NBN phosphorylation regulates the accumulation of MRN and ATM at sites of DNA double-strand breaks.

In response to ionizing radiation, the MRE11/RAD50/NBN complex re-distributes to the sites of DNA double-strand breaks (DSBs) where each of its individual components is phosphorylated by the serine-threonine kinase, ATM. ATM phosphorylation of NBN is required for the activation of the S-phase checkpoint, but the mechanism whereby these phosphorylation events signal the checkpoint machinery remains unexplained. Here, we describe the use of direct protein transduction of the homing endonuclease, I-PpoI, into human cells to generate site-specific DSBs. Direct transduction of I-PpoI protein results in rapid accumulation and turnover of the endonuclease in live cells, facilitating comparisons across multiple cell lines. We demonstrate the utility of this system by introducing I-PpoI into isogenic cell lines carrying mutations at the ATM phosphorylation sites in NBN and assaying the effects of these mutations on the spatial distribution and temporal accumulation of NBN and ATM at DSBs by chromatin immunoprecipitation, as well as timing and extent of DSB repair. Although the spatial distribution of NBN and ATM recruited to the sites of DSBs was comparable between control cells and those expressing phosphorylation mutants of NBN, the timing of accumulation of NBN and ATM was altered. Serine-to-alanine mutations that blocked phosphorylation resulted in delayed recruitment of both NBN and ATM to DSBs. Serine-to-glutamic acid substitutions that mimicked the phosphorylation event resulted in both increased and prolonged accumulation of both NBN and ATM at DSBs. The repair of DSBs in cells lacking full-length NBN was significantly delayed compared with control cells, whereas blocking phosphorylation of NBN resulted in a more modest delay in repair. These data indicate that following the induction of DSBs, phosphorylation of NBN regulates its accumulation, and that of ATM, at sites of DNA DSB as well as the timing of the repair of these sites.

Deletion and insertion in vivo somatic mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene of human T-lymphocytes.

Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In a population of 172 healthy people (average age, 34; mutant frequency, 10.3 x 10(-6)), deletion/insertion mutations constituted 41% (89) of the 217 independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among +/- 1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions of 3-200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by -1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both endpoints were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), possibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 deletions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation.

Spectrum of somatic mutation at the hypoxanthine phosphoribosyltransferase (hprt) gene of healthy people.

Understanding the significance of somatic mutations requires knowledge of the mutations that occur in vivo in healthy people. The molecular characterization of mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene in 217 independent T-lymphocyte mutants from 172 donors, including smoking and non-smoking males and females, reveals a broad spectrum of in vivo somatic mutation occurring in a population of healthy people. Identification of the DNA alteration in individual mutant clones was accomplished using either one or a combination of multiplex polymerase chain reaction analysis of genomic DNA, sequencing of cDNA, and genomic DNA sequencing. The total spectrum consists of 59% (128/217) base substitutions: 126 simple and two tandem CC>TT base substitutions; 39% (85/217) deletion/insertion type mutations: 30 frameshifts, 26 small (3-200 basepairs) and 27 large deletions, and two duplications; and the remaining 2% (4/217) complex mutations involving the deletion of one to 11 basepairs which are replaced by 1 to 10 basepairs. No significant difference was detected between the base substitution spectra for the smokers and the non-smokers. Analysis of the number of mutations occurring at any one base position led to the identification of three hotspots for mutations at basepairs 197, 508 and 617, in the hprt gene coding region. Spontaneous deamination of CpG may be implicated in the creation of basepair 508 as a hotspot since all mutations detected are C>T transitions resulting in the nonsense mutation, TAG. At basepairs 197 and 617 both G>T transversions and G>A transitions were found indicating that at least two mechanisms were involved in creating mutations at these positions. Comparison of the mutation spectra from two populations can provide insight into the origin of the mutations. This study provides an excellent base for comparison of mutation spectra in other human populations.

Corneal endothelial changes in type I and type II diabetes mellitus.

Forty-six corneas from 25 patients who had had type II (adult-onset) diabetes for more than ten years were examined by specular microscopy with quantitative morphometric analyses of individual endothelial cells. Thirty-four corneas from 21 age-matched nondiabetic subjects were examined for comparison. We also examined 31 corneas from 17 patients with type I (juvenile-onset) diabetes and compared them to 41 corneas from 23 age-matched normal volunteers. The corneal endothelium in type II diabetes showed no difference in cell density but demonstrated a significantly higher coefficient of variation, a decrease in the percentage of hexagonal cells, and a low figure coefficient compared to an age-matched nondiabetic population. Type I diabetes produced similar cell changes, but these changes occurred in the earlier decades. Moreover, we detected a significantly higher rate of cell loss in type I diabetes, resulting in a significant decrease in cell density in the fourth and fifth decades. These results clearly indicate that the diabetic endothelium is morphologically abnormal. The observed anatomic changes result in a less stable and more vulnerable cell layer, possibly explaining some of the persistent clinical changes in the diabetic cornea after surgical trauma.

Diabetic keratopathy as a manifestation of peripheral neuropathy.

In a study of 102 patients (64 women and 38 men; 63 whites and 39 nonwhites; 77 with adult-onset disease and 25 with juvenile-onset disease), the data, after being adjusted for age, showed that diabetic peripheral neuropathy was associated with diabetic keratopathy. The strongest predictor of both keratopathy and corneal fluorescein staining was vibration perception threshold in the toes (P less than .01); the severity of keratopathy was directly related to the degree of diminution of peripheral sensation. Other predictors of keratopathy were reduced tear break-up time (P less than .03), the type of diabetes (P less than .01), and metabolic status, shown by fasting C-peptide levels (P less than .01). No significant relationships were found between keratopathy and tear glucose levels, endothelial cell densities, corneal thickness, or duration of disease.

Damage to the epithelial basement membrane in the corneas of diabetic rabbits.

Epithelial healing problems and basement membrane abnormalities have been observed in the corneas of patients with diabetes mellitus. In this study the rates of corneal epithelial wound healing following transcorneal freezing (with a 6-mm-diameter probe cooled in liquid nitrogen) were compared in diabetic (alloxan-induced) and nondiabetic rabbits. Also compared was the extent of injury to the epithelial basement membrane between the two groups. The overall rate of wound healing was faster in the diabetic animals; the wounds closed at 40 hours after freezing in diabetic animals and at 45 hours after in the nondiabetic controls. The lamina densa of the basement membrane was removed by the freezing procedure in two thirds of the diabetic animals but not in any of the controls. The results of this study indicate that epithelial healing problems in diabetes may be related to damage to the basement membrane, with resulting poor adhesion of regenerating epithelial cells.

Diabetic corneal neuropathy.

Corneal epithelial lesions can be found in approximately one-half of asymptomatic patients with diabetes mellitus. These lesions are transient and clinically resemble the keratopathy seen in staphylococcal keratoconjunctivitis. Staphylococcal organisms, however, can be isolated in equal percentages from diabetic patients without keratopathy. Diabetic peripheral neuropathy was found to be related to the presence of diabetic keratopathy after adjusting for age with analysis of covariance. The strongest predictor of both keratopathy and corneal fluorescein staining was vibration perception threshold in the toes (P less than 0.01); and the severity of keratopathy was directly related to the degree of diminution of peripheral sensation. Other predictors of keratopathy were: reduced tear breakup time (P less than 0.03), type of diabetes (P less than 0.01), and metabolic status as indicated by c-peptide fasting (P less than 0.01). No significant relationships were found between the presence of keratopathy and tear glucose levels, endothelial cell densities, corneal thickness measurements, the presence of S epidermidis, or with duration of disease. It is our conclusion that asymptomatic epithelial lesions in the nontraumatized diabetic cornea can occur as a manifestation of generalized polyneuropathy and probably represent a specific form of corneal neuropathy.

Dream affect and the menstrual cycle.

Ten women subjects exhibited no evidence of waking mood change or differential dream recall as a function of their menstrual cycles. These findings indicate that, with the use of simple screening procedures, women as well as men can and should be used as subjects in research on sleep and dreams.

Measurement and purification of human chromosomes by flow cytometry and sorting.

The 24 human chromosome types of normal diploid fibroblast cell strain were classified into 15 groups by high-resolution flow cytometry on the basis of 33258 Hoechst fluorescence. Chromosomes associated with each group were flow sorted onto microscope slides and identified by quinacrine banding analysis. DNA cytophotometry of metaphase chromosomes from the same cell strain supported and extended this identification. Four of the groups purified were due to chromosomes of a single type--namely, chromosomes 5, 6, 13, and 17. Eight additional groups were also separated and found to contain the following chromosomes: 1 and 2; 3 and 4; 7, 8, and X; 9--12; 14 and 15; 16 and 18; 20 and Y; and 19, 21, and 22. The average purity for the 12 sorted fractions was 78%.