Detection of replicative integrity in small colonic biopsies using the BrdUrd comet assay.

The alkaline single-cell gel electrophoresis or comet assay is a relatively simple method of measuring DNA single-strand breaks and alkali-labile sites in individual cells. Previously, we have used a combination of this with bromodeoxyuridine labelling of DNA and immunolocalisation of the BrdUrd to show that DNA replicative integrity can be assessed in single cultured cells. This study demonstrates the application of the technique to single cells derived from small human colonic biopsies isolated at routine endoscopy. A high level of reproducibility within replicate comet slides and between comet slides prepared from various colonic sites within a single patient is shown. Preliminary results demonstrate that defects in replication can be detected in tumour and premalignant colonic tissue adjacent to the tumour, suggesting that alterations in replicative integrity are an early event in neoplasia, appearing in premalignant mucosal cells. This development deems the BrdUrd comet assay suitable as an ex vivo molecular end point that can be measured easily in tissue collected by biopsy at routine colonic endoscopy. Thus, the BrdUrd comet assay has the potential to facilitate trial investigations of diet- or environment-related factors that may affect replicative integrity in the colon and provides a novel biomarker for colon carcinogenesis.

Molecular, cytogenetic and genetic abnormalities in MDS and secondary AML.

Myelodysplasia (MDS) is a clonal disease, which increases with age, suggesting that multiple steps are required for the evolution of the condition. Approximately 30% of MDS evolve into acute myelogenous leukemia (AML). In this review, we intend to delineate the genetic events, which may drive this sequence and therefore we will focus primarily on cytogenetic abnormalities where the genes have been identified and oncogenes and suppressor genes that have been implicated. In terms of the biological mechanisms, which characterise this process, it is generally thought that the MDS cell has impaired differentiation, and has increased apoptosis. As the disease progresses in addition, the cells have increased proliferation. As the disease evolves, the population of cells, which predominate remain immature, have decreased apoptosis and in many cases, upregulate anti-apoptotic genes and have deregulated proliferation as the number of blast cells increase. Etiological factors, which contribute to the development of leukemia, include therapeutic agents administered for a primary malignancy. The cytogenetic abnormalities, predisposition factors and genes involved in secondary leukemia will also be reviewed.

Spore coat formation and timely sporulation depend on cellulose in Dictyostelium.

Cellulose is a major component of the extracellular coat that surrounds the terminally-differentiated spore of Dictyostelium. It is sandwiched between two layers of proteins that derive from prespore vesicles by exocytosis. Strains unable to synthesize cellulose due to null mutations in the gene encoding the catalytic subunit of cellulose synthase (dcsA) failed to make detergent-resistant spores but produced small, highly refractile, round spore-like cells up to a day late. Although these cells resembled spores in appearance, they were unstable, only transiently ellipsoid in shape, and sensitive to hypo-osmotic shock, drying, or detergents. Differentiation of these pseudo-spores was induced in the normal time frame by activation of the cAMP-dependent protein kinase or co-development with wild type cells, and coat proteins were secreted by the dcsA-null cells at the same time as wild type cells. A substantial fraction of secreted coat proteins was loosely associated with the surface of the mutant cells, resembling the precoat posited to form early during normal sporulation. Transmission electron microscopy revealed that the precoat had little ultrastructural organization in the absence of cellulose. Thus, cellulose in the coat appears to be required for the organization of the pre-coat precursors as well as the stability, dormancy, and shape of the spore.

Alternative effects of RAS and RAF oncogenes on the proliferation and apoptosis of factor-dependent FDC-P1 cells.

Despite the fact that RAF-1 lies immediately downstream of p21RAS in the MAP kinase-signalling cascade, recent evidence in non-haematopoietic environments suggest that RAS and RAF can transduce signals through alternative pathways specific to a particular cell type. Since mutational activation of RAS occurs at high frequency in human leukaemia, we have investigated the contribution of signalling from mutant RAF in mediating the transforming effects of the N-RAS oncogene in the growth factor-dependent cell line, FDC-P1. Independent activation of N-RAS extended the period of exponential growth leading to an increased saturating density under optimal growth conditions. Under conditions of growth factor withdrawal, cells expressing mutant RAS, but not control cells, demonstrated protection against apoptotic death. Although RAF promoted cell proliferation in a similar manner to that observed in FDCP-RAS cells, expression of mutant RAF was not as effective at protecting these cells against apoptotic death following growth factor withdrawal. The results suggest that RAS utilises RAF-dependent signals in promoting the proliferation of FDC-P1 cells but the anti-apoptotic effects of this oncogene are mediated through a RAF- and BCL-2-independent pathway.

The bromodeoxyuridine comet assay: detection of maturation of recently replicated DNA in individual cells.

The single-cell gel electrophoresis (Comet) assay is a relatively simple method of measuring DNA single strand breaks and alkali-labile sites in individual cells. We have combined this with bromodeoxyuridine (BrdUrd) labeling of DNA and immunolocalization of the BrdUrd to assess DNA replicative integrity on a single-cell basis. We show that the existence of strand discontinuities in recently replicated domains of DNA, caused during semiconservative replication or exacerbated by the arrest of replicative polymerases at UV irradiation- or chemical-induced lesions, can be detected in individual cells. Data obtained from BrdUrd-Comets are consistent with biochemical data derived with a range of techniques showing that DNA replication involves the creation of strand breaks or gaps adjacent to recently replicated material, and that DNA damage prolongs the duration of such discontinuities where DNA polymerases are stalled opposite lesions (R. T. Johnson et al, The Legacy of Cell Fusion, pp. 50-67, Oxford: Science Publications, 1994; R. B. Painter, J. Mol. Biol., 143: 289-301, 1980.). Compared with standard biochemical techniques, the BrdUrd-Comet assay is simple and suitable for the accurate and automatable assessment of replicative integrity in very small numbers of mammalian cells, such as may be obtained by biopsy.

Growth and fatness at three to six years of age of children born small- or large-for-gestational age.

OBJECTIVE: To compare young children 3 to 6 years of age who were born small-for-gestational age (SGA; <10th percentile for gestational age) or large-for-gestational age (LGA; >/=90th percentile) with those who were born appropriate-for-gestational age (10th-89th percentile) to determine whether there are differences in growth and fatness in early childhood associated with birth weight status. DESIGN AND METHODS: National sample of 3192 US-born non-Hispanic white, non-Hispanic black, and Mexican-American children 3 to 6 years of age (36-83 months) examined in the third National Health and Nutrition Examination Survey and for whom birth certificates were obtained. On the birth certificates, length of gestation from the mother's last menstrual period was examined for completeness, validity, and whether the pattern of missing (n = 141) and invalid data (n = 147) on gestation was random. Gestation was considered invalid when >44 weeks, or when at gestations of

Growth of infants and young children born small or large for gestational age: findings from the Third National Health and Nutrition Examination Survey.

OBJECTIVES: To compare the growth profiles of infants and young children born small for gestational age (SGA, < 10th percentile birth weight for gestation) or large for gestational age (LGA, > or =90th percentile) with those appropriate for gestational age, and to document the expected growth patterns through early childhood based on national health examination survey data. SAMPLE: Infants and children, 2 to 47 months of age, who were born in the United States and examined using the Third National Health and Nutrition Examination Survey (1988-1994). MAIN OUTCOME MEASURES: Measurements of growth status based on normalized distributions (z scores or standard deviation units [SDUs] for weight, length, and head circumference. RESULTS: Prevalence rates were as follows: SGA infants, 8.6%; appropriate for gestational age infants, 80.9%; and LGA infants, 10.5%. Infants who were SGA appeared to catch up in weight in the first 6 months, but thereafter maintained a deficit of about -0.75 SDUs compared with infants who were appropriate for gestational age. The weight status of LGA infants remained at about +0.50 SDUs through 47 months of age. Length and head circumference were also associated with birth weight status, averaging over -0.60 SDUs for SGA infants and +0.43 SDUs for LGA infants. CONCLUSIONS: Birth weight status is related to growth rates in infancy and early childhood, which underscores the importance of considering child growth relative to birth status when using growth charts. Small for gestational age infants remain shorter and lighter and have smaller head circumferences, while LGA infants grow longer and heavier and have larger head circumferences.

Muscularity and fatness of infants and young children born small- or large-for-gestational-age.

OBJECTIVE: There is growing interest in the extent to which body composition, both short- and long-term, differs in infants and children born at the extremes of birth weight. This is because a growing number of studies have linked low birth weight and fetal growth restriction to the chronic diseases in adulthood that often are obesity-related, and there is also evidence to suggest that heavy infants may be at increased risk for obesity in later life, again with the attendant obesity-related chronic diseases. Our objective was to compare anthropometric indices of body composition of infants and young children born small-for-gestational-age (SGA, <10th percentile) or large-for-gestational age (LGA, >/=90th percentile) with those of normal birth weight status (appropriate-for-gestational-age, AGA) in a US sample. DESIGN: National sample of US-born non-Hispanic white, non-Hispanic black, and Mexican-American infants and young children, 2 to 47 months of age, examined in the third National Health and Nutrition Examination Survey (NHANES III, 1988-1994), for whom birth certificates were obtained. The primary outcomes were normalized anthropometric indices (z scores or standard deviation units [SDU]) of nutritional status and body composition (mid-upper arm circumference, triceps and subscapular skinfolds, mid-upper arm muscle and mid-upper arm fat areas (UFA), and the arm fat index). The outcomes thus were scaled to permit comparison across chronologic ages. RESULTS: The prevalence of SGA was 8.6%, appropriate-for-gestational-age 80.9%, and LGA 10.5%. From ages 2 to 47 months, for infants and young children born SGA, there was a persistent overall deficit in muscularity (mid-upper arm circumference and mid-upper arm muscle area) of approximately -0.50 SDU, but less of a deficit in fatness, particularly at the youngest ages. For infants and young children born LGA, there was a surfeit in muscularity of approximately 0.45 SDU, with less of a surfeit in fatness, particularly at the youngest ages. Across all ages, the mean UFA showed a statistically significant deficit for SGA children (-0.27 +/- 0.10 SDU) and surfeit for LGA children (0.24 +/- 0.08 SDU). At individual ages for UFA and at individual and all ages combined for skinfold thicknesses, there were no significant differences in level of subcutaneous fatness in the three birth-weight-for-gestational-age groups. There was a tendency in the first year for the arm fat index (% arm fat) to be significantly higher for SGA infants, but the effect did not persist after the first year. CONCLUSION: SGA infants remain smaller and LGA infants larger in size through early childhood, but the discrepancies in weight are primarily attributable to differences in lean body mass (muscularity). Fatness is less affected. Thus, based on the fatness indicators used, at any given weight for infants and children 2 to 47 months of age, percent body fat appears to be relatively higher for children who were SGA at birth and lower in those who were LGA at birth. These differences in body composition for SGA infants support the evidence documenting a link between disturbances in intrauterine growth and chronic disease associated with subsequent adiposity in adulthood.