Heterozygous PALB2 c.1592delT mutation channels DNA double-strand break repair into error-prone pathways in breast cancer patients.

Hereditary heterozygous mutations in a variety of DNA double-strand break (DSB) repair genes have been associated with increased breast cancer risk. In the Finnish population, PALB2 (partner and localizer of BRCA2) represents a major susceptibility gene for female breast cancer, and so far, only one mutation has been described, c.1592delT, which leads to a sixfold increased disease risk. PALB2 is thought to participate in homologous recombination (HR). However, the effect of the Finnish founder mutation on DSB repair has not been investigated. In the current study, we used a panel of lymphoblastoid cell lines (LCLs) derived from seven heterozygous female PALB2 c.1592delT mutation carriers with variable health status and six wild-type matched controls. The results of our DSB repair analysis showed that the PALB2 mutation causes specific changes in pathway usage, namely increases in error-prone single-strand annealing (SSA) and microhomology-mediated end-joining (MMEJ) compared with wild-type LCLs. These data indicated haploinsufficiency regarding the suppression of error-prone DSB repair in PALB2 mutation carriers. To the contrary, neither reduced HR activities, nor impaired RAD51 filament assembly, nor sensitization to PARP inhibition were consistently observed. Expression of truncated mutant versus wild-type PALB2 verified a causal role of PALB2 c.1592delT in the shift to error-prone repair. Discrimination between healthy and malignancy-presenting PALB2 mutation carriers revealed a pathway shift particularly in the breast cancer patients, suggesting interaction of PALB2 c.1592delT with additional genomic lesions. Interestingly, the studied PALB2 mutation was associated with 53BP1 accumulation in the healthy mutation carriers but not the patients, and 53BP1 was limiting for error-prone MMEJ in patients but not in healthy carriers. Our study identified a rise in error-prone DSB repair as a potential threat to genomic integrity in heterozygous PALB2 mutation carriers. The used phenotypic marker system has the capacity to capture dysfunction caused by polygenic mechanisms and therefore offers new strategies of cancer risk prediction.

siRNA screening identifies differences in the Fanconi anemia pathway in BALB/c-Trp53+/- with susceptibility versus C57BL/6-Trp53+/- mice with resistance to mammary tumors.

BALB/c mice heterozygous for Trp53 develop a high proportion of spontaneous mammary tumors, a phenotype distinct from other mouse strains. BALB/c-Trp53+/- female mice, thus, resemble the hereditary Li-Fraumeni syndrome (LFS) characterized by early-onset of breast cancer, even though LFS involves TP53 mutations, which may involve not only loss- but also gain-of-function. Previous analysis of tumors in BALB/c-Trp53+/- females showed frequent loss of heterozygosity involving the wild-type allele of Trp53 and displayed characteristics indicative of mitotic recombination. Critical involvement of DNA double-strand break (DSB) repair dysfunction, particularly of homologous recombination (HR), was also noticed in the etiology of human breast cancer. To better define functional alterations in BALB/c-Trp53+/- mice, we applied a fluorescence-based DSB repair assay on mouse embryonic fibroblasts (MEFs) from BALB/c-Trp53+/- versus C57BL/6J-Trp53+/- mice. This approach revealed deregulation of HR but not non-homologous end-joining (NHEJ) in BALB/c-Trp53+/-, which was further confirmed for mammary epithelial cells. Screening of a small interfering RNA-library targeting DSB repair, recombination, replication and signaling genes, identified 25 genes causing differences between homologous DSB repair in the two strains upon silencing. Interactome analysis of the hits revealed clustering of replication-related and fanconi anemia (FA)/breast cancer susceptibility (BRCA) genes. Further dissection of the functional change in BALB/c-Trp53+/- by immunofluorescence microscopy of nuclear 53BP1, Replication protein A (RPA) and Rad51 foci uncovered differences in crosslink and replication-associated repair. Chromosome breakage, G2 arrest and biochemical analyses indicated a FA pathway defect downstream of FancD2 associated with reduced levels of BRCA2. Consistent with polygenic models for BRCA, mammary carcinogenesis in BALB/c-Trp53+/- mice may, therefore, be promoted by a BRCA modifier allele in the FA pathway in the context of partial p53 loss-of-function.

Holographic grating formation in poly(spiropyran I-glutamate).

An argon-ion laser interferometer (0.3638 or 0.488 microm) wrote photoreversible holographic grating (fringe spacing 0.39-100 microm) on spin-cast thin films (5 microm thick) of poly(spiropyran l-glutamate).

Kinetic study of the helix to coil dark reaction of poly(spiropyran-L-glutamate).

An investigation of kinetics of the helix to coil dark reaction of light adapted poly(spiropyran-L-glutamic acid) (PSLG) dissolved in hexafluoroisopropanol was performed. The reaction was associated with the spiropyran (SP) to merocyanine (MC) ring opening. The ring opening reaction monitored with UV/VIS spectroscopy showed first order kinetics. Chromophore and polypeptide backbone circular dichroism data fit to an expression consistent with a single intermediate series mechanism. By FTIR, we monitored the polypeptide alpha-helix amide I, the MC chromophore--C = C--stretch and the protonated unmodified carboxylate C = O stretch bands. During the first step of the series mechanism, changes in the hydrogen bonding of the unmodified carboxylate groups occurred, suggesting breakup of polypeptide aggregates. The second step of the proposed series mechanism was dominated by the helix to coil transition and the ring opening of SP to MC. The CD spectrum of MC in the dark adapted PSLG was red shifted and had a narrower bandwidth than the UV/VIS spectrum. The kinetic and spectroscopic data suggested that a fraction (population I) of the MC chromophores experienced optical activity induced by the chiral polypeptide environment, while the remainder of the MC chromophores (population II) were solvated and enantiomeric.

[Pharmacokinetics and biotransformation of the analgesic flupirtine in the rat and dog]. / Untersuchungen zur Pharmakokinetik und Biotransformation des Analgetikums Flupirtin bei Ratte und Hund.

Pharmacokinetics and biotransformation of 14C-labelled ethyl-N-[2-amino-6-(4-fluorophenylmethylamino)pyridin-3-yl]carbama te maleate (flupirtine maleate, D 9998 maleate) was studied in rats and dogs. The drug was rapidly and completely absorbed after peroral administration in both species. The kinetics of the plasma levels after intravenous administration show a short distribution phase followed by an elimination phase with half-lives between 2 and 3 h. Similar half-lives were observed after peroral administration: 2.2. h in the rat and 2.6 h in the dog. Renal excretion amounts to 20% (rat) and 36% (dog) after i.v. administration, and to 22% (rat) and 35% (dog) after p.o. administration. The major part of the dose is excreted via the feces. The drug is reversibly distributed to the tissues. Similar concentrations appear in the well perfused organs. A brain/plasma concentration ratio of greater than or equal to 1 was found and is a favourable prerequisite for a centrally acting analgesic. Insight in the biotransformation pathways of 14C-flupirtine maleate was obtained by structure determination of urinary metabolites. The urinary radioactivity of the rat consisted practically exclusively of p-fluoro-hippuric acid that is generated by an oxydative metabolic degradation of flupirtine. Dog urine, too, contains this metabolite, however, accompanied by the drug excreted unchanged and by a further metabolite structurally still very similar to flupirtine. The latter metabolite is formed via acetylation of an in vivo hydrolysis product of flupirtine and retains 1/4 of the analgesic potency of flupirtine. Regarding the patterns of excretion and of biotransformation the dog represents an intermediate between rat and man.

[Effect of tinofedrine on oxygen, glucose and lactate metabolism in dogs and rats]. / Einflub von Tinofedrin auf den Sauerstoff-, Glukose- und Laktat-Stoffwechsel des Hundes und der Ratte.

(+)-(R)-alpha(S)-1-[(3-3-Di-3-thienylallyl) amino]-ethyl-benzyl alcohol hydrochloride (tinofedrine hydrochloride) is a newly synthetized hemodynamic and cerebrally active substance. The results were obtained with dogs by synchronous measurement of vertebral and local cerebral blood flow, and assessment of O2, glucose and lactate metabolisms, In rats the cerebral glucose uptake index was taken as a scale for the glucose extraction. With dogs i.v. administration of tinofedrine causes, besides a marked and long-lasting rise of the cerebral blood flow, a strong increase of the glucose extraction. Compared to glucose up-take, the O2 increase is much smaller and lactate delivery unchanged. This indicates, besides a metabolic degradation, the utilization of the obtained glucose as a depot substate. Also in rats tinofedrine causes a significant glucose extraction, expressed as a remarkable rise of the brain uptake index.

[The influence of tinofedrine on cerebral energy metabolism in normotensive, hypotensive and hypoxemic rats after carotis ligation (author's transl)]. / Einfluss von Tinofedrin auf den zerebralen Energiestoffwechsel von normotonen, hypotonen und hypoxämischen Ratten nach Carotis-Verschluss.

l-(+)-alpha-(1-[(3,3-Di-3-thienylallyl)amino]-ethyl)-benzyl alcohol hydrochloride (tinofedrine hydrochloride, D 8955, Novocebrin), a new cerebrally active substance, synthetized in our research laboratories, was investigated for its action on the disturbed cerebral metabolism in rats. By variation of the arterial blood pressure (normal, 100 and 70 mmHg), and O2-concentration for the artificial ventilation of 30 or 15 vol% and carotis ligation for 15, 30 and 60 min different degrees of cerebral disturbances could be induced. The glucose concentration and energy state of the brain were used as criteria of the degree of cerebral disturbance. After intravenous injection of tinofedrine such a disturbed cerebral state could be normalized partially or completely.

[Studies on the pharmacokinetics and metabolism of tinofedrine]. / Untersuchungen zu Pharmakokinetik und Metabolismus von Tinofedrin.

Pharmacokinetics and biotransformation of l-(+)-alpha-(1-[(3,3-di-3-thienylallyl)-amino]-ethyl)-3-benzyl alcohol hydrochloride (tinofedrine hydrochloride, D 8955, Novocebrin) were investigated in rat and dog by means of the 3H-labelled drug. After i.v. administration similar biphasic time courses of the plasma levels are seen in the rat and the dog. The elimination is linear with a half-life of 3.0 and 3.8 h, respectively. After peroral administration 3H-tinofedrine is absorbed much more rapidly by the dog than by the rat. Radioactivity is subsequently eliminated from plasma by both species at nearly identical speed, which corresponds to that after i.v. administration. Quantitative analysis of the organ distribution after i.v. administration in the rat reveals a distinct affinity of 3H-tinofedrine for the lung. All tissue bindings, however, after both routes of administration are reversible. After preoral administration the drug is absorbed nearly quantitatively in the dog and in the rat with 50--63%. Only unchanged tinofedrine can be detected in the blood of the mesenteric veins. Tinofedrine is completely metabolized. In the rat and in the dog three different conjugates are formed, two of which are excreted with the bile. During their way through the intestine they are split again into tinofedrine, which is found in the feces. 3H-Tinofedrine after i.v. and peroral administration is mainly excreted fecally by the rat and the dog. This is caused by the great extent of the biliary excretion of the metabolites.

[Studies on pharmacokinetics and biotransformation of reproterol in animal and man (author's transl)]. / Untersuchungen zur Pharmakokinetik und Biotransformation von Reproterol bei Tier und Mensch

Investigations on the pharmacokinetics and biotransformation in the rat, dog, rabbit and in humans were performed with 3H-or 14C-labelled 7-(3-[2-(3,5-dihydroxyphenyl)-2-hydroxy-ethlamino]-propyl)-theophylline (reproterol, Bronchospasmin). Following i.v. administration of reproterol, a similar course of the plasma levels as shown in both rat and dog. After oral administration to the rat, elimination occurs within 2 h following application; in the dog, however, a relatively constant plasma level persists for up to 24 h, which is then reduced during an elimination half-life of 12.4 h. Following i.v. as well as oral administration to the rabbit, phases of distribution and elimination persist over a considerable length of time. Plasma levels following oral administration remain relatively constant during a time period of 8--30 h, after which they decrease with a half-life of 70 h. Renal elimination in the dog and rabbit after i.v. application seems to be the main route of excretion (dog 57%, rabbit 66%), while in the rat there is 58% fecal elimination. Absorption ratios following oral administration amount to 13% in the rabbit and 18% in the rat and dog. The absorption ration in the rat following intratracheal application reaches 90%. This was particularly important in view of the anticipated use of reproterol as an aerosol. Tests on the quantitative organic distribution further showed that in the rat, the lund tissue has a particular affinity to reproterol. In man following i.v. administration, reproterol is rapidly distributed and eliminated. The highest plasma level reached within 2 h after oral administration, correlates well with the initial plasma level following i.v. administration. A great similarity was shown for the reproterol metabolism in rat, dog, rabbit and man. With complete metabolization, the same main metabolite is always formed.