The number and localisation of adenine nucleotide-binding sites in beef-heart mitochondrial ATPase (F1) determined by photolabelling with 8-azido-ATP and 8-azido-ADP

1. When irradiated 8-azido-ATP becomes covalently bound (as the nitreno compound) to beef-heart mitochondrial ATPase (F₁) as the triphosphate, either in the absence or presence of Mg²⁺, label covalently bound is not hydrolysed.

2. In the presence of Mg²⁺ the nitreno-ATP is bound to both the and β subunits, mainly (63%) to the subunits.

3. After successive photolabelling of F₁ with 8-azido-ATP (no Mg²⁺) and 8-azido-ADP (with Mg²⁺) 4 mol label is bound to F₁, 2 mol to the and 2 mol to the β subunits.

4. When the order of photolabelling is reversed, much less 8-nitreno-ATP is bound to F₁ previously labelled with 8-nitreno-ADP. It is concluded that binding to the -subunits hinders binding to the β subunits.

5. F₁ that has been photolabelled with up to 4 mol label still contains 2 mol firmly bound adenine nucleotides per mol F₁.

6. It is concluded that at least 6 sites for adenine nucleotides are present in isolated F₁.     

PD-L1基因敲除小鼠构建及初步表型验证

目的 程序性死亡配体-1(PD-L1)是免疫调节途径的重要因子,是抗肿瘤免疫疗法中重要的靶标之一。利用CRISPR/Cas9技术成功构建PD-L1基因敲除小鼠模型,并初步分析其表型。方法 构建Cas9和sgRNA载体,并转录获得RNA,通过显微注射方式将RNA注射到C57BL/6小鼠受精卵中,经过鉴定获得F₀代阳性小鼠。F₀代小鼠与野生型C57BL/6小鼠交配获得F₁代杂合子小鼠,再通过F₁代小鼠自交获得F₂代纯合子小鼠品系。随后通过Real-Time PCR和流式实验分别检测PD-L1基因在mRNA和蛋白质水平上的表达情况。结果 Real-Time PCR和流式实验检测结果显示与野生型C57小鼠相比,PD-L1纯合子小鼠的PD-L1 mRNA相对表达水平和细胞上的蛋白质表达均有显著性下降,仅测定到本底的信号,证实已成功构建PD-L1基因敲除小鼠品系,为PD-L1体内基因功能研究提供了新的小鼠模型。     

Spermatogenesis parameters and testosterone production at puberty as predictors of testicular functional activity in mice (<Emphasis Type="Italic">Mus musculus</Emphasis>)

The aim of this study was to investigate fertility-associated parameters of spermatogenesis and androgenic status in male laboratory mice at puberty and to assess their prognostic significance in the realization of the definitive testicular function. In three inbred murine strains, BALB/cLac, CBA/Lac and PT, the serum testosterone level, its testicular concentration, epididymal sperm count (sperm reserve) and portion of sperm with abnormal head morphology were evaluated on days 45 (puberty) and 90 (adulthood) of postnatal development. CBA/Lac males were characterized by a lower epididymal sperm count vs. other strains at both ages indicative of poorer spermatogenesis. At the same time, CBA/Lac males had a lower portion of sperm with abnormal head morphology, and this could be considered as a compensatory reaction aimed at improving sperm fertility. Distinct inter-strain differences in the portion of sperm with morphologically abnormal heads were established at both ages, while the inter-strain ratio remained invariable (BALB/cLac > PT > CBA/Lac). Thus, the level of abnormal spermatogenesis in the pubertal period may have a predictive significance for the definitive testicular activity in adult mice. No inter-strain and age-dependent changes were found in serum and testicular testosterone levels except for the PT strain, in which both testosterone levels rose from puberty to adulthood, suggesting a shift of the pubertal testosterone peak towards later times. Our data show that in male laboratory mice the genetic peculiarities of the testicular function manifest themselves during puberty and persist until adulthood.     

野生小家鼠与实验小白鼠杂交世代的血红蛋白电泳分析

两种基因型不同的亲本之间的杂交试验是遗传学家和育种工作者熟悉的手段。杂种优势通常在高度近交系间的杂交子代中是常见的,也是育种学家赖以培养新品种的依据。 实验小白鼠(Mus musculus albino)是从野生小家鼠(Mus musculus)培育而来的。Klein(1975)和Moriwaki等(1979)认为现在广泛应用的实验小白鼠很可能来源于中国和日本的小家鼠。但是这2种近亲系小鼠的杂交试验至今未见报道。     

斑马鱼hoxa1a基因调控颅面骨骼发育的功能研究*

hox基因编码一类高度保守的转录因子家族,人类HOXA1的突变会导致阿萨巴斯卡发育不良综合征 (Athabascan brainstem dysgenesis syndrome, ABDS),使人出现因颅骨异常导致的面部畸形和面部麻痹等症状。利用模式生物斑马鱼研究其同源基因hoxa1a的功能机制。首先利用CRISPR/Cas9技术对斑马鱼hoxa1a进行基因编辑,获得了hoxa1a基因突变,T7E1酶切结果显示F₀酶切效率平均为70%。F₁进一步筛选到两种突变类型,分别是插入了8 bp和删除了7 bp的杂合突变体。杂合子自交得到hoxa1a F₂纯合突变体,并且测序验证hoxa1a基因突变成功。5 dpf时,hoxa1a纯合突变体出现颅面发育异常。阿尔新蓝软骨染色和茜素红硬骨染色结果表明,hoxa1a突变体中颅骨发育异常、筛骨板断裂,鳃弓发育出现缺损。成功在斑马鱼中构建ABDS疾病模型,表明hoxa1a突变会造成斑马鱼颅面骨骼发育异常,为其功能机制研究奠定了基础,为人类ABDS疾病的致病机制研究提供了新的思路。     

Rhodamine 6G, inhibitor of both H-ejections from mitochondria energized with ATP and with respiratory substrates

Rhodamine 6G inhibited ATP hydrolysis by oligomycin-sensitive ATPase, purified from rat liver mitochondria, in good accord with the dose-response curve for its inhibition of energy transduction of ATP synthesis in mitochondria, but it did not inhibit ATP hydrolysis by purified F₁. Rhodamine 6G also inhibited both H⁺-ejections from mitochondria energized with respiratory substrates and with ATP.

The present findings show that the inhibitory effect of rhodamine 6G on energy transduction is not due to a modification of the transport system for adenine nucleotides, P_i, and respiratory substrates, and that the inhibition sites of rhodamine 6G are on components related with H⁺-ejection by redox components and also on F₀.     

灌溉施肥水平对盐渍化农田水盐分布及玉米产量的影响

水资源缺乏和过量施肥影响着干旱半干旱盐渍化地区农业的发展.研究不同灌溉和施肥量对土壤水盐分布和青贮玉米产量的影响,可为该区确定适宜的灌溉和施肥量提供依据.试验于2015和2016年在大同盆地的盐渍化农田进行,设3种灌溉水平:土壤水分上限分别为田间持水率(θ_f)的100%(W₁)、90%(W₂)和80%(W₃),根据各处理灌溉前的土壤平均实际含水率计算灌水量;2015年设4种施肥水平:900(F₁)、750(F₂)、600(F₃)和450 kg·hm^-2(F₄),2016年设F₁、F₂和F₃共3种.试验用化肥为缓释复合肥,总养分含量48%,其中N:P₂O₅:K₂O的比例为30:12:6.结果表明: 土壤表层电导率随施肥量的增加而增大,施肥水平对平均电导率(EC)和含水率的影响在0～10 cm土层显著,与F₁相比,F₂的0～10 cm土层平均EC在2015年和2016年分别降低25.6%～42.7%和6.4%～7.7%.20～80 cm土层的水分含量随施肥量的增加而降低,与F₁相比,2015年F₂、F₃和F₄处理20～80 cm土层平均土壤含水率分别增加5.9%、16.7%和16.7%,2016年F₂和F₃分别增加13.3%和16.7%.产量在两年中均表现为F₁和F₂高于F₃和F₄,W₃低于W₁和W₂; F₁和F₂的产量差异不明显;与W₁相比,W₂的产量减少低于15 %.因此,施复合肥600～750 kg·hm^-2(氮肥含量180～270 kg·hm^-2),且灌溉水平为W₁和W₂时,可以保证该地区盐渍化土壤种植玉米获得较高的产量,并且不会造成根系层的盐分积累.     

甲氰菊酯和阿维菌素对柑橘全爪螨的亚致死效应

通过叶碟饲养的方法, 利用生命表技术,研究了甲氰菊酯和阿维菌素亚致死剂量LC₂₀处理柑橘全爪螨若螨后,对试验种群当代(F₀)和后代(F₁、F₂代)生长发育及繁殖的影响.结果表明: 甲氰菊酯LC₂₀处理若螨后,当代雌成螨产卵量显著增加;F₁、F₂代的产卵前期缩短,后代雌性比例增大,且均与对照差异显著;同时,F₁和F₂代种群内禀增长率(r_m)和周限增长率(λ)增大,世代历期(T)和种群加倍时间(Dt)缩短,且F₂代与对照相比差异显著.用阿维菌素LC₂₀处理若螨后,当代种群雌成螨产卵量显著下降; F₁和F₂代的产卵量也显著下降,但后代雌性比例增大,产卵前期显著缩短;F₁和F₂代的种群r_m和λ增大,T和Dt缩短,且F₂代比F₁代更为明显.总体来看,甲氰菊酯和阿维菌素亚致死浓度LC₂₀对柑橘全爪螨的影响并不完全相同,甲氰菊酯能够促进当代种群的发展,而阿维菌素对当代种群有一定的抑制作用;但两种杀螨剂亚致死浓度处理柑橘全爪螨对后代种群都有一定的促进作用.研究结果对柑橘全爪螨综合防治策略的制定有一定的指导意义.     

Enhancement of Squamous Cell Development in Cultured Skin by Cyclic Adenine Nucleotide and Prostaglandins

The present study tests the hypothesis that agents known to elevate the level of intracellular cyclic adenine nucleotide may direct different epithelial cells onto a pathway of epidermoid (squamous) development and differentiation. We report here that the mixture of dibutyryl cyclic AMP (dbcAMP), prostaglandins E₁, E₂ and B₁, (PG E₁, E₂, B₁), and papaverine (pap) enhances the rate of normal squamous cell development in organ-cultured skin of chick embryos. The three components may act synergistically to elevate the level of intracellular cyclic adenine nucleotide. We recently reported that the same group of agents induces abnormal development (squamous metaplasia) and aberrant differentiation (keratin production) in the normally cuboidal epithelium of cultured whole mammary glands of mice [1]. Thus, dbcAMP, PG E₁, E₂, B₁, and pap are effective in enhancing normal squamous cell development and also in inducing squamous metaplasia de novo in the epithelial components of two different organs of embryonic and adult animals of two classes of vertebrates. The combined findings are suggestive that cyclic adenine nucleotide together with the prostaglandins may act generally on diverse types of epithelia to bring about squamous cell development and a differentiation marked by keratin production.     

草鱼(♀)×赤眼鳟(♂)杂交F1倍性分析和性腺发育特点

为探讨草鱼(Ctenopharyngodon idella♀)×赤眼鳟(Squaliobarbus curriculus♂)杂交F₁的倍性和生育性能, 对杂交F₁的染色体核型和红细胞核DNA含量进行分析和测定, 并运用组织学方法对繁殖期和非繁殖期共124尾杂交F₁的性腺发育特点进行研究。结果显示: (1)杂交F₁染色体2n=48, 核型公式2n=26 m+20 sm+2 st, NF=94, 未发现端部着丝点染色体、随体和次缢痕等结构; (2)杂交F₁相对DNA含量为对照组鸡血的1.29倍, 其绝对DNA含量为(3.21±0.61) pg/N, DNA指数为0.92; (3)经解剖观察和切片鉴定, 杂交F₁卵细胞分为两种类型: 一类卵细胞第Ⅰ—Ⅴ时相均发育正常, 可完成排卵并产生后代, 为正常发育型; 另一类卵细胞发育到第Ⅴ时相后, 卵黄颗粒减少, 油滴增加, 呈无卵黄空洞状, 为异常发育型。杂交F₁精巢包括初级精母细胞、次级精母细胞和精子细胞, 无成熟精子; 部分精小囊内精细胞量极少, 多为结缔组织或脂肪组织; 生产实践中暂未发现可以排精的杂交F₁个体。研究表明, 草鱼与赤眼鳟杂交获得的杂交F₁为二倍体, 部分雌性可育, 雄性可能为败育群体。     

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.   Objective : Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods : Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl₂-treated non-irradiated mice, HgCl₂-treated non-irradiated mice infused with male BMCs 1 day after HgCl₂, and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results : Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl₂, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions : Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl₂, despite establishment of chimerism and cell proliferation in bone marrow and spleen.     

Absence of in vivo mutagenic potency of heated and fried oils in mice.

The mutagenic potency of heated and fried oils (under laboratory conditions), viz. peanut, sesame and coconut oils were studied employing two well validated in vivo mammalian assays, viz. bone marrow micronucleus assay and the sperm morphology assay in albino mice. In the bone marrow micronucleus assay oral doses of fresh, heated and fried oils (7.5 ml and 30.0 ml/kg body weight) did not induce any increase in the incidence of micronucleated polychromatic erythrocytes. Intragastric administration of oils at the highest dose (30.0 ml/kg body weight/day) for 5 consecutive days in male mice did not produce any morphological effect either on the testicular or epididymal weights and histology. No significant alterations were observed in the caudal sperm counts at any of the sampling intervals and also there was no treatment related increase in the incidence of sperm head abnormalities. It is concluded that controlled thermal treatment as well frying of these oils did not produce appreciable amount of mutagens which could be detected under in vivo situations.     

Proteins required for the binding of mitochondrial ATPase to the mitochondrial inner membrane

1. Isolated F₁ (mitochondrial ATPase) binds to urea-treated submitochondrial particles suspended in sucrose/Tris/EDTA with a dissociation constant of 0.1 μM.

2. About one-third of the F₁ and the oligomycin-sensitivity conferring protein (OSCP) are lost during preparation of submitochondrial particles prepared at high pH (A particles). None is lost from particles treated with trypsin (T particles).

3. After further treatment with alkali of urea-treated particles, binding of F₁ requires the addition of OSCP. Maximum binding is reached when both OSCP and Fc₂ are added. The concentration of F₁-binding sites in the presence of both OSCP and Fc₂ is about the same as that in TU particles.

4. After further extraction with silicotungstate of urea- and alkali-treated particles, OSCP no longer induces binding of F₁, unless Fc₂ is also present. Fc₂ induces binding in the absence of OSCP but with a lower binding constant and, in contrast to results under all the other conditions studied in this paper, the ATPase activity is oligomycin insensitive.

5. It is tentatively concluded that OSCP is the binding site for F₁ and Fc₂ is the binding site for OSCP.     

Potential use of hirudin in diabetic cataract: a study of galactose mediated human lens epithelial cells injury

Osmotic stress, together with weakened antioxidant defense mechanisms, is attributed to the changes observed in human diabetic cataract. The use of hirudin, an antithrombic agent, in the pathogenesis of human cataracts has not been studied so far. Since the epithelium is the metabolic unit of the lens, the effect of recombinant hirudin variant III (rHV3) on galactose-induced morphological changes and antioxidant status of human lens epithelial line SRA01/04 in culture was evaluated in this study. The human lens epithelial cells (hLECs) were cultured in D/F₁₂ medium (normal group), D/F₁₂ medium + 50 mM d-galactose (control group) or D/F₁₂ medium + 50 mM d-galactose + rHV3 (test group) for 24 or 72 h. The cells were observed under the light, fluorescence and transmission electron microscope for any morphological changes, while the cell viability was assessed by methylthiazol tetrazolium (MTT) assay. The cells in flasks were harvested for the estimation of various antioxidant parameters. Cell morphology, viability, malondialdeyde, glutathione and antioxidant enzymes were significantly altered in the control group as compared with the normal group. Administration of rHV3 confers significant protection against these changes in the human lens epithelial cells. These results demonstrated that rHV3 could effectively protect galactose-induced hLEC injury and suggested that it could have potential use in diabetic cataracts.     

Age-independent,gray matter-localized,brain-enhanced oxidative stress in male fischer 344 rats: brain levels of F(2)-isoprostanes and F(4)-neuroprostanes

While studies showed that aging is accompanied by increased exposure of the brain to oxidative stress, others have not detected any age-correlated differences in levels of markers of oxidative stress. Use of conventional markers of oxidative damage in vivo, which may be formed ex vivo and/or eliminated by endogenous metabolism, may explain these conflicting results. Recently, F₂-isoprostanes and F₄-neuroprostanes, peroxidation products of arachidonic acid and docosahexaenoic acid, respectively, have been identified as sensitive and reliable markers of oxidative injury. Therefore, this study was designed to quantify brain levels of F₂-isoprostanes and F₄-neuroprostanes and their precursors in 4, 10, 50, and 100 week old male Fischer 344 rats. Data show that levels of F₂-isoprostanes and F₄-neuroprostanes were comparable in all animal age groups. However, levels of F₄-neuroprostanes were approximately 20-fold higher than those of F₂-isoprostanes in all age groups, despite the fact that brain levels of docosahexaenoic acid were only twice as high as those of arachidonic acid. Based on our findings, it is concluded that aging is not accompanied by enhanced brain susceptibility to oxidative stress. Furthermore, the metabolically active gray matter of the brain, where docosahexaenoic acid is abundant, appears more susceptible to oxidative stress than the white matter.     

水稻核不育系4个柱头性状的遗传分析

为改良水稻(Oryza sativa)核不育系柱头性状提供遗传信息, 调查了粳型核不育系7001S、籼型核不育系Z913S及其杂交、自交获得的F₁、F₂和F_2:3群体的4个柱头性状, 分析了4个性状间的相关性, 并利用主基因+多基因遗传模型对2个世代4个性状进行遗传分析。结果表明, 4个性状两两间呈极显著正相关, 相关系数介于0.274-0.897之间。除F_2:3群体中花柱长度和柱头外露率分别表现出受2对加性-显性主基因和1对负等效加性-显性主基因+多基因控制外, F₂和F_2:3群体的柱头长度、花柱长度、柱头-花柱总长度以及柱头外露率均表现出受2对主基因和多基因控制, 且F_2:3群体中控制花柱长度的主基因表现出加性-显性效应, 其余均表现出加性-显性-上位性效应。2个世代中4个性状均以主基因遗传为主。     

Increased sperm abnormalities due to dietary restriction

The effect of dietary restriction on sperm-head morphology in BDF₁ mice was studied. The food intake of the ice was restricted to 2.0 or 1.5 g/day during the whole experimental period, while control animals were fed ad libitum. The average food intake of control mice was 4.2 ± 0.5 g/day. The frequencies of abnormal sperm in food-restricted mice remained at the basal level for the first 2 weeks. In week 3, the frequencies of abnormal sperm increased only in mice on the severely restricted diet (1.5 g/day). In week 5, the frequencies of abnormal sperm increased significantly in both restricted groups, and a negative correlation between the food intake and the frequency of abnormal sperm was observed. These results suggest that sperm abnormalities are not always the results of exogenous mutagen-induced damage     

草鱼(♀)×赤眼鳟(♂) F1及其亲本CAST基因cDNA全长克隆与结构差异

钙蛋白酶抑制蛋白(Calpastatin, CAST)在肌肉生长和肉质特征形成中发挥重要作用。为探究草鱼(Ctenopharyngodon idellus) (♀)×赤眼鳟(Squaliobarbus curriculus) (♂)正交F₁的肉质相关分子基础, 通过RACE(Rapid-amplification of cDNA ends) 技术分别克隆了草鱼(♀)×赤眼鳟(♂) F₁及其亲本的CAST基因cDNA全长, 并利用生物信息学方法分析比较了三种鱼的CAST结构差异。结果表明: 草鱼(♀)、赤眼鳟(♂)及草鱼(♀)×赤眼鳟(♂) F₁的CAST基因cDNA全长分别为3036、3165和3086 bp, 编码901、893和904个氨基酸; 预测蛋白质分子量分别为93.72、92.77和94.02 kD; 推测的理论等电点分别为5.92、6.01和6.02。草鱼(♀)×赤眼鳟(♂) F₁ CAST与草鱼(♀)和赤眼鳟(♂)核苷酸序列相似性分别为94.52%和90%。三种CAST蛋白均包括4个含有典型七肽的钙蛋白酶抑制结构域。草鱼(♀)、赤眼鳟(♂)和F₁ CAST氨基酸残基中分别存在73、82和75个潜在的磷酸化修饰位点。蛋白三级结构分析显示草鱼(♀)、赤眼鳟(♂)和F₁ CAST中分别含有24、12和20个β-折叠, 且均呈链状结构。综合可知, F₁ CAST在序列相似度、磷酸化位点数、蛋白质结构及进化地位与草鱼(♀)均更接近。该研究结果为阐明草鱼(♀)×赤眼鳟(♂)正交F₁肉质形成机理提供了分子基础。     

杉木解酚菌的研究

从杉木林土壤中筛选到5株高效解酚菌(F₂、F₃、F₄、F₇、F₁₅),结果表明,F₄、Fd,F₂、F₁₅5d能将600rng·L^-1的阿魏酸降解完,F₇的降解率为91.3%;F₄、F₃、F₂、F₇d,F₁₅、4d能将600mg·L^-1的对羟基苯甲酸完全降解;5株解酚菌对邻香草醛的降解能力较弱,除F₂5d能将300mg·L^-1的邻香草醛完全降解外,其余4株5d的降解率为83%～96%.同时发现,F₄能抑制杉苗生长,而其余4株则能在不同条件下促进杉苗生长,将5种菌按等比例混合使用,在土壤添加酚酸的情况下,仍能促进菌根苗Z和非菌根苗生长,杉木苗干重增长率为8.9%～168%.     

Meiosis in male PL/J mice: a genetic model for gametic aneuploidy

Sperm from mice of the PL/J strain have a high frequency of sperm-head morphology abnormalities. Fluorescence in situ hybridization (FISH) methods revealed that PL/J sperm are also characterized by a high frequency of aneuploidy. The traits of abnormal sperm head morphology and aneuploidy are associated with numerous meiotic abnormalities. Spermatocytes of PL/J mice exhibit chromosome asynapsis during meiotic prophase as well as reduced crossing over, revealed by analysis of both MLH1 foci in pachytene spermatocytes and chiasmata seen at the first meiotic metaphase. During the first meiotic division, roughly one-third of the PL/J spermatocytes exhibit aberrant spindle morphology, with abnormalities including monopolar spindles, split spindle poles, and incomplete spindle formation and centrosomal abnormalities. F1 progeny of a cross between PL/J and C57BL/6J did not exhibit a high frequency of either sperm aneuploidy or sperm head morphology aberrations, as would be expected if the PL/J traits were dominant. Among progeny of a backcross of F1 mice to PL/J, none of 16 males assessed exhibited elevated frequencies of sperm head morphology abnormalities. Four of the individuals exhibited elevated sperm aneuploidy, but not at the levels of the PL/J parents. Thus, it is likely that the aberrant PL/J traits are due to several genes and/or modifiers affecting the generation of both sperm aneuploidy and abnormal sperm head morphology.