FLOW CYTOMETRIC DETECTION OF SUBHAPLOID NUCLEI IN HUMAN SPERM AS A MEASURE OF DNA FRAGMENTATION AND APOPTOSIS.

The use of assisted reproductive technologies (ARTs) is increasing worldwide. In order to predict the rate of pregnancy after ART the DNA fragmentation index (DFI) of ejaculated spermatocytes may be a better marker than conventional semen quality parameters. Spermatocytes with fragmented DNA are associated with apoptotic stages and are characterized by a low DNA content. The subhaploid nuclei of DNA-damaged spermatocytes can be easily detected by flow cytometry. We here analyzed the percentage of subhaploid nuclei of semen samples from 163 patients aged 26 to 74 years who consulted one of the ten centres for reproductive medicine which routinely send sperm samples to our laboratory in order to determine special sperm parameters. The percentage of subhaploid nuclei indicating the DFI of spermatocytes did not correlate with age and sperm volume, but inversely correlated with sperm concentration and the percentage of motile spermatocytes. This is in concordance with previous studies which demonstrated that DNA damage of spermatozoa correlates with conventional semen quality parameters. Since DNA-damaged spermatocytes are associated with an impaired outcome of assisted conception technologies, this method could help to monitor sperm quality of subfertile men after measures to increase sperm quality and to improve selection criteria of cryopreserved sperm samples in assisted reproduction medicine.

Rapid detection of methicillin-resistant staphylococci by real-time PCR directly from positive blood culture bottles.

For the rapid detection of methicillin-resistant staphylococci directly from blood cultures containing gram-positive cocci in clusters, we implemented a real-time (LightCycler) polymerase chain reaction (PCR) specific for the Staphylococcus aureus nuc gene encoding nuclease and the mecA gene encoding methicillin resistance. For the 475 positive blood cultures tested, the assay turned out to have 100% sensitivity and 100% specificity for the identification of methicillin-susceptible (n = 108) and methicillin-resistant (n = 34) S. aureus. When coagulase-negative staphylococci (CoNS) were included, the overall sensitivity for the detection of methicillin resistance was 93% and the specificity was 99%. Real-time PCR for nuc and mecA from blood culture bottles with staphylococci yields therefore a rapid (2-3 h) identification of S. aureus and CoNS including methicillin resistance.

Metallo-beta-lactamase expressing multi-resistant Acinetobacter baumannii transmitted in the operation area.

Outbreaks of Acinetobacter baumannii demonstrating multiple antibiotic resistance, including meropenem resistance, have been described as severe therapeutic problems. Here we describe a monoclonal outbreak of infection and colonization with multidrug-resistant A. baumannii over a two-month period. Resistance to meropenem was mediated by expression of a metallo-beta-lactamase enzyme. Four of 14 patients showed clinical signs of infection and two died. Contamination of the environment, water, or instruments were excluded as causes of the outbreak. All patients, except one, underwent surgery in a specific operation theatre where surgery of contamination class IV (infected, dirty) was performed. Although individual surgeon error was eliminated, analyses of the patients' histories suggested that bacterial transmission had occurred during surgery. Five patients showed signs of A. baumannii infection and two of these patients suffered from large abdominal wounds infected with a high density of A. baumannii requiring repeated revisions. Presumably, these revisions favoured the transmission of A. baumannii, which is remarkably resistant to various environmental stresses including soaps, disinfectants and dry conditions. No case of meropenem-resistant A. baumannii had been observed in the hospital before the outbreak. Interestingly, the resistant bacteria appear to have been imported by a patient returning from West Africa. This indicates that, similar to MRSA, multiresistant A. baumannii may be introduced by patients from foreign hospitals. The outbreak was stopped in the following months by reinforcing standard procedures and by taking all necessary precautions such as patient isolation, and finally only one new case was detected.

Metrizamide gradient centrifugation of histone-DNA complexes.

Mouse DNA was complexed with different combinations of histones. Most of these complexes were separated in metrizamide gradient into two fractions--heavy and light. At the protein: DNA ratio of 0.8 a complex of DNA with five histones contained 60% of DNA in the heavy fraction whereas in the absence of histone H1, this fraction contained only 40% of DNA. Complexes of DNA with the H2A + H2B histone pair banded in metrizamide as one, light fraction at the density of 1.140 g/cm3. DNA reconstituted with the H3 + H4 histone pair formed in metrizamide two peaks of 1.240 g/cm3 and 1.140 g/cm3. Among complexes of DNA with single histones H2A, H2B, H3 and H4 only the complex DNA-H4 formed a component having in metrizamide a density of 1.200 g/cm3, similar to the density of purified chromatin.

[Interaction of SV40 T-antigen with tumor cell chromatin]. / Vzaimodeistvie T-antigena virusa SV40 s khromatinom opukholevykh kletok.

The T-antigen of SV40 virus can be found in purified chromatin prepared from virus-induced tumour cells of the Syrian hamster. After treatment of chromatin or isolated nuclei with micrococcal nuclease this protein is detected in the high molecular weight and oligonucleosomal fractions. Data from sedimentation analysis and gel electrophoresis suggest that the T-antigen is predominantly linked with the oligonucleosomal fraction and in a lesser degree with mononucleasomes containing linker DNA and histone H1. A small amount of the T-antigen is found in the mononucleosome complex devoid of histone H1; however, the ratio of the T-antigen to DNA in this case is about 30 times less than that in the oligonucleosomal fraction. In order to investigate the nature of T-antigen binding to nucleosomes, the interaction between the T-antigen and nucleosomes from normal rat liver was studied under restricted binding of the antigen to DNA (pH 8.0). The T-antigen was effectively bound to the nucleosomes and coprecipitated with them in 5 mM MgCl2. It was shown that the T-antigen was adsorbed on columns packed with immobilized histones H1 and nucleosomal histones without H1; the former eluted at 0.15 - 0.25 M NaCl, the latter - at 0.35 - 0.5 M NaCl. The possibility of T-antigen interaction with cellular DNA and protein components of chromatin (primarily to H1) is discussed.

Complex of DNA with chromatin proteins investigated by isopycnic centrifugation in metrizamide.

Complexes of mouse main band DNA with a fraction of non-histone proteins (NHP), having a high affinity for DNA, in the absence or presence of histones have been investigated by gradient centrifugation in metrizamide. Two types of complexes were formed at an input ratio of NHP to DNA between 1 and 2.5. In metrizamide gradients a majority of DNA was found in the light complex (at the density of 1.14-1.16 g/cm3) even at the very high NHP to DNA ratio. When histones were present in the reaction mixture, most of the DNA was found in the heavy complex (1.19-1.21 g/cm3). The electrophoretic profiles of the proteins recovered from the heavy and light complexes were different; some fractions of nonhistone proteins were present only in the heavy component.