Effect of UV irradiation on transduction by coliphage T1.

Lysates of the virulent coliphage T1 transduce seven markers between strains of Escherichia coli with reproducilble efficiencies which range from 10(-7) to 10(-5). The ability of a UV-irradiated lysate to transduce Arg(+), Str(R), Trp(+), Lac(+), and Pro(+) is 90% and Bio(+) is 99% inactivated by doses which inactivate plaque formation of T1 by six orders of magnitude. A dose of irradiation which causes a 1- to 2-log drop in the titer of T1 stimulated Gal(+) transduction by two- to three-fold; no other marker tested was stimulated. Irradiation causes dislinkage of some co-transducible markers but not others.     

Angiotensin II induces MMP-2 in a p47phox-dependent manner

Activated matrix metalloproteinases (MMPs) in patients with acute coronary syndromes may contribute to plaque destabilization. Since reactive oxygen species (ROS) induce MMP-2 and angiotensin II (ANG II) enhances NADPH-oxidase-dependent ROS formation, we assessed whether ANG II induces MMP-2 in a NADPH-oxidase-dependent manner. MMP-2 mRNA expression and activity were analyzed in wildtype and p47phox-deficient (p47phox-/-) murine smooth muscle cells (SMC). To address a clinical implication, sections of human atherosclerotic arteries were stained for MMP-2, p47phox, ANG II, AT1-receptor, and alpha-smooth muscle cell actin (alpha-SMC actin). MMP-2 protein expression and activity from these arteries were compared to those without atherosclerosis. ANG II enhances mRNA synthesis and activity of MMP-2 in a p47phox-dependent manner. Immunohistochemical analyses revealed a co-localization of MMP-2 with p47phox, ANG II, AT1-receptor, and alpha-SMC actin. MMP-2 protein expression and gelatinolytic activity are increased in atherosclerotic arteries. Thus, activation of the renin-angiotensin system may contribute to plaque destabilization via ROS-dependent induction of MMP-2.     

Inhibition of proteasome function induces programmed cell death in proliferating endothelial cells.

Proteolysis mediated by the ubiquitin-proteasome system has been implicated in the regulation of programmed cell death. Here we investigated the differential effects of proteasomal inhibitors on the viability of proliferating and quiescent primary endothelial cells in vitro and in vivo. Subconfluent, proliferating cells underwent carbobenzoxy-L-isoleucyl-gamma-t-butyl-L-glutamyl-L-alanyl-L-leucinal (PSI) -induced apoptosis at low concentrations (EC(50)=24 nM), whereas at least 340-fold higher concentrations of PSI were necessary to obtain the same effect in confluent, contact-inhibited cells. PSI-mediated cell death could be blocked by a caspase-3 inhibitor (Ac-DEVD-H), but not by a caspase-1 inhibitor (Ac-YVAD-H), suggesting that a caspase-3-like enzyme is activated during PSI-induced apoptosis. When applied to the embryonic chick chorioallantoic membrane, a rapidly expanding tissue, PSI induced massive apoptosis also in vivo. PSI treatment of the CAM led to the formation of areas devoid of blood flow due to the induction of apoptosis in endothelial and other cells and to the collapse of capillaries and first order vessels. Our results demonstrate that proteasomal inhibitors such as PSI may prove effective as novel anti-angiogenic and anti-neoplastic substances.     

Massenspektrometrische untersuchungen an äther-estern der 2,3-dihydroxypropionsäure

The mass spectra of two homologous series of 2,3-dialkoxypropionic acid alkylesters with ester moieties ranging from 8 to 22 C-atoms and ether chains having 12 or 16 C-atoms have been studied and were discussed. Fragmentation starts by splitting off one of the two alkoxy groups. Consequently, two courses of fragmentation could be observed. This was proved by the mass spectrum of the newly synthetized DL-2-tetradecyl-3-hexadecyloxypropionic acid dodecylester.     

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.     

Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells

The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C (RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant. We thus, provide direct evidence for a role of mammalian Rad51C in HR processes. The reduced HR events described here help to explain the deficient phenotypes observed in Rad51C mutants and support an accessory role of Rad51C in Rad51-mediated recombination.     

Transduction of bacteriophage lambda by bacteriophage T1.

When bacteriophage T1 was grown on bacteriophage lambda-lysogenic cells, phenotypically mixed particles were formed which had the serum sensitivity, host range, and density of T1 but which gave rise to lambda phage. T1 packaged lambda genomes more efficiently both when the length of the prophage was less than that of wild-type lambda and when the host cell was polylysogenic. Expression of the red genes of lambda or the recE system of Escherichia coli during T1 growth enhanced pickup of lambda by T1, whereas packaging was reduced in recB cells. If donors were singly lysogenic, the expression of transduced lambda genomes as a PFU required lambda-specified excisive recombination, whereas lambda genomes transduced from polylysogens required only lambda- or E. coli-specified general recombination to give a productive infection.     

Reproductive barriers between two sympatric beetle species specialized on different host plants

Knowledge on interspecific pre‐ and post‐zygotic isolation mechanisms provides insights into speciation patterns. Using crosses (F₁ and backcrosses) of two closely related flea beetles species, Altica fragariae and A. viridicyanea, specialized on different hosts in sympatry, we measured: (a) the type of reproductive isolation and (b) the inheritance mode of preference and host‐specific performance, using a joint‐scaling test. Each species preferred almost exclusively its host plant, creating strong prezygotic isolation between them, and suggesting that speciation may occur at least partly in sympatry. Reproductive isolation was intrinsic between females of A. fragariae and either A. viridicyanea or F₁ males, whereas the other crosses showed ecologically dependent reproductive isolation, suggesting ecological speciation. The genetic basis of preference and performance was at least partially independent, and several loci coded for preference, which limits the possibility of sympatric speciation. Hence, both ecological and intrinsic factors may contribute to speciation between these species.     

Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines

In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.     

Cytogenetic harvesting of commonly used tumor cell lines

Tumor cell lines are widely used both as disease models and, increasingly, as genomic resources for the ascertainment of new cancer genes. Cytogenetic analysis remains a major route to uncovering the cancer genome. However, cancer cell lines vary inexplicably in their harvesting preferences, which must, therefore, be determined by trial and error. This article describes harvesting protocols optimized empirically for 550 commonly used, mainly human, cancer cell lines together with evidence-based procedures to assist in determining conditions for unlisted cell lines and subsidiary protocols for cytogenetic analysis using G-banding and fluorescence in situ hybridization.