Homologous recombination repairs secondary replication induced DNA double-strand breaks after ionizing radiation

Ionizing radiation (IR) produces direct two-ended DNA double-strand breaks (DSBs) primarily repaired by non-homologous end joining (NHEJ). It is, however, well established that homologous recombination (HR) is induced and required for repair of a subset of DSBs formed following IR. Here, we find that HR induced by IR is drastically reduced when post-DNA damage replication is inhibited in mammalian cells. Both IR-induced RAD51 foci and HR events in the hprt gene are reduced in the presence of replication polymerase inhibitor aphidicolin (APH). Interestingly, we also detect reduced IR-induced toxicity in HR deficient cells when inhibiting post-DNA damage replication. When studying DSB formation following IR exposure, we find that apart from the direct DSBs the treatment also triggers formation of secondary DSBs peaking at 7-9 h after exposure. These secondary DSBs are restricted to newly replicated DNA and abolished by inhibiting post-DNA damage replication. Further, we find that IR-induced RAD51 foci are decreased by APH only in cells replicating at the time of IR exposure, suggesting distinct differences between IR-induced HR in S- and G2-phases of the cell cycle. Altogether, our data indicate that secondary replication-associated DSBs formed following exposure to IR are major substrates for IR-induced HR repair.     

Allelic dimorphism-associated restriction of recombination in Plasmodium falciparum msp1

Allelic dimorphism is a characteristic feature of the Plasmodium falciparum msp1 gene encoding the merozoite surface protein 1, a strong malaria vaccine candidate. Meiotic recombination is a major mechanism for the generation of msp1 allelic diversity. Potential recombination sites have previously been mapped to specific regions within msp1 (a 5' 1-kb region and a 3' 0.4-kb region) with no evidence for recombination events in a central 3.5-kb region. However, evidence for the lack of recombination events is circumstantial and inconclusive because the number of msp1 sequences analysed is limited, and the frequency of recombination events has not been addressed previously in a high transmission area, where the frequency of meiotic recombination is expected to be high. In the present study, we have mapped potential allelic recombination sites in 34 full-length msp1 sequences, including 24 new sequences, from various geographic origins. We also investigated recombination events in blocks 6 to 16 by population genetic analysis of P. falciparum populations in Tanzania, where malaria transmission is intense. The results clearly provide no evidence of recombination events occurring between the two major msp1 allelic types, K1-type and Mad20-type, in the central region, but do show recombination events occurring throughout the entire gene within sequences of the Mad20-type. Thus, the present study indicates that allelic dimorphism of msp1 greatly affects inter-allelic recombination events, highlighting a unique feature of allelic diversity of P. falciparum msp1.     

Vaccines to treat cancer--an old approach whose time has arrived

There are extensive DNA changes in tumor cells and the genes of tumor cells continuously mutate at a high rate. While this can provide therapeutic targets, it makes it unlikely that an agent that is selective for a single target will work against all cells in a tumor. However, it may be possible to use tumor epitopes as sentinels to engage adaptive and innate immunological mechanisms and create a tumor destructive environment effective also against variant cells that have lost a given antigen or their ability to present it. We hypothesize that therapeutic tumor vaccines, in combination with the targeting, to tumors, of costimulatory molecules such as anti-CD137scFv, or lymphokines such as GMCSF, will expand anti-tumor responses for therapeutic benefit when used as an adjunct to surgery and chemotherapy.     

Dominant Mutations in GRHL3 Cause Van der Woude Syndrome and Disrupt Oral Periderm Development

Mutations in interferon regulatory factor 6 (IRF6) account for ∼70% of cases of Van der Woude syndrome (VWS), the most common syndromic form of cleft lip and palate. In 8 of 45 VWS-affected families lacking a mutation in IRF6, we found coding mutations in grainyhead-like 3 (GRHL3). According to a zebrafish-based assay, the disease-associated GRHL3 mutations abrogated periderm development and were consistent with a dominant-negative effect, in contrast to haploinsufficiency seen in most VWS cases caused by IRF6 mutations. In mouse, all embryos lacking Grhl3 exhibited abnormal oral periderm and 17% developed a cleft palate. Analysis of the oral phenotype of double heterozygote (Irf6^+/−;Grhl3^+/−) murine embryos failed to detect epistasis between the two genes, suggesting that they function in separate but convergent pathways during palatogenesis. Taken together, our data demonstrated that mutations in two genes, IRF6 and GRHL3, can lead to nearly identical phenotypes of orofacial cleft. They supported the hypotheses that both genes are essential for the presence of a functional oral periderm and that failure of this process contributes to VWS.     

Enhanced persistence in the colonic microbiota of Escherichia coli strains belonging to phylogenetic group B2: role of virulence factors and adherence to colonic cells

Escherichia coli segregates into four phylogenetic groups, A, B1, B2 and D. B2 and D strains usually possess virulence factors, cause most extra-intestinal infections and have superior capacity to persist in the infantile colonic microbiota. Here, we investigated 24 resident and 37 transient E. coli strains from the colonic microbiota of 13 Swedish schoolgirls sampled in the 1970s with respect to phylogenetic group identity, carriage of virulence factor genes, O and K antigens and mannose-sensitive and -resistant adherence to the colonic cell line HT-29. Resident strains more often belonged to phylogenetic group B2 than transient strains (38% vs 5% p=0.004). In contrast, transient strains more often than resident strains belonged to group A (57% vs 29%, p=0.04) or B1 (24% vs 13%, p=0.33). Most B2 strains belonged to uropathogenic O serogroups, carried genes for P fimbriae, K5 capsule and hemolysin and adhered in higher numbers to HT-29 cells via mannose-resistant mechanisms than strains from the other groups. Further, among strains carrying genes for P or S fimbriae, those belonging to group B2 adhered in highest numbers. In logistic regression, genes for P fimbriae and aerobactin predicted persistence in the colonic microbiota (p=0.050 and 0.056, respectively), while B2 origin did not reach significance as an independent variable (p=0.16). Our results indicate that virulence factors carried by group B2 strains contribute to their strong colonizing capacity. These factors may actually be regarded as fitness factors in the human gut.     

Restriction map of virulence plasmid in Yersinia enterocolitica O:3

Restriction map of the 72-kb virulence plasmid isolated from Yersinia enterocolitica O:3 was generated using EcoRI, BamHI, HindIII, and XbaI restriction enzymes. The mapping was done after cloning all of the 13 BamHI fragments of the plasmid in Escherichia coli. In addition, the restriction enzyme analysis revealed two types of virulence plasmids (types I and II) in Y. enterocolitica O:3. No functional differences between the strains bearing type I or type II plasmid were observed.     

Autoimmunity to glial antigens in vitro.

Lymph nodes cells and spleen cells in a 50:50 mixture from Fischer 344 rats were cultured on syngeneic glial cells and fibroblasts. In the glia cultures, but not in the fibroblast cultures, the lymphocytes were stimulated to a vivid blast transformation and mitotic activity with a peak after 7 days, after which they reverted to small- and medium-sized lymphocytes. The stimulated lymphoid cells were not cytotoxic to glial cells when tested in a microcytotoxicity assay. A fraction of the lymphoblasts and their progeny (approximately 4%) took a positive intracytoplasmic stain for γ-globulin immunoglobulin G after direct immunofluorescence. Efforts to demonstrate immunoglobulin produced and released into the culture medium by the stimulated cells were negative. The findings may indicate that among the lymphoid cells responding to the glial antigens under these conditions, there are suppressor cells that abrogate the killer cell effect.     

Cellular immunity and blocking serum activity in chimeric mice

Mice (9 CBA × T6, 6 T6, and 7 CBA) were irradiated and repopulated with foreign (BALB/c) bone marrow. Lymph node cells from 16 of 18 repopulated mice not showing signs of graft versus host disease (GVH), were cytotoxic to host type fibroblasts but not to BALB/c fibroblasts, and sera from the same mice could block lymphocyte mediated cytotoxicity. No blocking was seen with sera from three mice which had signs of GVH. LNC from the latter three mice were cytotoxic to recipient fibroblasts.It is suggested that the blocking effect detected in vitro may protect against GVH in vivo, but the relative importance of the blocking phenomenon as compared to other mechanisms is not yet settled.     

Sequence analyses of fimbriae subunit FimA proteins on Actinomyces naeslundii genospecies 1 and 2 and Actinomyces odontolyticus with variant carbohydrate binding specificities

Background  

Actinomyces naeslundii genospecies 1 and 2 express type-2 fimbriae (FimA subunit polymers) with variant Galβ binding specificities and Actinomyces odontolyticus a sialic acid specificity to colonize different oral surfaces. However, the fimbrial nature of the sialic acid binding property and sequence information about FimA proteins from multiple strains are lacking.