Identification of the core motif of the BRCA2 C-terminal RAD51-binding domain by comparing canine and human BRCA2

Mammary tumors are the most common tumors in women and non-spayed female dogs. One of the reasons for mammary tumors is mutations of the tumor suppressor gene, BRCA2. BRCA2 participates in homologous recombination repair by interacting with the RAD51 recombinase. BRCA2 has two RAD51-binding domains, consisting of BRC repeats and the C-terminal RAD51-binding domain, respectively. Although several studies have addressed the function of the C-terminal RAD51-binding domain of human BRCA2, the amino acid sequences required for the RAD51-interaction activity remain unclear. In this study, the C-terminal RAD51-binding domains of canine and human BRCA2 were compared; the canine domain displayed a weaker interaction with RAD51. This difference was attributed to the C-terminal portion of the domain via a comparison between canine and human domains. Furthermore, peptides shorter than those previously reported displayed RAD51-interacting activity, and a core motif of this domain consisting of 25 amino acids was identified. Since a mutation (S3323N) was reported in the core motif of this domain, the effect of this mutation was evaluated. The mutant exhibited similar RAD51-binding activity as that of the wild-type protein, suggesting that the mutation was functionally neutral. These data suggested that the C-terminal portion of the BRCA2 C-terminal RAD51-binding domain influenced its RAD51-interaction activity, and a minimum core motif of 25 amino acids was identified in this domain. These data may help clarify BRCA2 function, as well as the tumorigenic effects of BRCA2 mutation.     

Comparison of N-terminal pro-atrial natriuretic peptide and three cardiac biomarkers for discriminatory ability of clinical stage in dogs with myxomatous mitral valve disease

Plasma N-terminal pro-atrial natriuretic peptide (NT-proANP) concentration increases with progression of myxomatous mitral valve disease (MMVD) in dogs. This multicentre, prospective study compared plasma NT-proANP, N-terminal pro-brain natriuretic peptide (NT-proBNP), ANP, and cardiac troponin I (cTnI) concentrations in dogs with MMVD for their characteristics and discriminatory ability to detect cardiac dilatation and congestive heart failure (CHF). Thirty-six healthy dogs and 69 dogs with MMVD were included. Clinical variables were obtained via physical examination, thoracic radiography, and echocardiography. The discriminatory ability of each cardiac biomarker (CB) to determine the presence or absence of cardiac dilatation (event 1) and CHF (event 2) was evaluated using the receiver operating characteristic curves. Plasma NT-proANP, NT-proBNP, and ANP concentrations showed a significant association with the left atrium/aorta ratio (P<0.01). The area under the curve of plasma NT-proANP and NT-proBNP concentrations were 0.72 and 0.75, respectively in event1 and 0.72 and 0.76, respectively in event2. Plasma NT-proANP and NT-proBNP concentrations showed sensitivity 80.0 and 80.0%; specificity 67.6 and 64.7% in event1 (cutoff value; 8,497.81 pg/ml and 1,453.00 pmol/l, respectively) and sensitivity 85.7 and 81.0%; specificity 60.4 and 64.6% in event2 (cutoff value; 8,684.33 pg/ml and 1,772.00 pmol/l, respectively). In dogs with MMVD, plasma NT-proANP, NT-proBNP, and ANP concentrations increase with left atrial enlargement. Particularly, plasma NT-proANP and NT-proBNP concentrations appeared to be equally useful in the discriminatory ability to detect cardiac dilatation and CHF.     

中国广东家蚕微粒子孢子在Antheraea eucalypti细胞系的感染增殖

将中国广东的Ｎｏｓｅｍａ ｂｏｍｂｙｃｉｓ ＣＧＳ,用０２ｍｏｌ／ＬＫＯＨ 处理后,接种感染Ａ．ｅｕｃａｌｙｐｔｉ 细胞系。孢子的发芽率达８７％ ,在Ａ．ｅｕｃａｌｙｐｔｉ 细胞初期感染率为２７ ％ 。发育各阶段具２ 核,可观察到短极丝孢子和二次感染体形成,孢子芽母细胞按二分裂形成２ 个双核的孢子芽,具典型 Ｎｏｓｅｍａ 属特征。Ｎｏｓｅｍａｂｏｍｂｙｃｉｓ ＣＧＳ 生物学特性、血清学类型与日本Ｎｏｓｅｍａ ｂｏｍｂｙｃｉｓ ＮＩＳ００１ 大致相同,但孢子大小、裂殖体的形态、在Ａ．ｅｕｃａｌｙｐｔｉ 细胞寄生的细胞感染率、每个细胞的产孢数,２ 种微孢子虫却有差异。     

Experimental Final Hosts of Metagonimus Hakubaensis (Trematoda: Heterophyidae) and Their Suitability to the Fluke

Seven laboratory mammal and bird species were orally inoculated with 200–1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.     

Sugar expressions on the vaginal epithelium in pregnant mice

Sugar expressions were examined on the epithelium of both the middle portion of the vagina and the vaginal portion of the cervical canal (CC) in pregnant mice to understand the pathogenesis of bacterial infection in the female reproductive organ by using a panel of lectins. As a result, N-acetylglucosamine was positive before pregnant day (P) 7 but negative after P10 and at diestrus on both the vagina and the CC. In addition, some differences in sugar expressions were seen between them. These results suggest that sugar expressions on the mucosal surface would change not only site-specifically but also time-dependently, and these sugar differences indicate the possibility of the alteration of the settled bacterial species on the vaginal mucosa in pregnancy.     

不同时间尺度环境因子对黑河下游胡杨(Populus euphratica)径向生长的影响

本研究以黑河下游额济纳旗成熟的河岸胡杨林为对象,分析不同年龄胡杨(Populus euphratica)的年轮宽度序列与不同时间尺度环境因子的关系。结果表明:①在年尺度上,气温是影响胡杨径向生长的主要因子,但不同年龄胡杨对气温的响应有所不同。②在季节尺度上,休眠季气温越高越有利于壮龄胡杨的径向生长,而生长季气温越高越不利于老龄胡杨的径向生长。③在月尺度上,前一年3月和当年9月的平均气温越高,对壮龄胡杨的径向生长越有利,而当年10月平均气温越高,对老龄胡杨的径向生长越不利;前一年7月径流量的增加对老龄胡杨的径向生长有利,而当年2月径流量的增加对壮龄胡杨径向生长有利。综上所述,在季节和年等相对较长的时间尺度上,气温是影响胡杨径向生长量的主要因素,随着时间尺度的缩小,胡杨对环境因子的响应更为细化,并且表现出一定的滞后效应。径流量作为重要的环境因子在月尺度上才突显出来,前一年7月和当年2月的径流补给对胡杨径向生长有极大的促进作用。因此,在黑河统一调度管理过程中,可通过适度调增2月和7月洪水的下泄量来满足胡杨径向生长对水分的需求。     

IMPACT OF TISSUE INHOMOGENEITY ON DOSE DISTRIBUTION IN THE CANINE CARPAL AND TARSAL REGIONS FOR COBALT AND 6 MV PHOTONS

We quantified the effect of tissue inhomogeneity on dose distribution in a canine distal extremity resulting from treatment with cobalt photons and photons from a 6 MV accelerator. Monitor units for a typical distal extremity treatment were calculated by two methods, using equally weighted, parallel-opposed fields. The first method was a computed tomography (CT)-based, computerized treatment plan, calculated without inhomogeneity correction. The second method was a manual point dose calculation to the isocenter. A computerized planning system was then used to assess the dose distribution achieved by these two methods when tissue inhomogeneity was taken into account. For cobalt photons, the median percentage of the planning target volume (PTV) that received <95% of the prescribed dose was 4.5% for the CT-based treatment plan, and 26.2% for the manually calculated plan. For 6 MV photons, the median percentage of the PTV that received <95% of the prescribed dose was <1% for both planning methods. The PTV dose achieved without using inhomogeneity correction for cobalt photons results in potentially significant under dosing of portions of the PTV.     

SYNERGISTIC INTERACTIONS BETWEEN INHIBITORS OF GROWTH AND PHOTOSYNTHESIS

Summary. The herbicidal activity on Echinochloa crus-galli (L.) Beauv. var. oryzicola Ohwi of the photosynthesis inhibitors chlorbromuron and simetryne decreased rapidly with increasing plant age. The decreased activity is explained on the basis of a proposed ‘growth-dilution’ hypothesis, which is also relevant to the synergism observed with combinations of these two herbicides and various growth inhibitors. In an attempt to make use of the synergistic action for practical weed control in paddy rice (Oryza sativa L.) a test was made to determine the effects on crop and weeds of granular formulations of two herbicide combinations, namely 5% w/w 2-(2-chlorobenzylthio)-5-propyl-l,3,4-oxadia-zole (benoxazole) plus l-5% w/w of either chlorbromuron or simetryne. Both formulations were applied at doses of 30 and 40 kg product/ha and the benoxazole+ simetryne mixture gave very promising results. Interactions synergiques entre les inhibiteurs de croissance et la photosynthése I. L'hypothése du rolentissement de la croissance Résumé. L'action herbicide sur Echinochloa crus-galli (L.) Beauv. var. oryzicola Ohwi d'inhibiteurs de la photosynthése, tels que le chlorbromuron et la simétryne, décroît rapidement à mesure que l'age des plantes augmente. Cette diminution d'activité a été expliquée en proposant une hypothise basée sur le ralentissement de la croissance, hypothése qui s'appuie également sur le synergisme observé avec les combinaisons de ces deux herbicides et divers inhibiteurs de croissance. En vue d'utiliser l'action synergique dans la pratique pour le désherbage du riz (Oryza sativa L.), un essai a été réalisé pour determiner les effets, sur la récolte et sur les mauvaises herbes, de formulations des deux herbicides associés, comprenant 5% (en poids) de 2-(2-chlorobenzylthio)-5- propyle-l,3,4-oxadiazole (bénoxazole) plus 1,5% (en poids), soit de chlorbromuron, soit de simétryne. Les deux formulations ont été appliquées a des doses de 30 et 40 kg/ha et le mélange bénoxazole+simétryne a donné des résultats trés prometteurs. Synergistische Wechselmrkungen zwischen Wachstums- und Photosynthesehemmstoffen. I. Die Theorie des ‘Verdünnungseffektes durch Wachstum” Zusammenfassung. Die herbizide Wirkung der Photosynthesehemmstoffe Chlorbromuron und Simetryn gegenüber Echinochloa crus-galli (L.) Beauv. var. oryzicola Ohwi nahm mit zunehmendem Alter der Pflanzen schnell ab. Dies wurde mit einer Theorie der ‘Verdünnungswirkung durch Wachstum’ erklärt, die ausserdem die Begründung für den Synergismus lieferte, den man bei Kombination dieser zwei Herbizide mit verschiedenen Wachstumshemmstoffen beobachtete. Um diese synergistische Wirkung für die praktische Unkrautbekämpfung in Reis (Oryza sativa L.) nutzbar zu machen, wurde ein Versuch angelegt. Damit sollte die Wirkung granulierter Formulierungen zweier Herbizidkombinationen, nämlich 5% w/w 2-(2-Chlorbenzylthio)-5-propyl- 1,3,4-oxadiazol (Benoxazol) plus 1,5% w/w Chlorbromuron oder Simetryn, auf Kultur früchte und Unkräuter ermittelt werden. Beide Formulierungen wurden mit 30 und 40 kg/ha angewandt. Die Benoxazol-Simetryn-Mischung ergab dabei vielversprechende Ergebnisse.     

Effects of oxidative stress caused by tert-butylhydroquinone on cytotoxicity in MDCK Cells

Antioxidant and oxidative stress effects of prooxidants are generally dose-dependent, and these effects depend on the prooxidant species and cell type. However, the cellular response to oxidant challenge is a complicated interplay of events involving cellular expression of phase II detoxification enzymes and cellular metal metabolism. This study demonstrates the effect of tert-butylhydroquinone (t-BHQ)-induced oxidative stress on MDCK cells. Cell toxicity tests were carried out using the crystal violet (CV) assay with the following prooxidants: t-BHQ, diethyl maleate (DEM), hydrogen peroxide (H(2)O(2)), diquat (DQ) and β-naphthoflavone (β-NF). Except for β-NF, these prooxidants showed dose-dependent cytotoxicity besides the most potent t-BHQ cytotoxicity. Only t-BHQ and DEM caused significant time-dependent expression of ferritin protein as an antioxidant, which segregates Fe(2+), causing the Fenton reaction. t-BHQ and DEM increased formation of lipid peroxidation, but DQ showed a tendency to decrease lipid peroxidation levels. In XTT assay, even when substantial cell death was observed in the CV assay, t-BHQ appeared to increase cell viability by enhancing XTT reduction, likely through the production of NADPH. Although curcumin, which induces cytoprotective phase II enzymes and chelates metal irons, decreased cell viability, it inhibited t-BHQ cytotoxicity. These results indicate that t-BHQ exhibits strong cytotoxicity against MDCK cells, an effect mitigated by curcumin, and that t-BHQ-induced oxidative stress activates the pentose phosphate pathway.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.