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A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

Systemic inflammatory responses in patients undergoing extracorporeal membrane oxygenation (ECMO) contribute significantly to ECMO-associated morbidity and mortality. In recent years, the number of type 2 diabetes mellitus patients has increased, and the number of these patients undergoing ECMO has also increased. Type 2 diabetes mellitus is a high-risk factor for complications during ECMO. We studied the effects of ECMO on inflammatory response in a diabetic rat ECMO model. Twenty-eight rats were divided into 4 groups: normal SHAM group (normal rats: n = 7), diabetic SHAM group (diabetic rats: n = 7), normal ECMO group (normal rats: n = 7), and diabetic ECMO group (diabetic rats: n = 7). We measured the plasma levels of cytokines, tumor necrosis factor-α, and interleukin-6. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine (Cr), and liver-type fatty acid binding protein (L-FABP) were examined in the rat cardiopulmonary bypass model to ascertain organ damage. In addition, the lung wet-to-dry weight (W/D) ratio was measured as an index of pulmonary tissue edema. A pathologic evaluation of kidneys was conducted by hematoxylin-eosin (HE) and periodic-acid-methenamine-silver (PAM) staining. In the diabetic ECMO group, levels of cytokines, AST, ALT, LDH, and L-FABP increased significantly, reaching a maximum at the end of ECMO in comparison with other groups (p &lt, 0.05). In addition, hematoxylin-eosin and periodic acid-methenamine-silver staining of renal tissues showed marked injury in the ECMO group (normal ECMO and diabetic ECMO groups). Furthermore, when the normal ECMO and diabetic ECMO groups were compared, severe organ injury was seen in the diabetic ECMO group. There was remarkable organ injury in the diabetic ECMO group. These data demonstrate that diabetes enhances proinflammatory cytokine release, renal damage, and pulmonary edema during ECMO in an animal model.

Loss of muscle mass and strength are progressing with aging. Exercise is a beneficial method to prevent physical dysfunction, and habitual exercise can improve the muscle quality. Therefore, we evaluated the effects of long-term habitual exercise&rsquo, s impact on sarcopenia utilizing the senescence-accelerated mice prone8 (SAMP8) model. Notably, 27 w SAMP8 were used in this study. Mice were classified into 28 (28 w) and 44 weeks old. The 44-week group was divided into the sedentary group (44 w) and a group exercising for 16 weeks (44 w + Ex). The 44 w + Ex performed habitual exercise from 28 to 44 weeks. Additionally, grip strength tests were performed with mice aged 28 and 44 weeks. Muscles were harvested and measured muscle weight at 44 w. Gastrocnemius decreased in 44 w, but was unchanged in 44 w + Ex. There was a trend for lower muscle grip strength in the 44 w group, but there was no change in 44 w + Ex. The phosphorylation levels of Akt and p70S6K as a protein synthesis marker were decreased in 44 w. Cytochrome c oxidase subunit IV (CoxIV) mRNA and protein levels decreased in 44 w. These results suggested that long-term habitual exercise attenuates muscle mass and strength decline, possibly through maintenance of muscle protein synthesis and mitochondrial maintenance.

Muscle mass and strength decrease with aging, but habitual exercise can maintain muscle health. β-Hydroxy-β-methyl butyrate calcium (HMB) and black ginger (BG) are anti-oxidants that have been reported to improve muscle protein metabolism and energy production; these molecules may have synergistic effects. The senescence-accelerated mouse-prone 8 (SAMP8) model is a useful model of muscle aging. Therefore, in this study, we explored how the combination of habitual exercise, HMB, and BG affected muscle aging. We used 28-week-old SAMP8 mice divided into five groups: control, exercise (Ex), Ex+BG, Ex+HMB, and Ex+BG+HMB (Ex+Comb). Mice were required to run on a treadmill for 16 weeks at 5 days per week. In 44-week-old mice, grip strength tests and dissection were conducted. Muscle weight was measured, and the gastrocnemius muscle was subjected to quantitative polymerase chain reaction and immunoblotting. Muscle mass and strength were preserved in the Ex+Comb group, and mitochondrial function was preserved through suppressing oxidative stress. Muscle protein synthesis signaling was improved in the Ex+Comb group. Autophagy and the ubiquitin system were normalized by Ex+Comb treatment. Overall, habitual exercise and HMB plus BG treatment maintained muscle health by suppressing oxidative stress, preserving mitochondrial function, and maintaining muscle protein metabolism in SAMP8 mice.

Loss of muscle mass and strength are progressing with aging. Exercise is a beneficial method to prevent physical disfunction and habitual exercise improve the muscle quality. Therefore, we evaluated the effects of a long-term habitual exercise on the senescence-accelerated mice prone8 (SAMP8). 27wk SAMP8 were used in this study. Mice were classified into 28 (28w) and 44 weeks old. The 44-week group was divided into the sedentary group (44w) and a group exercising for 16 weeks (44w+Ex). The 44w+Ex performed habitual exercise from 28 to 44 weeks. Additionally, grip strength tests were performed with mice aged 28 and 44 weeks. Mice were dissected and collected muscle samples and measured muscle weight at 44w. Gastrocnemius was decreased in 44w but were unchanged in 44w+Ex. Grip strength in 44w was lower trend, but there was no change in 44w+Ex. The phosphorylation levels of Akt and p70S6K as a protein synthesis marker were decreased in 44w. Cytochrome c oxidase subunit IV(COXIV) mRNA and protein levels decreased in 44w. These results suggested that long-term habitual exercise attenuated muscle mass and strength decline through improving muscle protein synthesis and mitochondrial function. In conclusion, long-term habitual exercise attenuated muscle mass and strength decline.

We previously characterized a 177-kDa allergen, M-177, from Dermatophagoides farinae. Thereafter, a counterpart to M-177 for Euroglyphus maynei was cloned as Eur m 14, and its sequence revealed that two environmental allergens, Mag 1 and Mag 3, are digested fragments of M-177. The aims of this study were to clone the cDNA of Der f 14 corresponding to M-177 and to elucidate the allergenic capacities of the N-terminal fragment of Der f 14 (Der f 14-N). Recombinant allergens were produced as trigger-factor-fused proteins in Escherichia coli. Der f 14-N showed the highest IgE-binding frequency among Der f 14-derived fragments in patients allergic to house dust mite by enzyme-linked immunosorbent assay. Der f 14-N showed the highest capacity to induce cell proliferation in murine lymphocyte and human peripheral mononuclear cells among Der f 14-derived fragments. Der f 14-N induced IL-13, IFN-γ and IL-17 production more than Der f 1 and Der f 2 in mouse, and induced IL-5 and IFN-γ production at levels comparable to those of Der f 1 and Der f 2 in some patients. The high prevalence of IgE binding to the Der f 14-N indicates that it could be an important mite allergen.

SMC5/6 function in genome integrity remains elusive. Here, we show that SMC5 dysfunction in avian DT40 B cells causes mitotic delay and hypersensitivity toward DNA intra‐ and inter‐strand crosslinkers (ICLs), with smc5 mutants being epistatic to FANCC and FANCM mutations affecting the Fanconi anemia (FA) pathway. Mutations in the checkpoint clamp loader RAD17 and the DNA helicase DDX11, acting in an FA‐like pathway, do not aggravate the damage sensitivity caused by SMC5 dysfunction in DT40 cells. SMC5/6 knockdown in HeLa cells causes MMC sensitivity, increases nuclear bridges, micronuclei, and mitotic catastrophes in a manner similar and non‐additive to FANCD2 knockdown. In both DT40 and HeLa systems, SMC5/6 deficiency does not affect FANCD2 ubiquitylation and, unlike FANCD2 depletion, RAD51 focus formation. SMC5/6 components further physically interact with FANCD2‐I in human cells. Altogether, our data suggest that SMC5/6 functions jointly with the FA pathway to support genome integrity and DNA repair and may be implicated in FA or FA‐related human disorders., The structural maintenance complex SMC5/6 interacts with and functions jointly with key components of the Fanconi anemia pathway to facilitate DNA repair and preserve genome stability.

Azo dyes, including Sudan I, Orange II and Orange G, are industrial dyes that are assumed to have genotoxic potential. However, neither the type of DNA damage induced nor the structural features responsible for toxicity have been determined. We used a panel of DNA-repair-pathway-deficient mutants generated from chicken DT40 cells to evaluate the ability of these azo dyes to induce DNA damage and to identify the type of DNA damage induced. We compared the structurally related azo dyes Sudan I, Orange II and Orange G to identify the structural features responsible for genotoxicity. Compared with wild type cells, the double-strand break repair defective RAD54-/-/KU70-/- cells were significantly more sensitive to Sudan I, but not to Orange II or Orange G. The quantum-chemical calculations revealed that Sudan I, but not Orange II or Orange G, has a complete planar aromatic ring structure. These suggest that the planar feature of Sudan I is critical to the inducing of double-strand breaks. In summary, we used a DNA-repair mutant panel in combination with quantum-chemical calculations to provide a clue to the chemical structure responsible for genotoxicity.

The high prevalence of house dust mite (HDM) allergy is a growing health problem worldwide, and the characterization of clinically important HDM allergens is a prerequisite for the development of diagnostic and therapeutic strategies. Here, we report a novel HDM allergen that belongs structurally to the highly conserved Rid/YjgF/YER057c/UK114 family (Rid family) with imine deaminase activity. Isolated HDM cDNA, named der f 34, encodes 128 amino acids homologous to Rid-like proteins. This new protein belongs to the Rid family and has seven conserved residues involved in enamine/imine deaminase activity. Indeed, we demonstrated that purified Der f 34 had imine deaminase activity that preferentially acted on leucine and methionine. Native Der f 34 showed a high IgE binding frequency as revealed by two-dimensional immunoblotting (62.5%) or ELISA (68%), which was comparable with those of a major HDM allergen Der f 2 (77.5 and 79%, respectively). We also found that Der f 34 showed cross-reactivity with another prominent indoor allergen source, Aspergillus fumigatus. This is the first report showing that the Rid family imine deaminase represents an additional important pan-allergen that is conserved across organisms.

ALC1 (amplified in liver cancer 1), an SNF2 superfamily chromatin-remodeling factor also known as CHD1L (chromodomain helicase/ATPase DNA binding protein 1-like), is implicated in base-excision repair, where PARP (Poly(ADP-ribose) polymerase) mediated Poly(ADP-ribose) signaling facilitates the recruitment of this protein to damage sites. We here demonstrate the critical role played by ALC1 in the regulation of replication-fork progression in cleaved template strands. To analyze the role played by ALC1 as well as its functional relationship with PARP1, we generated ALC1-/-, PARP1-/-, and ALC1-/-/PARP1-/- cells from chicken DT40 cells. We then exposed these cells to camptothecin (CPT), a topoisomerase I poison that generates single-strand breaks and causes the collapse of replication forks. The ALC1-/- and PARP1-/- cells exhibited both higher sensitivity to CPT and an increased number of chromosome aberrations, compared with wild-type cells. Moreover, phenotypes were very similar across all three mutants, indicating that the role played by ALC1 in CPT tolerance is dependent upon the PARP pathway. Remarkably, inactivation of ALC1 resulted in a failure to slow replication-fork progression after CPT exposure, indicating that ALC1 regulates replication-fork progression at DNA-damage sites. We disrupted ATPase activity by inserting the E165Q mutation into the ALC1 gene, and found that the resulting ALC1-/E165Q cells displayed a CPT sensitivity indistinguishable from that of the null-mutant cells. This observation suggests that ALC1 contributes to cellular tolerance to CPT, possibly as a chromatin remodeler. This idea is supported by the fact that CPT exposure induced chromatin relaxation in the vicinity of newly synthesized DNA in wild-type but not in ALC1-/- cells. This implies a previously unappreciated role for ALC1 in DNA replication, in which ALC1 may regulate replication-fork slowing at CPT-induced DNA-damage sites.

DNA double strand breaks (DSBs) induced by etoposide, an inhibitor of DNA topoisomerase II, are repaired mainly by non-homologous end joining (NHEJ). Unexpectedly, it was found that at high doses of etoposide, proteins involved in NHEJ, such as KU70/80, DNA-PKcs and ARTEMIS/SNM1C, trigger apoptosis rather than repair of DSBs. Because ARTEMIS is a member of the SNM1 protein family that includes SNM1A and APOLLO/SNM1B, this study examined whether SNM1A and/or APOLLO are also involved in etoposide-induced apoptosis. Using SNM1A(-/-) and APOLLO(-/-) cells, it was found that both SNM1A and APOLLO participate in etoposide-induced apoptosis. Although cell viability monitored by MTT assay did not differ between SNM1A(-/-)/APOLLO(-/-)/ARTEMIS(-/-), SNM1A(-/-)/APOLLO(-/-), and single gene knockout cells, DNA fragmentation monitored by TUNEL assay differed between these cells, suggesting that the three SNM1 family nucleases function independently, at least during the induction of apoptotic DNA fragmentation.

舗装道路の路面間隙における雑草の埋土種子集団の有無と埋土種子の供給源を明らかにするために，栃木県小山市から青森県上北郡七戸町までの国道4号線を対象に，路面間隙および道路周辺の雑草植生と，間隙内から採取した土壌に含まれる雑草種子の種類，数および生存率を調査した。 その結果，1）舗装道路の路面間隙に埋土種子集団が形成されていること，2）メヒシバ（Digitaria ciliaris（Retz.）Koeler），エノコログサ（Setaria viridis（L.）Beauv.），タンポポ類（Taraxacum spp.），カタバミ類（Oxalis spp.）は比較的に大きな埋土種子集団を形成していること，3）オオバコ（Plantago asiatica L.）やヨモギ（Artemisia princeps Pampan.）などの雑草は路面間隙の優占種でありながら，埋土種子集団を形成していないこと，4）埋土種子の供給源は草種によって異なり，メヒシバのように路面間隙と道路周辺の両方から種子が供給されている雑草とノゲシ（Sonchus oleraceus L.）のように路面間隙と道路周辺以外からも種子が供給されている雑草があること，5）路面間隙内の雑草種子の生存率はオランダミミナグサ（Cerastium glomeratum Thuill.），カタバミ（Oxalis corniculata L.），ノミノツヅリ（Arenaria serpyllifolia L.），セイタカアワダチソウ（Solidago altissima L.），ナガハグサ（Poa pratensis L.）およびノボロギク（Senecio vulgaris L.）種子で高く，メヒシバおよびタンポポ類の種子で低いことが明らかになった。

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.

RECQL1 and RECQL5 as well as BLM reportedly interact with TOP3α whose defect is lethal for the cell. Therefore in this study, we characterized recql5 / recql1 / blm triple mutants from DT40 cells to determine whether the triple mutants show a top3α disrupted cell-like phenotype. The triple mutants are viable. Moreover, both blm / recql1 and recql5 / blm cells, and recql5 / recql1 / blm cells grew slightly slower than blm cells, that is, triple mutant cells grew almost the same rate as either of the double mutant cells. The blm cells showed sensitivity to methyl methanesulfonate (MMS) and ultraviolet light (UV), about a 10-fold increase in sister chromatid exchange (SCE), and about a 3-fold increase in damage-induced mitotic chiasma compared to wild-type cells. The triple mutants showed the same sensitivity to MMS or UV and the same frequency of damage-induced mitotic chiasma compared to those of blm cells, indicating that unlike BLM, RECQL1 and RECQL5 play a little role in the repair of or tolerance to DNA damages. However, recql5 / blm cells showed higher frequency of SCE than blm cells, whereas the RECQL1 gene disruption had no effect on SCE in blm cells and even in recql5 / blm cells.

Two kinds of PCR-product cDNAs that encode premature lysozyme peptides (Rs-Lys1 and Rs-Lys2) were cloned from workers of a Japanese damp-wood termite, Reticulitermes speratus. The Rs-Lys1 and Rs-Lys2 cDNAs encoded deduced sequences of 170 and 164 amino acids, respectively. Alignment of these sequences with those of other insect lysozymes showed that the cDNAs encode lysozyme homologues with putative signal peptides, insertions eight amino acids long, and a relatively long C-terminus (13-17 amino acids). A maximum likelihood tree, constructed using the cDNA sequences, indicated that the termite lysozymes are related to those of mosquitoes and lepidopterans. Southern-blotting analysis identified single copies of these lysozyme genes in the termite. Reverse transcript (RT)-PCR and in situ hybridization experiments showed that Rs-Lys1 and Rs-Lys2 are expressed in the salivary glands of worker termites. Here, we discuss the possible digestive function of these lysozymes.

Summary Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood-feeding termite, Reticulitermes speratus (Kolbe) (Isoptera, Rhinotermitidae) were studied to examine the possibility that termites digest symbiont bacteria transferred by trophallaxis. Total lysozyme activity was found predominantly in the salivary gland and to a minor extent in the digestive tracts. However, specific lysozyme activity was high in the foregut as well as in the salivary gland. The similarity of the lysozyme pH profile of the salivary gland and of the foregut suggested that the foregut lysozyme came from the salivary gland. Major protease activity having the optimum pH of 7.5 was found in the midgut. Total free amino acid amount and concentration in the midgut was higher than elsewhere in the digestive tract. The possibility that lysozyme secreted from the salivary gland into the foregut digests hindgut bacteria transferred by trophallaxis was discussed.

SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. We performed multiple amino acid alignments and examined the relationships between the SLC10 proteins. The extracellular N-terminus of SLC10A4 was predicted to be relatively longer at the amino acid level than those of SLC10A1, SLC10A2 and SLC10A6. We examined the relationship between the N-terminus and transport activity of SLC10A4. Rat Slc10a4 is predominantly expressed in rat cholinergic neurons; therefore, TE671 cells expressing the acetylcholine receptor and acetylcholinesterase were used. After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. Substrates were added to the cells, and their uptake was quantified by liquid chromatography tandem mass spectrometry. Lithocholic acid (LCA) and taurocholic acid (TCA) uptake and cell death effects of LCA were increased by thrombin treatment. After RNA interference treatment for SLC10A4, bile acid uptake was also quantified. In consequence, increases in the LCA and TCA uptake did not occur. Therefore, SLC10A4 may have low activity but becomes activated by proteases, including thrombin, following cleavage. We have demonstrated that SLC10A4 appears to be a protease-activated transporter and transports bile acids.

Background: Environmental control to reduce the amount of allergens in a living place is thought to be important to avoid sensitization to airborne allergens. However, efficacy of environmental control on inactivation of airborne allergens is not fully investigated. We have previously reported that positively- and negatively-charged plasma cluster ions (PC-ions) reduce the IgE-binding capacity of crude allergens from Japanese cedar pollen as important seasonal airborne allergens. Cat (Felis domesticus) and fungus (Aspergillus fumigatus) are also important sources of common airborne allergens in living spaces throughout the year, and early sensitization with those allergens is considered to be a risk factor for future development of allergic rhinitis, pollinosis and asthma. The aim of this study is to examine whether the PC-ions reduce the IgE-binding capacity of a cat major allergen (Fel d 1) and fungal allergens in an experimental condition. Methods: Fel d 1, crude fungal extract, or a fungal major allergen Asp f 1, was treated with PC-ions for 6 h in an experimental cylindrical apparatus. Sham-treated allergens were prepared in the same experimental apparatus without generation of PC-ions. The degradation of the PC-ions-treated Fel d 1 was analyzed by SDS-PAGE, and the IgE-binding capacity of the PC-ions-treated allergens was analyzed by ELISA inhibition assay. Results: Exposure of Fel d 1, crude fungal extract and Asp f 1 to PC-ions significantly decreased protein content of Fel d 1 or Asp f 1, respectively. SDS-PAGE analysis suggested that the decreased Fel d 1 content upon exposure with PC-ions was attributable to protein degradation. ELISA inhibition indicated that the PC-ions treatment significantly impaired IgE-binding capacities of Fel d 1, crude fungal allergens, and Asp f 1 compared to sham treatment. Discussion: Our data suggest that treatment with PC-ions not only reduce indoor cat and fungal allergens, but also impair their allergenicity. Conclusion: These results suggest that environmental control with PC-ions is useful for inactivation of indoor cat and fungal allergens., This study was financially supported in part by the SHARP Corporation.

SUMO conjugation is a reversible posttranslational modification that regulates protein function. SENP1 is one of the six SUMO-specific proteases present in vertebrate cells and its altered expression is observed in several carcinomas. To characterize SENP1 role in genome integrity, we generated Senp1 knockout chicken DT40 cells. SENP1(-/-) cells show normal proliferation, but are sensitive to spindle poisons. This hypersensitivity correlates with increased sister chromatid separation, mitotic slippage, and apoptosis. To test whether the cohesion defect had a causal relationship with the observed mitotic events, we restored the cohesive status of sister chromatids by introducing the TOP2α(+/-) mutation, which leads to increased catenation, or by inhibiting Plk1 and Aurora B kinases that promote cohesin release from chromosomes during prolonged mitotic arrest. Although TOP2α is SUMOylated during mitosis, the TOP2α(+/-) mutation had no obvious effect. By contrast, inhibition of Plk1 or Aurora B rescued the hypersensitivity of SENP1(-/-) cells to colcemid. In conclusion, we identify SENP1 as a novel factor required for mitotic arrest and cohesion maintenance during prolonged mitotic arrest induced by spindle poisons.

The distributions of lysozyme and protease activities and of amino acids was measured in the guts of five species of higher termites, Macrotermes annandalei, Odontotermes formosanus, Pericaproitermes nitobei, Termes comis and Nasutitermes takasagoensis. Lysozyme activity was found only in M. annandalei, P. nitobei and N. takasagoensis. Protease activity was high in the midgut of all species but negligible elsewhere in the gut. Amino acid concentration was highest in the midgut of all species of workers.

Replication checkpoint protein Rad17 senses DNA lesions during DNA replication and halts progression of replication fork. The cells derived from Bloom syndrome individuals show some defects in DNA replication. In order to investigate the functional relationship between the replication checkpoint protein Rad17 and BLM, which is the product of the causative gene of Bloom syndrome, we generated BLM/RAD17 double knockout (blm/rad17) cells using chicken DT40 cells. The blm/rad17 cells showed exaggerated growth defects as determined by analysis of their growth curves and plating efficiency compared to those of either of the single gene mutants. These defects seem to be due to an increase in DNA lesions that cause spontaneous cell death, suggesting that Rad17 and BLM execute different functions in the progression of replication forks. We also demonstrate that targeting integration was dramatically compromised by a lack of Rad17. In addition, the elevated frequency of sister chromatid exchange (SCE) due to homologous recombination in BLM knockout (blm) cells was greatly reduced by disruption of the RAD17 gene. Thus, in addition to its role in the replication checkpoint, Rad17 appears to play a role in homologous recombination.

We investigated the function of the interaction between WRN (Werner syndrome gene product) and Ku70 and between WRN and DNA-PKcs, which are components of the DNA-PKcs/Ku70/Ku80 complex, by generating KU70(-/-)/WRN(-/-) and DNA-PKcs(-/-/-)/WRN(-/-) double-gene knockout chicken DT40 cells. When treated with camptothecin (CPT), an inhibitor of DNA topoisomerase I, WRN(-/-) cells showed higher sensitivity than wild-type cells, whereas KU70(-/-) and DNA-PKcs(-/-/-) cells showed hyper-resistance. Disruption of KU70 or DNA-PKcs suppressed the sensitivity of WRN(-/-) cells to CPT, rendering them as resistant to CPT treatment as KU70(-/-) and DNA-PKcs(-/-/-) cells. On the other hand, CPT sensitivity of BLM(-/-) cells, which are defective in a RecQ helicase similar to WRN, was enhanced by deletion of KU70. The implications for the function of WRN in the non-homologous end-joining pathway of DNA repair involving Ku70 and DNA-PKcs, which may be the cause of lethality in the presence of CPT, will be discussed.

It has recently been claimed that our β-bungarotoxin preparation contained three contaminants, including a postsynaptic toxin. We have extended our purification procedure and found no evidence of such contaminants.

The neurotoxin, β-bungarotoxin (β-BuTX) purified from Bungarus multicinctus snake venom exerts various effects on frog and rat motor nerve terminals. Exposure to β-BuTX triggers, in the terminals, impulses which are rapidly distributed to the whole motor unit and elicit contractions of the muscle. β-BuTX raises the frequency of spontaneous release of acetylcholine quanta and inhibits the release evoked by nerve impulses, all of which leads to complete cessation of transmitter release. Subsequently, the motor axon disappears from the endplate and the Schwann cell establishes extensive synaptic contact with the muscle fibre. After only 30 min in β-BuTX, some endplates attain a stage of denervation similar to that seen several days after transecting the motor nerve. The neuromuscular effects of β-RuTX may result from its phospholipase activity and from an increase in the level of ionized calcium inside the terminals.