A CHEK2 genetic variant contributing to a substantial fraction of familial breast cancer

CHEK2 (previously known as "CHK2") is a cell-cycle-checkpoint kinase that phosphorylates p53 and BRCA1 in response to DNA damage. A protein-truncating mutation, 1100delC in exon 10, which abolishes the kinase function of CHEK2, has been found in families with Li-Fraumeni syndrome (LFS) and in those with a cancer phenotype that is suggestive of LFS, including breast cancer. In the present study, we found that the frequency of 1100delC was 2.0% among an unselected population-based cohort of 1,035 patients with breast cancer. This was slightly, but not significantly (P=.182), higher than the 1.4% frequency found among 1,885 population control subjects. However, a significantly elevated frequency was found among those 358 patients with a positive family history (11/358 [3.1%]; odds ratio [OR] 2.27; 95% confidence interval [CI] 1.11-4.63; P=.021, compared with population controls). Furthermore, patients with bilateral breast cancer were sixfold more likely to be 1100delC carriers than were patients with unilateral cancer (95% CI 1.87-20.32; P=.007). Analysis of the 1100delC variant in an independent set of 507 patients with familial breast cancer with no BRCA1 and BRCA2 mutations confirmed a significantly elevated frequency of 1100delC (28/507 [5.5%]; OR 4.2; 95% CI 2.4-7.2; P=.0002), compared with controls, with a high frequency also seen in patients with only a single affected first-degree relative (18/291 [6.2%]). Finally, tissue microarray analysis indicated that breast tumors from patients with 1100delC mutations show reduced CHEK2 immunostaining. The results suggest that CHEK2 acts as a low-penetrance tumor-suppressor gene in breast cancer and that it makes a significant contribution to familial clustering of breast cancer-including families with only two affected relatives, which are more common than families that include larger numbers of affected women.     

Preventive oncologic check-ups in the District Sumperk, 1975-1986: the description of a database

From 1975 through 1986, repeated preventive oncologic check-ups were carried out in the District Sumperk. In the course of the check-ups, 49,426 persons were examined. The examinations were aimed at surveillance of initial stadium of cancers as well as at detection of other serious chronic diseases. The results of the check-ups form a large database suitable for a number of epidemiological analyses. In this article, methods of the data collection are described, individual data items are characterized and their summary is presented in tables. Utilization of the database is proposed in specified types of epidemiological studies.     

Antineutrophil cytoplasmic antibodies directed against bactericidal/permeability-increasing protein detected in children with cystic fibrosis inhibit neutrophil-mediated killing of Pseudomonas aeruginosa

Antineutrophil cytoplasmic antibodies (ANCA) directed against bactericidal/permeability-increasing protein (BPI) were repeatedly found in cystic fibrosis (CF) patients. We analyzed the effect of BPI-ANCA in inhibiting neutrophil-mediated killing of Pseudomonas aeruginosa. The bactericidal effect expressed as percentage of killed bacteria after 1 h incubation with neutrophils was 55% when the neutrophils were pretreated with normal human serum, ranged from 49 to 63% with the sera from control BPI-ANCA-negative groups and sharply decreased to the mean 30.5% (range 8-51%) in the presence of BPI-ANCA. Furthermore, the effect mediated by BPI-ANCA was dose dependent and reflected the titer of BPI-ANCA in tested sera.     

Pectate hydrolases of parsley (Petroselinum crispum) roots

The presence of various enzyme forms with terminal action pattern on pectate was evaluated in a protein mixture obtained from parsley roots. Enzymes found in the soluble fraction of roots (juice) were purified to homogeneity according to SDS-PAGE, partially separated by preparative isoelectric focusing and characterized. Three forms with pH optima 3.6, 4.2 and 4.6 clearly preferred substrates with a lower degree of polymerization (oligogalacturonates) while the form with pH optimum 5.2 was a typical exopolygalacturonase [EC 3. 2.1.67] with relatively fast cleavage of polymeric substrate. The forms with pH optima 3.6, 4.2 and 5.2 were released from the pulp, too. The form from the pulp with pH optimum 4.6 preferred higher oligogalacturonates and was not described in plants previously. The production of individual forms in roots was compared with that produced by root cells cultivated on solid medium and in liquid one.     

Protective effects of nocardia delipidated cell mitogen on the mucosa of the small intestine after irradiation of germ-free piglets.

The radioprotective effect of the bacterial immunomodulator Nocardia delipidated cell mitogen (NDCM) on intestinal mucosa and disaccharidase activities was studied in irradiated germ-free piglets. Three-week-old germ-free (GF) piglets were intragastrically pretreated with 1 mg NDCM per 1 kg body weight. The piglets were whole-body irradiated with 2.5 Gray five days after the NDCM pretreatment and sacrificed eight days after irradiation. In the non-irradiated group of GF piglets, NDCM application stimulated lactase activity and markedly increased sucrase activity. This stimulatory effect of NDCM disappeared after irradiation and the piglets exhibited a normal activity of lactase in the jejunal brush-border membrane vesicles, while the sucrase activity decreased to the level found in irradiated controls. NDCM-pretreated intestinal mucosa contained some infrequent lymphocytes which disappeared from the control irradiated tissue. It also exhibited less injury of the epithelium and stroma cells.     

Generation and Phenotypic Analysis of a Transgenic Line of Rabbits Secreting Active Recombinant Human Erythropoietin in the Milk

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60–178 and 60–162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [³H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.     

TOPBP1 regulates RAD51 phosphorylation and chromatin loading and determines PARP inhibitor sensitivity

Topoisomerase IIβ-binding protein 1 (TOPBP1) participates in DNA replication and DNA damage response; however, its role in DNA repair and relevance for human cancer remain unclear. Here, through an unbiased small interfering RNA screen, we identified and validated TOPBP1 as a novel determinant whose loss sensitized human cells to olaparib, an inhibitor of poly(ADP-ribose) polymerase. We show that TOPBP1 acts in homologous recombination (HR) repair, impacts olaparib response, and exhibits aberrant patterns in subsets of human ovarian carcinomas. TOPBP1 depletion abrogated RAD51 loading to chromatin and formation of RAD51 foci, but without affecting the upstream HR steps of DNA end resection and RPA loading. Furthermore, TOPBP1 BRCT domains 7/8 are essential for RAD51 foci formation. Mechanistically, TOPBP1 physically binds PLK1 and promotes PLK1 kinase–mediated phosphorylation of RAD51 at serine 14, a modification required for RAD51 recruitment to chromatin. Overall, our results provide mechanistic insights into TOPBP1’s role in HR, with potential clinical implications for cancer treatment.     

TRIP12 and UBR5 Suppress Spreading of Chromatin Ubiquitylation at Damaged Chromosomes

Histone ubiquitylation is a prominent response to DNA double-strand breaks (DSBs), but how these modifications are confined to DNA lesions is not understood. Here, we show that TRIP12 and UBR5, two HECT domain ubiquitin E3 ligases, control accumulation of RNF168, a rate-limiting component of?a pathway that ubiquitylates histones after DNA breakage. We find that RNF168 can be saturated by increasing amounts of DSBs. Depletion of TRIP12 and UBR5 allows accumulation of RNF168 to supraphysiological levels, followed by massive spreading of ubiquitin conjugates and hyperaccumulation of ubiquitin-regulated genome caretakers such as 53BP1 and BRCA1. Thus, regulatory and proteolytic ubiquitylations are wired in a self-limiting circuit that promotes histone ubiquitylation near the DNA lesions but at the same time counteracts its excessive spreading to undamaged chromosomes. We provide evidence that this mechanism is vital for the homeostasis of ubiquitin-controlled events after DNA breakage and can be subverted during tumorigenesis.     

Sperm cells induce distinct cytokine response in peripheral mononuclear cells from infertile women with serum anti-sperm antibodies

Background and Aims

Anti-sperm antibodies in can markedly reduce the likelihood of natural conception. The etiology of this anti-sperm immunity in human females is unknown. We compared the cytokine response of peripheral blood mononuclear cells (PBMCs) from infertile patients with or without anti-sperm antibodies (ASA) and fertile women.

Methodology/Principal Findings

We cultivated the PBMCs together with sperm antigens (whole cells or cell lysate), and screened the supernatants for 40 cytokines by antibody array. When stimulated with whole sperm cells, the PBMCs from patients with ASA produce less IL-3, IL-11, IL-13, ICAM-1, GCSF and more IL-2, IL-4 and IL-12p70 as compared to healthy women. PBMCs from patients with ASA produce typically less IL-13, IL-7, IL-17 and MIG, and more MIP-1β and IL-8, as compared to PBMCs from patients without ASA. In response to sperm cell lysate, PBMCs from infertile women without ASA respond initially by increase in production of growth factors (GCSF, GM-CSF and PDGF-BB) followed by increase in chemokines (e.g. IL-8, MCP-1 and MIP-1β).

Conclusions

Cellular immune responses to sperm antigens, measured by production of cytokines, differ among infertile women with ASA, infertile women without ASA and healthy women. This difference could play an important role in the initial steps of the infertility pathogenesis.     

Effect of bacterial monoassociation on brush-border enzyme activities in ex-germ-free piglets: comparison of commensal and pathogenic Escherichia coli strains

This study was designed to investigate the effect of monoassociation of germ-free piglets with Escherichia coli strains on the development of intestinal brush-border enzyme activities. Piglets were delivered by hysterectomy, reared for seven days under germ-free conditions and fed milk formula diet. One group was maintained germ-free, the other four groups were monoassociated on day eight with one of four E. coli strains: non-pathogenic O86 or O83 and G58-1, or pathogenic 933D. The development of brush-border digestive enzyme functions in the small intestine was evaluated after 15 days. Germ-free controls exhibited slower developmental declines of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and delayed increases of sucrase and glucoamylase compared to conventionally grown animals. Association of germ-free piglets with the non-pathogenic E. coli strains O86 and O83 resulted in increased enterocyte differentiation along the length of the small intestine, accompanied by declining activities of lactase, gamma-glutamyltranspeptidase and alkaline phosphatase, and elevated activities of maturational markers such as sucrase and glucoamylase. Maturational changes also occurred along the villus-crypt axis, as revealed by histochemical localization of aminopeptidase N on the villi tips in piglets colonized with E. coli O83. Interestingly, colonization with the pathogenic E. coli strain 933D stimulated changes in the main differentiation enzyme markers lactase, sucrase and glucoamylase to an extent comparable with those produced by the non-pathogenic and probiotic E. coli strains. In conclusion, germ-free piglets represent a valuable tool to study the consequences of colonization of the immature sterile gut with defined strains of bacteria.