Induction of DNA double-strand breaks by restriction enzymes in X-ray-sensitive mutant Chinese hamster ovary cells measured by pulsed-field gel electrophoresis

This investigation was designed to determine whether the cytotoxic effects of different restriction endonucleases are related to the number and type of DNA double-strand breaks (DSBs) they produce. Chinese hamster ovary (CHO) K1 and xrs-5 cells, a radiosensitive mutant of CHO K1, were exposed to restriction endonucleases HaeIII, HinfI, PvuII and BamHI by electroporation. These enzymes represent both blunt and sticky end cutters with differing recognition sequence lengths. The number of DSBs was measured by pulsed-field gel electrophoresis (PFGE). Two forms of PFGE were employed: asymmetric field-inversion gel electrophoresis (AFIGE) for measuring the kinetics of DNA breaks by enzyme digestion and clamped homogeneous gel electrophoresis (CHEF) for examining the size distributions of damaged DNA. The amount of DNA damage induced by exposure to all four restriction enzymes was significantly greater in xrs-5 compared to CHO K1 cells, consistent with the reported DSB repair deficiency in these cells. Since restriction endonucleases produce DSBs alone as opposed to the various types of DNA damage induced by X rays, these results confirm that the repair defect in this mutant involves the rejoining of DSBs. Although the cutting frequency was directly related to the length of the recognition sequence for four restriction enzymes, there was no simple correlation between the cytotoxic effect and the amount of DNA damage produced by each enzyme in either cell line. This finding suggests that the type or nature of the cutting sequence itself may play a role in restriction enzyme-induced cell killing.     

Wortmannin inhibits repair of DNA double-strand breaks in irradiated normal human cells

Recently the concept of dissociative identity disorder (formerly known as multiple personality disorder) has attracted increasing public and scientific interest. However, it is rarely diagnosed in the clinical setting. the reported case of a 47-year-old woman with a history of child abuse demonstrates the problems of differential diagnosis. A number of psychopathologic symptoms pointed to a multiple personality disorder, but in the follow-up psychotic symptoms such as delusions, possible hallucinations and bizarre behavior clearly emerged. The differential diagnosis of dissociative identity disorder includes paranoid schizophrenia, as in the case described, borderline personality disorder, hysteria, simulation and the false memory syndrome. Finally, social and cultural factors have to be considered.     

Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.     

Reorientation time of DNA molecules in pulsed-field gel electrophoresis

The precise determination of the influence of an electric field strength E on the resolution of DNA molecules during a pulsed-field gel electrophoresis shows that the maximal molecular size Nmax of still resolved DNA molecules is described by the equation Nmax = k tau E alpha, where k is a coefficient, tau is a pulse time, and alpha is an exponent (calculated as approximately 3/2). We assume that the best estimation of the reorientation time tau R for each DNA fragment is such a pulse time in which this DNA molecule is the largest separated one.     

Estimation of DNA single-strand breaks by single cells gel electrophoresis in tumor cells

Receptor-like protein tyrosine phosphatase beta (RPTP beta) shows structural and functional similarity to cell adhesion molecules (CAMs). It binds to several neuronal CAMs and extracellular matrix (ECM) proteins that combine to form cell-recognition complexes. Here, the authors discuss the implications of such complexes for intercellular signaling, and the regulation of RPTP activity by cell-cell and cell-ECM contact.     

Analysis of Bordetella pertussis isolates from an epidemic by pulsed-field gel electrophoresis

We examined genetic variation among 78 clinical isolates of Bordetella pertussis, including 54 strains recovered during a 1986 pertussis epidemic. A total of 16 pulsed-field gel electrophoresis (PFGE) profiles, generated with each of three different enzymes (XbaI, SpeI, and DraI), were obtained from the epidemic and sporadic isolates included in the study. Indistinguishable profiles were seen among strains unrelated temporally or geographically, as well as among strains isolated sporadically from the same geographic areas. All isolates from the epidemic had indistinguishable PFGE profiles. The PFGE pattern of the epidemic strains was shared with only 1 of 25 strains isolated independently of the outbreak. This isolate was cultured from a specimen from a laboratory scientist who had been working with the epidemic strains, further implicating the usefulness of PFGE for the epidemiologic study of clinical strains of B. pertussis. Differences in PFGE profiles for single epidemic strains occurred occasionally upon repeated passage on agar medium, suggesting that subculturing of initial isolates should be minimized before pulsed-field analysis.     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

In vitro induction of primary DNA double-strand breaks in leukemic and normal blood cells by ultraviolet irradiation

The yield of UV-induced DNA double-strand breaks was studied for white blood cells ("light" fraction) derived from peripheral blood, and from patients with lymphomas, chronic lymphoid leukemia (CLL), and chronic myeloid leukemia (CML). The method employed was constant-field electrophoresis of plug-embedded DNA in agarose gel. Characteristic dose-response curves were obtained for various cell populations. Lymphoid cells, both from healthy subjects and CLL patients, revealed less damage to DNA under UV-irradiation, whereas CML cells were much more affected. Possible interpretation of these results includes species-specific differences in UV-induced DNA damage, as well as sufficient DNA crosslinking, thus interfering with DNA dsbs detection in irradiated cells.     

Single-strand breaks in oligodeoxyribonucleotides induced by fission neutrons and gamma radiation and measured by gel electrophoresis: protective effects of aminothiols

In order to approach the detailed structure-function relationships of aromatase, we studied the inhibitory and inactivatory potencies of several steroidal androstenedione analogues (1: 4-hydroxyandrostenedione, 2: 4-acetoxyandrostenedione and 3: 7 alpha-(4'-amino)phenylthio-4-androstene-3, 17-dione) and non-steroidal imidazole derivatives (4: ketoconazole, 5: miconazole and 6: fadrozole) on equine aromatase in placental microsomes, a well established mammalian model. Human placental microsomes and the purified enzyme from equine testis were also used to compare inhibition by 1 and 2. In equine microsomes, all compounds tested exhibited a competitive inhibition, with Ki values of 4.1, 26 and 1.8 nM for 1, 2 and 3, and of 2400, 1.4 and 4 nM for 4, 5, and 6, respectively. The Km for androstenedione, the substrate mainly used in these studies, was 1.8 +/- 0.13 nM. The three non-steroidal derivatives did not inactivate equine aromatase, but 1 and 2 acted as comparable inactivators to a much higher degree than 3. Compound 1 inhibited in a similar manner (89-94%) purified or equine and human microsomal aromatases, whereas 2 inhibited microsomal aromatase more efficiently in the horse than in man (92% and 33% inhibition, respectively). There was only a 40% inhibition with 2 on the purified equine enzyme, which is no more in the natural membrane environment. The comparisons between equine and human microsomal aromatases allow precise functional and structural differences to be observed with these enzymes.     

Genetic relationship between blood and nonblood isolates from bacteremic patients determined by pulsed-field gel electrophoresis

A total of 148 isolates from 55 bacteremic patients were examined by pulsed-field gel electrophoresis. Genetically different nonblood strains were isolated from 13.9% of patients with bacteremia caused by gram-positive cocci and 42.1% with Pseudomonas aeruginosa bacteremia, indicating that antibiograms of a single nonblood P. aeruginosa isolate are not always informative for treatment of bacteremia.     

Typing of human Campylobacter jejuni isolates in Finland by pulsed-field gel electrophoresis

A total of 69 pulsed-field gel electrophoresis (PFGE) types were identified among 176 Campylobacter jejuni isolates from Finnish patients. In two geographic areas studied, five predominant PFGE types comprised over 40% of the isolates. One-third of the isolates had unique PFGE types. In small outbreaks, identical PFGE patterns were demonstrated, indicating a common source of infection.     

Investigation of a pseudo-outbreak of Nocardia asteroides infection by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA PCR

Molecular strain typing by pulsed-field gel electrophoresis and by randomly amplified polymorphic DNA analysis was used to investigate a cluster of four Nocardia asteroides isolates associated with the BACTEC 460 TB system. An instrument motor drive misalignment resulted in inadequate needle sterilization and cross-contamination of BACTEC vials. This pseudo-outbreak illustrates the importance of proper BACTEC 460 needle sterilization and maintenance and confirms the usefulness of molecular typing methods for epidemiologic investigations.     

Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Several different genomic fingerprints can be obtained from various commercially-important species of Bifidobacterium using pulsed-field gel electrophoresis (PFGE) following digestion of DNA with XbaI and SpeI. Four different genomic finger printings were discernible for reference strains of Bifidobacterium animalis, five for B. bifidum, three for B. breve, five for B. infantis and three for B. longum. Standard commercially-available industrial strains of B. animalis are identical to the reference strain ATCC 27536, previously isolated from chicken feces. There was more genomic heterogeneity among industrial strains of B. longum, in that only one gave profiles similar to the type strain of this species (ATCC 15707). The other 14 commercially-available strains of B. longum (mainly isolated from Japanese commercial preparations) were divided into four new molecular types based on their PFGE patterns. The PFGE method indicated that only five distinct strains of B. longum and one strain of B. animalis are used in commercial preparations. Additionally, the use of polymerase chain reaction amplification of portions of 16S rDNA provides a highly specific technique to discriminate between the species B. breve, B. infantis and B. longum.     

Radiation-induced DNA damage and repair in radiosensitive and radioresistant human tumour cells measured by field inversion gel electrophoresis

Radiation-induced DNA damage induction and repair was measured in two human squamous carcinoma cell lines with differing radiosensitive. Experiments were carried out with field inversion gel electrophoresis (FIGE), adapted to measure DNA double strand break (DSB) induction and repair in unlabelled cells. The sensitivity of the method was increased by introducing a hybridization membrane into the agarose gel. Damaged DNA accumulated on one spot on the membrane resulting in high local concentrations. This DNA was quantified using radioactively-labelled total human DNA as a probe. Dose response experiments for damage induction correlated well with the results using prelabelled cells. Linear DNA damage induction curves were observed with a sensitivity for the post-labelling method of 1 Gy. No differences in DSB induction were found, however, between the radiosensitive SCC61 and the radioresistant SQ20B cell line. Repair experiments were carried out with trypsinized cells with different doses and repair temperatures. The 10, 25 and 50 Gy doses resulted in 6, 13 and 50% of the DNA migrating out of the plug at 0 h. For both the cell lines 75-85% of the initial damage was repaired within 1 h at 37 degrees C at all three radiation doses, i.e. no significant differences were observed in repair rates or extent between the two cell lines. At 24 degrees C repair was slower than at 37 degrees C, and at 0 degree C no repair was observed. In summary, radiosensitivity differences at physiological temperatures could not be explained by differences in either induction or repair of DNA damage as measured by pulsed field gel electrophoresis.     

Hydroxyl radicals and DNA double-strand breaks in X-irradiated E. coli

We have used glycerol to study the relationship between hydroxyl radicals, one of the primary radiolytic products, and the production of DNA double-strand breaks in selected E. coli strains. Our results suggest that when bacteria are irradiated at doses up to about 120 Gray, hydroxyl radicals produce DNA lesions, but not double-strand breaks.     

A charge-coupled-device camera image analysis system for quantifying DNA distributions in agarose gels after pulsed-field gel electrophoresis

In recent reports of the so-called "floral variant" of follicular lymphoma, an unusual variant of follicular lymphoma mimicking progressive transformation of germinal centers, questions have been raised regarding whether this process represents a malignant lymphoma. We studied 19 examples of the floral variant of follicular lymphoma and report our light microscopic, immunohistochemical, and molecular diagnostic findings. Morphologic changes consisted of effacement of normal lymph node architecture by follicles composed of atypical lymphocytes. The follicles were surrounded by prominent mantle zones that invaginated irregularly into the follicle centers, often imparting a "floral" appearance. Sufficient material was available for immunophenotypic or genotypic studies in 15 biopsies. Twelve of 15 cases studied by immunohistochemistry demonstrated phenotypes supporting a diagnosis of lymphoma. Five demonstrated light-chain restriction; one was an immunoglobulin-negative B-cell neoplasm; and six, in which only formalin-fixed, paraffin-embedded tissue was available, demonstrated overexpression of the bcl-2 protein. Southern blot analysis revealed evidence of clonal immunoglobulin heavy-chain gene rearrangement in all five cases tested. Overall, 12 of the 15 biopsies studied with these techniques showed immunologic or genotypic support for malignant lymphoma. The results of this study demonstrate that the floral variant of follicular lymphoma does indeed represent a malignant lymphoma.     

Genotypic identification and phylogenetic analysis of the spotted fever group rickettsiae by pulsed-field gel electrophoresis

Using pulsed-field gel electrophoresis, we studied the chromosomes of spotted fever group rickettsiae. We digested the DNA of 16 species currently known to belong to this group with SmaI, EagI, and BssHII. The genome size of 13 rickettsiae was between 1,200 and 1,300 kb. "Rickettsia massiliae" and "R. helvetica" genome sizes were 1,370 and 1,397 kb, respectively, and that of R. bellii was 1,660 kb. It was possible to obtain distinctive patterns for each species, but in R. conorii, 10 isolates exhibited the same profiles, showing that pulsed-field gel electrophoresis is a good interspecies identification tool. We achieved a phylogenetic analysis of these bacteria by using the Dice coefficient and UPGMA and Package Philip programming. We established a dendrogram of the genetic relationships between the different species showing the existence of a cluster in the spotted fever group rickettsiae including R. conorii, R. rickettsii, R. parkeri, R. sibirica, "R. africae," "R. slovaca," Thai tick typhus rickettsia, and Israeli tick typhus rickettsia. We located three genes previously cloned and sequenced (genes encoding the R. rickettsii surface proteins of 120 and 190 kDa and the R. prowazekii citrate synthase gene), using Southern hybridization. The genes encoding citrate synthase and the surface protein of 190 kDa were usually located on the same band, and it is hypothesized that they are relatively close on the chromosome.     

Phage typing combined with pulsed-field gel electrophoresis and random amplified polymorphic DNA increases discrimination in the epidemiological analysis of Salmonella enteritidis strains

In adult rabbits, mid-diaphyseal segments of the radius or ulna were excised to produce defects greater than the critical size for spontaneous bone repair. The defects were enveloped in sleeves composed of nonbiodegradable expanded polyfluoroethylene (ePTFE), pore size 30, 60, 90 microns, and compared with sleeves of three biodegradable materials. Bone morphogenetic protein and associated noncollagenous bone matrix protein (BMP/NCP) or recombinant human morphogenetic protein (rhBMP-2) were implanted inside the sleeves. Albumin was implanted for a control system. Without intracompartmental BMP, only about 10%-15% of the defect was repaired by bone growth extending from the bone ends into the sleeves composed of ePTFE, pore size 30 microns. With sleeves with pore size 60 or 90 microns and intracompartmental BMP/NCP, 54%-96% regeneration occurred within 8 weeks after the operation. Sleeves of biodegradable nonimmunogenic materials such as polyorthoester (POE) and polylactic-polyglycollic acids (PLA/PGA) permitted 86%-98% restoration of bone continuity, but only when BMP was present in the lumen. With puncture holes (0.5 mm in diameter), implants of BMP/NCP in the 30-micron PTFE sleeve produced transmembrane external callus formation and bone regeneration to 147%. Sleeves composed of aorta first calcified, then induced complete intracompartmental bone regeneration. Atelocollagen sleeves incited a low-grade inflammatory cell reaction and did not promote complete regeneration. Under conditions presently undisclosed segments of the ulna bridged with ePTFE, were incompletely paired, even with intracompartmental BMP/NCP. Puncture holes of 0.5 mm admitted ingrowth of capillaries and introduced local conditions favorable for the response to BMP/NCP. BMP/NCP may promote proliferation of nutrient vessels and differentiation of bone marrow stroma cells between the open bone ends. For further investigation, the hypothesis to be examined is that the optimum response to BMP/NCP and rhBMP-2 would emerge in compartments containing first a high concentration gradient and second proliferating perivascular cells.     

A novel mutation of presenilin 1 in familial Alzheimer's disease in Israel detected by denaturing gradient gel electrophoresis

Germline mutations in the presenilin 1 (PS1) gene apparently account for the majority of early-onset, familial Alzheimer's disease (AD). Using a mutation-screening strategy (denaturing gradient gel electrophoresis; DGGE), we analyzed a large family with early onset AD and seizures. The patients in this family showed a novel missense mutation in exon 5 of the PS1 gene (A to T change in codon 120, altering glutamine to aspartic acid). This novel mutation is located within the second hydrophilic domain of the molecule, a region not particularly involved in previously described germline mutations, and is of unknown biological significance. These results also demonstrate that DGGE can be used effectively to screen for mutations within this gene.