the same degree of closure as the ambient treated plants. UV-damage and growth responses could alter the diversity of the meadow ecosystem and these effects could be exacerbated by water loss.

Outdoor ultraviolet polychromatic action spectra for growth responses of Bellis perennis and Cynosurus cristatus.

Polychromatic ultraviolet (UV) action spectra for various growth responses of the dicotyledon Bellis perennis L. (daisy) and the grass Cynosurus cristatus L. (crested dog's-tail) have been measured. The plants were grown in the natural environment and ambient daylight was supplemented with five different UV irradiances centred at eight different wavelengths (313, 318, 320, 322, 339, 348, 356 and 377 nm). Destructive growth analysis was performed on B. perennis and C. cristatus after 300 and 122 days respectively. Dose response curves were created to construct action spectra for individual responses. Different spectral responses were observed in these two plant types. B. perennis exhibited a substantial action maximum at 313 nm for the inhibition of aerial, root and total dry weight; a similar action maximum at 313 nm for the inhibition of leaf expansion was observed. Longer wavelengths were relatively ineffective on these growth parameters, with the exception of a small but statistically significant (P < 0.05) response to 320 nm radiation. By contrast, C. cristatus showed negligible response to 313 nm radiation, for inhibition of aerial, root and total dry weight but substantial responses to longer wavelengths, especially at 339 and 348 nm. These action spectra add weight to suggestions in the literature that UV-A has a role to play in responses in this region of the spectrum. The possible implications of these observations are discussed.

FISH analysis of a complex chromosome rearrangement involving nine breakpoints on chromosomes 6, 12, 14 and 16.

We report the prenatal diagnosis of an apparently balanced de novo complex chromosome rearrangement (CCR) which involved nine breakpoints on four different chromosomes. Fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) were performed as an adjunct to G-banding for characterization of the abnormal chromosomes. The 22-week female fetus showed minor dysmorphic features including dolichocephaly, broad fingernails, tibial bowing, clubfoot, thoracolumbar scoliosis and hypoplastic toenails. Autopsy revealed gall-bladder hypoplasia and an atrial septal defect. Chromosome analysis of fetal tissue confirmed the presence of the complex rearrangement.

Bilateral ulnar agenesis: case report and review of the literature.

Bilateral ulnar agenesis is a rare abnormality. A total of 36 cases are analyzed: 35 of these are documented in the literature and 1 stillborn male is presented in this study. Most patients had one of the three conditions: Al-Awadi/Raas-Rothschild syndrome, syndrome of ulnar aplasia with split hand/split foot deformity, or the Brachmann-de Lange syndrome. Fifty percent of all cases with bilateral ulnar agenesis were associated with lower limb defects and these cases, for the most part, also belonged to the aforementioned syndromes. Nonskeletal, internal organ malformations were identified in 34% of all patients. Nine patients presented with isolated bilateral ulnar agenesis. The Al-Awadi/Raas-Rothschild syndrome and the split hand/split foot deformity are heritable disorders. There was no evidence for genetic etiology in most of the other cases. Bilateral ulnar agenesis in our fetus was part of the Brachmann-de Lange syndrome with associated cardiac defect, diaphragmatic hernia, and umbilical artery agenesis.

Human fetal somatic and visceral morphometrics.

Because of the increasing prenatal use of high-resolution ultrasonography to assess fetal growth and because growth aberration is one of the principal manifestations of abnormal intrauterine development, a more precise definition of normal growth is desirable. Our data set from autopsies of fetuses/neonates (50-4,000 g), collected with systematic attention to precision of measurement, is analyzed and presented. Previous computations of fetal growth curves appear to overstate the variation about the mean. Through the identification and exclusion of outliers and the use of appropriate curve fitting techniques, the problem of overestimation of variation about the mean has been largely eliminated. The new growth standards we present can be used to assess aberration from proportional growth and correlate it with gestational circumstances and particular morphological features of the fetus.

Cardiovascular pathology in osteogenesis imperfecta type IIA with a review of the literature.

Lethal perinatal osteogenesis imperfecta (OI Type II) is a biochemically diverse collagen disorder characterized by short, crumpled long bones, beaded ribs, blue sclerae and thin, fragile skin. Cardiovascular abnormalities are rarely described. Using morphometry and light and electron (SEM and TEM) microscopy, we analyzed the hearts and great vessels from 2 fetuses with OI Type IIA and compared the findings with age-matched controls. The heart weights and atrioventricular valve (AVV) circumferences were reduced in OI. The chordae tendineae were short and fragile; both the AVVs and the chordae tendineae were hypercellular. TEM showed relatively little organized collagen in the chordae tendineae of OI fetuses. Furthermore, quantitative evaluation of collagen fibril size revealed a decrease in the cross-sectional diameter. There was also a marked decrease in the adventitial and intramural collagen of the intramyocardial arteries and great vessels in OI. Our study reports, for the first time, specific lesions in the cardiovascular systems of patients with OI Type II and reviews the cardiovascular pathology in other forms of OI.

Health promotion at the worksite. I. A process for establishing programs.

A 14-step process for establishing health promotion programs at the worksite is described. During a period of 3 years, it has been used to establish 58 such programs in 12 small industries that employ 4,200 persons in a rural county in Pennsylvania. Few problems have been encountered; the most serious is the very limited record-keeping system of industries. The process is flexible and can be readily generalized to different settings. The major strengths of the process are the sense of ownership it gives to employees, and the modesty of its costs and the costs of the resulting programs.

Weight loss competitions at the work site: impact on weight, morale and cost-effectiveness.

Three weight loss competitions were held in business/industrial settings. One competition was between three banks; the other two were within industries, either between employee teams selected at random or between divisions of the industry. Attrition in the competitions was less than 1 per cent and weight loss averaged 5.5 kg. Both employees and management reported positive changes in morale and employee/management relations, and both considered the competition important to the success of the program. The cost-effectiveness ratio ($ 2.93 per 1 per cent reduction in percentage overweight) is the best yet reported.

Practical management of children with apparent learning disabilities.

There should be a collaborative evaluation of a child with apparent learning disabilities. This article describes what the neurologist can expect from the other disciplines and how the services can be coordinated, using either the multidisciplinary services of a clinic or comprehensive assessment by a private practitioner.

Toxicity of Aroclor-R 1254 and its physiological activity in several estuarine organisms.

The occurrence of high concentrations of a PCB (Aroclor 1254) in the Pensacola estuary prompted field and laboratory studies by the Gulf Breeze Environmental Research Laboratory (EPA). Monitoring of the estuary indicates the chemical is present in all components--particularly in sediments and fishes. Residues appear to be diminishing in sediments. Toxicity tests show estuarine species sensitive at ppb concentrations in water, with a ciliate protozoan (Tetrahymena pyriformis W), (Fundulus similis), affected at or near 1.0 ppb. Tissue concentrations of Aroclor 1254 similar to those found in natural populations of shrimps from the contaminated estuary were successfully duplicated in laboratory experiments. Shrimps also concentrated the PCB from very low concentrations (0.04 ppb) in the water. Three estuarine species demonstrated pathologic changes at tissue and cellular level after chronic exposure to the chemical. Oysters (Crassostrea virginica) developed abnormal infiltration of leukocytes in the connective tissue, spot (Leiostomus xanthurus) developed fatty changes in their livers, and shrimp (Penaeus duorarum) developed crystalloids in hepatopancreatic nuclei.