Chromosome content and ultrastructure of radiation-induced micronuclei

Unrepaired or misrepaired radiation damage in mammalian chromosomescan result in micronucleus formation at the first cell division.This represents loss of genomic information which may causecell death. To improve our understanding of the mechanism ofradiation-induced micronucleus formation, we characterized micronucleusultrastructure and identified the origin of micronucleus DNA.Immunofluorescence microscopy showed that micronuclei were structurallysimilar to main nuclei since they contained nuclear lamins Aand C and were encapsulated by a network of vimentin intermediatefilaments. The contents of radiation-induced micronuclei werecharacterized using fluorescence in situ hybridization to probefor DNA originating from chromosomes 2, 7, 11 and 16. We postulatedthat if incorporation of DNA into micronuclei were random, thenthe probability of chromosomal DNA in micronuclei would be relatedto the target, i.e. chromosome size. Our results demonstratedthat incorporation of DNA from smaller chromosomes (11 and 16)was not different from expected values but incorporation ofDNA from the larger chromosomes (2 and 7) was significantlygreater than expected. Not all chromosomes in the human genome,therefore, were equally susceptible to genomic loss by micronucleusencapsulation. In conclusion, radiationinduced micronuclei havesimilar structural characteristics to main nuclei, chromosomedamage and/or repair after ionizing radiation may be non-random,and micronucleus formation may reflect this variability. ³To whom correspondence should be addressed     

Topography of basal glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose

The activity of the pentose phosphate shunt was assessed under basal conditions in subregions of the hippocampus by measuring the uptake and retention of [1-14C]glucose and [6-14C]glucose and their 14C-labelled metabolites. The relative and absolute retention of carbon-14 from each of the two compounds was nearly identical in all regions examined. For each compound, the highest accumulation of 14C occurred in the granule cell layer of the dentate gyrus and in the pyramidal cell layer. Relatively high retention of radioactivity was also found in the molecular layer of dentate gyrus and in the stratum lacunosum-molecular. The stratum radiatum and stratum oriens contained the lowest levels of radioactivity among hippocampal regions. The equal retention of radioactivity from [1-14C]glucose and [6-14C]glucose implies that pentose phosphate shunt activity is very low throughout the hippocampus under the conditions of this study. The uptake and retention of radioactivity was evaluated in different hippocampal regions 10 or 30 min following intravenous injection of [1-14C]glucose. Although there was significantly more radioactivity at 30 min than at 10 min, the same topographic pattern of radioactivity within the hippocampus was observed in rats after both survival periods, indicating that an equal fraction of the [1-14C]glucose utilized in different hippocampal regions is oxidized to 14CO2 under these conditions. Most regions of high glucose utilization in the hippocampus determined with [1-14C]glucose and [6-14C]glucose correspond to regions of intense histochemical staining for cytochrome oxidase reported in the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of production of type 1 pili among urinary tract isolates of Escherichia coli.

The piliation and hemagglutination properties of 54 consecutive Escherichia coli isolates from women with recurrent urinary tract infections were studied. Mannose-sensitive hemagglutination (MSHA) of guinea pig erythrocytes, characteristic of type 1-piliated bacteria, was produced by 75% of the isolates, 32% produced mannose-insensitive hemagglutination, and 14% produced no hemagglutination reaction. The production of type 1 pili was examined in those strains that produced MSHA only. Studies with antiserum prepared against purified pili suggested that at least three subtypes of type 1 hemagglutinins were represented among the isolates. All of the type 1-piliated isolates produced MSHA after serial subculture in static broth. After growth on agar, selected type 1-piliated isolates were subdivided into two groups. Many strains apparently suppressed piliation during growth on agar (regulated variants); all colonies became MSHA negative and were composed of nonpiliated cells as shown by electron microscopy. The loss of the MSHA phenotype often occurred after a single overnight passage on agar, and any remaining hemagglutinin was gradually lost with one to three additional passages. Seven strains, however, retained a significant hemagglutination titer after multiple subcultures on agar, and they produced colonies consisting of a mixed population of piliated and nonpiliated cells. These strains were apparently able to oscillate between states of pilus expression and nonexpression during growth on agar (random phase variants). When nonpiliated cells isolated from the mixed, random variant population were plated on agar, they gave rise to hemagglutination-positive colonies that consisted of both piliated and nonpiliated cells. The distinction between random variants and regulated variants was also observed in shaking broth cultures inoculated with nonpiliated cells. The random variants produced MSHA-positive cultures composed of piliated and nonpiliated cells, whereas the regulated strains remained nonpiliated. The results indicate that type 1 pili are a predominant adhesin of uropathogenic E. coli and that during growth on agar only about one-fourth of the type 1-piliated isolates regulate pilus expression by random phase variation.     

Properties of a Hemolysin Produced by Group B Streptococci

A hemolysin that appears to be responsible for the zones of beta-hemolysis surrounding colonies of group B streptococci (Streptococcus agalactiae) on blood agar plates has been isolated and partially purified. No soluble hemolysin was detectable in the supernatants of streptococcal cultures grown in several types of media. However, hemolytic activity was detected when streptococci were incubated with erythrocytes, and soluble hemolysin was isolated when bacterial suspensions were incubated in the presence of a variety of agents, including calf serum, albumin, Tween 80, and starch. Glucose and other fermentable carbohydrates stimulated hemolysin production, and metabolic inhibitors greatly reduced the titer of hemolysin that could be recovered, suggesting that cellular metabolism is necessary for hemolysin production or release. The soluble hemolysin was concentrated by ammonium sulfate precipitation and partially purified by gel filtration. Agents known to inhibit other streptococcal hemolysins, including phospholipids, trypan blue, proteases, and cholesterol, were tested for their effect on the group B hemolysin. Only the phospholipids inhibited hemolysin activity. The group B streptococcal hemolysin appears to be similar to, but distinct from, streptolysin S produced by Streptococcus pyogenes.     

Enhanced expression of intracellular adhesion molecule-1 and P-selectin in the diabetic human retina and choroid.

Elevated expression of intercellular adhesion molecule-1 (ICAM-1) as well as E- and P-selectin occurs on the vascular endothelium in a number of disease states and is thought to play an early critical role in the adhesion of circulating leukocytes to the endothelium. The goal of the present study was to investigate the immunolocalization of these molecules in the retina and choroid of postmortem human tissue sections from nondiabetic and diabetic subjects. Whereas ICAM-1 was localized primarily within the choriocapillaris of nondiabetic subjects, immunoreactivity in diabetics was significantly elevated throughout the choroidal vasculature and within retinal blood vessels (P < 0.05). In the choroid, P-selectin was most prominent in veins of the nondiabetic, whereas in diabetics, P-selectin was significantly elevated in arteries (P < 0.001) and veins (P < 0.05) and, in some cases, was also observed in choriocapillaris. P-selectin immunoreactivity was not observed in the retina of any subject. E-selectin immunoreactivity was not observed in choroid or retina in any subjects. Neutrophil numbers per square millimeter of tissue were significantly elevated in diabetic choroid (P < 0.05) and retina (P < 0.001). Our results demonstrate that ICAM-1 and P-selectin are constitutively expressed in the normal choroid and are upregulated in the choroidal vasculature in diabetes, but only ICAM-1 was upregulated in the retina of diabetic subjects. Increased cell adhesion molecule expression may contribute to the retinal and choroidal microangiopathy observed in diabetics by enhancing leukocyte adhesion and consequently the incidence of capillary obstruction and endothelial cell injury.     

Ethylenediaminetetraacetic acid (disodium salt)-labile bovine immunoglobulin M Fc binding to Brucella abortus: a cause of nonspecific agglutination.

It was demonstrated by a radioimmunoassay procedure that Brucella abortus agglutinins from noninfected cattle sera, absorbed to B. abortus antigen and eluted with ethylenediaminetetraacetic acid (EDTA), was immunoglobulin M that bound to that bacterium by its Fc portion. The EDTA-eluted immunoglobulin M agglutinated intact B. abortus cells but not erythrocytes treated with B. abortus lipopolysaccharide. The specificity of the EDTA-eluted immunoglobulin was for B. abortus, although a small titer to Yersinia enterocolitica serotype O:9 was observed. In contrast, immunoglobulin M purified from the serum of a cow injected 7 days previously with heat-killed B. abortus bound to the antigen by its Fab portion, was not labile to EDTA treatment, cross-reacted extensively with Y. enterocolitica serotype O:9, and agglutinated various other bacterial antigens and normal erythrocytes.     

Use of the Roche LightCycler instrument in a real-time PCR for Trichomonas vaginalis in urine samples from females and males

Trichomonas vaginalis is the agent of a highly prevalent sexually transmitted infection (STI) that can result in vaginitis, urethritis, and preterm birth. Traditional methods of diagnosis, including wet preparation, can be unreliable. In this study, we describe the adaptation of an existing PCR method for specific detection of T. vaginalis DNA into a rapid real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe chemistry. The FRET-based assay described demonstrated high sensitivity with a detection limit of 1.06 organisms, as well as a high specificity. A total of 253 urine samples collected prospectively from both men and women were tested for T. vaginalis DNA with both the FRET-based assay and a previously validated PCR assay. When the validated PCR assay was used as the "gold standard" and after discrepancies had been resolved, our FRET-based assay demonstrated an analytical sensitivity and specificity of 90.1 and 100%, respectively. Overall results suggest that FRET-based assays can provide rapid, accurate, and high-throughput detection of T. vaginalis and may prove useful in clinical settings and for large-scale screening programs.     

A computer-interfaced photometer and systematic spacing of duplicates to control within-plate enzyme-immunoassay variation

A Multiskan photometer for reading microtiter plate enzyme immunoassays was linked with a time sharing computer to facilitate control of assay variation and analysis of results. The interface that converted photometer output to RS-232-C format required changes to divide the output into segments short enough for input to the computer. To measure within-plate variation and investigate how the method of allocating sample duplicates to plate wells may affect the estimation of sample variance, uniformity tests were conducted with 47 plates. Coefficients of variation (CV) among wells within-plates ranged from 4.6 to 20.7% and in two-thirds of the plates exceeded 10%. Duplicates allocated to adjacent wells (method 1) gave consistently higher CV for sample means than duplicates allocated to opposite plate quadrants (method 2). In general, the CV by method 2 was about 30% smaller than that by method 1. Analysis of variance confirmed the effectiveness of the quadrant pattern of duplicate allocation as a method of controlling variation that arises from well position effects.     

Preparation of typing antisera specific for O antigens of Pseudomonas aeruginosa.

Results of serotyping 966 clinical isolates of Pseudomonas aeruginosa showed that 72% agglutinated specifically in one or another of the 16 typing antisera, but 28% agglutinated in two or more and often in as many as 10 antisera; this polyagglutinability correlated with a high incidence of cross-reactivity among the antisera. Absorption of each typing antiserum with either cell suspensions of five O-type strains or with a suspension of a particular polyagglutinable strain (SMC 247) abolished cross-reactivity in the typing antisera without significantly reducing titers against the homologous strains. All but four of the polyagglutinable strains agglutinated specifically in one or another absorbed antisera. The cross-reactions of unabsorbed antisera were interpreted to have been caused by antibodies directed not against specific O antigens but against thermostable specificities that remain undefined.     

Amplification of fluorescent in situ hybridisation signals in formalin fixed paraffin wax embedded sections of colon tumour using biotinylated tyramide.

Fluorescent in situ hybridisation (FISH) is a powerful tool for the evaluation of chromosomal alterations in formalin fixed paraffin wax embedded sections of colorectal cancer. However, initial experiments using a two-step detection system for digoxigenin labelled chromosome specific centromeric probes resulted in a complete lack of hybridisation signal from a number of colorectal tumour sections. This was due to high levels of background autofluorescence observed in this tissue, which masked any relatively weak hybridisations present. To overcome this problem, a biotinylated tyramide mediated amplification system was incorporated into the FISH detection protocol. This involves the use of horseradish peroxidase to activate the biotinylated tyramide, resulting in the deposition of a large number of biotin molecules at the site of bound peroxidase, which corresponds directly to the location of hybridised probe. Final detection was by means of a streptavidin-FITC conjugate. Using this technique, a panel of 11 colorectal tumour samples studied to date have shown strong, specific hybridisation signals to the nucleus of tumour cells. Amplification of FISH signals by biotinylated tyramide has the potential to improve weak hybridisation signals in cells from numerous sources, using a variety of probe types, including single copy gene probes as well as centromere specific probes.