Effects of the Missense Mutations in Canine BRCA2 on BRC Repeat 3 Functions and Comparative Analyses between Canine and Human BRC Repeat 3

Mammary tumors are the most common tumor type in both human and canine females. Mutations in the breast cancer susceptibility gene, BRCA2, have been found in most cases of inherited human breast cancer. Similarly, the canine BRCA2 gene locus has been associated with mammary tumors in female dogs. However, deleterious mutations in canine BRCA2 have not been reported, thus far. The BRCA2 protein is involved in homologous recombination repair via its interaction with RAD51 recombinase, an interaction mediated by 8 BRC repeats. These repeats are 26-amino acid, conserved motifs in mammalian BRCA2. Previous structural analyses of cancer-associated mutations affecting the BRC repeats have shown that the weakening of RAD51''s affinity for even 1 repeat is sufficient to increase breast cancer susceptibility. In this study, we focused on 2 previously reported canine BRCA2 mutations (T1425P and K1435R) in BRC repeat 3 (BRC3), derived from mammary tumor samples. These mutations affected the interaction of canine BRC3 with RAD51, and were considered deleterious. Two BRC3 mutations (K1440R and K1440E), reported in human breast cancer patients, occur at amino acids corresponding to those of the K1435R mutation in dogs. These mutations affected the interaction of canine BRC3 with RAD51, and may also be considered deleterious. The two BRC3 mutations and a substitution (T1430P), corresponding to T1425P in canine BRCA2, were examined for their effects on human BRC3 function and the results were compared between species. The corresponding mutations and the substitution showed similar results in both human and canine BRC3. Therefore, canine BRCA2 may be a good model for studying human breast cancer caused by BRCA2 mutations.     

An improved abnormal toxicity test by using reference vaccine-specific body weight curves and histopathological data for monitoring vaccine quality and safety in Japan

Vaccines differ from other pharmaceutical products. The quality and safety of batches are regulated to high standards by national regulatory authorities. Various quality control and safety tests have been developed, including the abnormal toxicity test (ATT), which is described in the World Health Organization (WHO) guidelines and in each country's pharmacopoeia. However, the criteria for abnormal results are not well defined in these guidelines. In addition, the animal grade to be used in ATT, classified on the basis of microbial colonization, was not designated in either guideline.In this study, we report a new and improved method of performing ATT, including statistical, histopathological analysis and hematological findings. It is based on the observation that there are body weight changes characteristic to each vaccine, and such standardized changes can be used as references for evaluating test vaccines. In addition, histopathological data are useful for determining vaccine quality and safety. Combined with histopathological examination, the improved ATT will be of great use for evaluating the consistency, quality and safety of different batches of vaccine. The results of these analyses were similar using either ‘clean’ or specific pathogen-free guinea pigs.     

Static magnetic field with a strong magnetic field gradient (41.7 T/m) induces c-Jun expression in HL-60 cells

We investigated the effects of 6- and 10-T static magnetic fields (SMFs) on the expression of protooncogenes using Western blot immunohybridization methods. We used a SMF exposure system, which can expose cells to a spatially inhomogeneous 6 T with a strong magnetic field (MF) gradient (41.7 T/m) and a spatially homogeneous 10 T of the highest magnetic flux density in this experiment. HL-60 cells exposed to either 6- or 10-T SMF for periods of 1 to 48 h did not exhibit remarkable differences in levels of c-Myc and c-Fos protein expression, as compared with sham-exposed cells. In contrast, c-Jun protein expression increased in HL-60 cells after exposure to 6-T SMF for 24, 36, 48, and 72 h. These results suggest that a homogeneous 10-T SMF does not alter the expression of the c-jun, c-fos, and c-myc protooncogenes. However, our observation that exposure to a strong MF gradient induced c-Jun expression suggests that a strong MF gradient may have significant biological effects, particularly regarding processes related to an elevation of c-jun gene expression.     

Identification of high excision capacity for 5-hydroxymethyluracil mispaired with guanine in DNA of Escherichia coli MutM,Nei and Nth DNA glycosylases

The oxidation and deamination of 5-methylcytosine (5mC) in DNA generates a base-pair between 5-hydroxymethyluracil (5hmU) and guanine. 5hmU normally forms a base-pair with adenine. Therefore, the conversion of 5mC to 5hmU is a potential pathway for the generation of 5mC to T transitions. Mammalian cells have high levels of activity of 5hmU-DNA glycosylase, which excises 5hmU from DNA. However, glycosylases that similarly excise 5hmU have not been observed in yeast or Escherichia coli. Recently, we found that E.coli MutM, Nei and Nth have DNA glycosylase activity for 5-formyluracil, which is another type of oxidation product of the thymine methyl group. In this study, we examined whether or not E.coli MutM, Nei and Nth have also DNA glycosylase activity that acts on 5hmU in vitro. When incubated with synthetic duplex oligonucleotides containing 5hmU:G or 5hmU:A, purified MutM, Nei and Nth cleaved the 5hmU:G oligonucleotide 58, 5 and 37 times, respectively, more efficiently than the 5hmU:A oligonucleotide. In E.coli, the 5hmU-DNA glycosylase activities of MutM, Nei and Nth may play critical roles in the repair of 5hmU:G mispairs to avoid 5mC to T transitions.     

Phylogeny and divergence of basal angiosperms inferred from<Emphasis Type="Italic"> APETALA3</Emphasis>- and<Emphasis Type="Italic"> PISTILLATA</Emphasis>-like MADS-box genes

The B-class MADS-box genes composed of APETALA3 (AP3) and PISTILLATA (PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5 region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from ~140–210 Ma, depending on the trees used and assumptions made.     

Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.     

Capacity of rete testicular and cauda epididymal boar spermatozoa to undergo the acrosome reaction and subsequent fusion with egg plasma membrane

The capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane was examined in rete testicular and cauda epididymal spermatozoa from boars. Sperm penetration assay using zona-free hamster eggs demonstrated that the penetration rates for rete testicular spermatozoa preincubated for induction of the acrosome reaction for 2 and 3 h were 55% and 97%, respectively. However, most of the eggs (93%) were penetrated with polyspermy by cauda epididymal cells preincubated for 2 h. Results obtained by the triple-stain technique revealed the percentages of acrosome-reacted spermatozoa in the rete testicular and cauda epididymal samples preincubated for 3 h to be 61% and 74%, respectively. These results indicate that many rete testicular spermatozoa possess the capacity to undergo the acrosome reaction and subsequent fusion with egg plasma membrane in vitro, which appears to be completely established only after sperm transit through at least the proximal part of the epididymis. © 1993 Wiley-Liss, Inc.     

A mutant endonuclease IV of Escherichia coli loses the ability to repair lethal DNA damage induced by hydrogen peroxide but not that induced by methyl methanesulfonate.

A mutant allele of the Escherichia coli nfo gene encoding endonuclease IV, nfo-186, was cloned into plasmid pUC18. When introduced into an E. coli xthA nfo mutant, the gene product of nfo-186 complemented the hypersensitivity of the mutant to methyl methanesulfonate (MMS) but not to hydrogen peroxide (H2O2) and bleomycin. These results suggest that the mutant endonuclease IV has normal activity for repairing DNA damages induced by MMS but not those induced by H2O2 and bleomycin. A missense mutation in the cloned nfo-186 gene, in which the wild-type glycine 149 was replaced by aspartic acid, was detected by DNA sequencing. The wild-type and mutant endonuclease IV were purified to near homogeneity, and their apurinic (AP) endonuclease and 3'-phosphatase activities were determined. No difference was observed in the AP endonuclease activities of the wild-type and mutant proteins. However, 3'-phosphatase activity was dramatically reduced in the mutant protein. From these results, it is concluded that the endonuclease IV186 protein is specifically deficient in the ability to remove 3'-terminus-blocking damage, which is required for DNA repair synthesis, and it is possible that the lethal DNA damage by H2O2 is 3'-blocking damage and not AP-site damage.     

Crystallographic study on the orthorhombic crystal of l-asparaginase from Escherichia coli HAP

The orthorhombic crystals of l-asparaginase from Escherichia coli A-1-3 KY3598 were characterized by the X-ray diffraction method as belonging to the space group P2₁22₁ with unit cell dimensions $\text{a = 116.7}\text{A}\text{?}\text{, b = 62.9}\text{A}\text{?}\text{and}\text{c = 86.6}\text{A}\text{?}$. The crystals contain one half of the tetrameric enzyme molecule per asymmetric unit, the smallest so far reported for this enzyme. Preliminary analysis of the Patterson map indicated that the molecule had at least pseudo 222 symmetry and established the position of the centers of the molecules in the unit cell.