Isolation of Subunits of Coupling Factor 1 from Maize and Spinach Chloroplasts and Properties of Combinations of Subunits with ATPase Activity

Subunits (, ß, ) and mixtures of subunits ( ß, , ß , ß ) were isolated without denaturationfrom a chloroform extract of chloroplast coupling factor 1 (CF₁)from maize (Zea mays var. Ushiku 5-4) and from spinach by fastprotein liquid chromatography (FPLC), on an anion-exchange columnof Mono-Q in the presence of n-octylglucoside (OG) and on achromatofocusing column of Mono-P. The ß -subunitcomplex (CF¹ ß ) was the minimum unit required forATPase activity, as was confirmed by the reconstituted complexof ß and subunits. An subunit isolated from maizeinhibited the ATPase activity of CF₁ ß from bothmaize and spinach. CF₁ ß was found to contain anOG-dependent Mg²⁺-ATPase. The ATPase activity of CF₁ ß required divalent cations, such as Mg²⁺ or Mn²⁺, for its expressionin the presence of OG; its optimum pH was 8.0 and it was markedlyinhibited by NaN₃. The enzyme hydrolyzed ATP in prefernece toGTP but not CTP, UTP, ADP, AMP or pNPP. Lineweaver-Burk plotsof its activity were curvilinear in the range of 0.6–0.7mM ATP.Mg²⁺. ¹Present address: Department of Biology, School of Education,Waseda University, Shinjuku-ku, Tokyo, 160 Japan. (Received February 15, 1989; Accepted April 20, 1989)     

玉米素对叶绿体耦联因子Mg~（2＋）－ATPase活力的调节

用２μｇ／ｍｌ玉米素溶液预处理叶绿体或在光活化前于活化液中加入２μｇ／ｍｌ玉米素溶液,观察到玉米素能促进叶绿体膜上耦联因子ＤＴＴ光活化Ｍｇ２＋－ＡＴＰａｓｅ及Ｍｇ２＋ＧＴＰａｓｅ的活力．且对ＧＴＰａｓｅ的促进比例常较ＡＴＰａｓｅ的大些。王米素对ＯＧ活化可溶性ＣＦ１Ｍｇ２＋－ＡＴＰａｓｅ活力同样表现出促进作用。用玉米素预处理ＣＦ１－β亚基（含微量ＣＦ１－α亚基）也观察到它能促进ＣＦ１－β亚基催化的Ｍｇ２＋－ＡＴＰａｓｅ活力。这些结果表明,玉米素在ＣＦ１上的作用部位至少有一个在β亚基或α．β亚基交界处调节其催化功能的。     

Genetic Study of the Loss and Restoration of Mutator Transposon Activity in Maize: Evidence against Dominant-Negative Regulator Associated with Loss of Activity

The Mutator system of transposable elements is characterized by a family of transposons called Mu transposons that share common termini and are actively transposing in Robertson's Mutator (Mu) lines of maize. Mu lines lose transposition activity during propagation by either outcrossing or inbreeding. This loss of transposition activity, which can occur at non-Mendelian frequencies, is in the form of loss of forward transposition activity resulting in a decrease in the generation of new mutations, as well as the loss of mutability of Mu transposon induced mutations, and it has been correlated with hypermethylation of the Mu elements. Previous studies have concluded that restoration of Mutator transposon activity by crossing inactive lines back to active lines is incomplete or transient, and depends upon the sex of the inactive parent. Further, it has been proposed that the inactive system is dominant to the active system, with the dominance possibly mediated through a negative regulatory factor that is preferentially transmitted through the female. In this study, we have examined the frequencies of loss and restoration of Mu transposon activity using a Mu line carrying an insertion in the bronze 1 locus. We find that transmission of Mu transposon activity to non-Mu plants can occur at high rates through males and females, but individual cases of decreased transmission through the male were observed. We also find that in crosses between inactive-Mu and active-Mu plants, reactivation was efficient as well as heritable, regardless of the sex of the inactive parent. Similar results were obtained whether the inactivation occurred in an outcross or a self. In all cases examined, loss of Mu transposon activity was correlated with hypermethylation of Mu elements, and reactivation was correlated with their demethylation. Our results indicate that an inactive Mu system does not exhibit dominance over an active Mu system. We conclude that contrary to current models, inactivation and its maintenance is not obligatorily associated with a dominant negative regulatory factor whether nuclear or cytoplasmic, and we propose a revised model to account for these and other observations.     

玉米bHLH类转录因子ABP7的启动子克隆及其活性分析

bHLH类转录因子广泛存在于植物中,在花的形态建成、光和激素信号转导等方面发挥着重要作用,但其表达调控机理尚待深入研究。从玉米中克隆了bHLH类转录因子ABP7,初步研究结果显示它可能参与了籽粒形成、激素应答等多个信号转导途径的调控。为进一步阐明ABP7基因上游调控途径,克隆了ABP7的5′侧翼序列,利用原生质体瞬时转化体系验证了其启动子活性,并对其顺式作用元件进行了分析。结果显示,位于ABP7基因起始密码子上游长为2 266 bp的DNA序列具有启动子活性,能够启动报告基因的表达,并且存在许多潜在的与GA、IAA和ABA激素信号转导和逆境胁迫响应相关的元件,为进一步鉴定ABP7基因发挥作用的信号途径、上游调控因子和分子机理以及ABP7基因在玉米生长发育和逆境应答中的功能奠定了基础。     

Characteristics of Membrane-Bound Lectin in Developing Phaseolus vulgaris Cotyledons

Cotyledons of developing Phaseolus vulgaris L. cv Greensleeves seeds were labeled for 2 to 3 hours with ³H-amino acids, and newly synthesized phytohemagglutinin (PHA) was isolated by affinity chromatography with thyroglobulin-Sepharose. The presence of 1% Tween in the homogenate increased the yield of radioactive PHA by 50 to 100%. Isopycnic sucrose gradients were used to show that this detergent-released PHA was associated with the endoplasmic reticulum (ER), and pulse-chase experiments showed that the half-life of the PHA in the ER was 90 to 120 minutes. Since PHA is transiently associated with the ER and accumulates in protein bodies, we postulate that this rapidly turning over pool of PHA in the ER represents protein en route to the protein bodies. The PHA in the ER has the same sedimentation constant as mature PHA and is capable of agglutinating red blood cells. The observations substantiate earlier claims that plant cells contain membrane-bound lectins. However, they also indicate that these lectins are not necessarily functional components of the membranes with which they are associated, but may represent transport pools of lectin normally localized in other cellular compartments.     

Proliferative Activity of Liver Growth Factor is Associated with an Improvement of Cigarette Smoke-Induced Emphysema in Mice

Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression.     

Friendship Network Characteristics Are Associated with Physical Activity and Sedentary Behavior in Early Adolescence

Introduction

There is limited understanding of the association between peer social networks and physical activity (PA), sedentary and screen-related behaviors. This study reports on associations between personal network characteristics and these important health behaviors for early adolescents.

Methods

Participants were 310 students, aged 11–13 years, from fifteen randomly selected Victorian primary schools (43% response rate). PA and sedentary behaviors were collected via accelerometer and self-report questionnaire, and anthropometric measures via trained researchers. Participants nominated up to fifteen friends, and described the frequency of interaction and perceived activity intensity of these friends. Personal network predictors were examined using regression modelling for PA and sedentary/screen behavior.

Results

Perceived activity levels of friends, and friendships with very frequent interaction were associated with outside-of-school PA and/or sedentary/screen time. Differences according to sex were also observed in the association between network characteristics and PA and sedentary time. A higher number of friends and greater proportion of same sex friends were associated with boys engaging in more moderate-to-vigorous PA outside of school hours. PA intensity during school-day breaks was positively associated with having a greater proportion of friends who played sports for girls, and a greater proportion of male friends for boys.

Conclusion

Friendship network characteristics are associated with PA and sedentary/screen time in late childhood/early adolescence, and these associations differ by sex. The positive influence of very active peers may be a promising avenue to strengthen traditional interventions for the promotion of PA and reduction in screen time.     

Inhibition of Membrane-Bound Adenosine Triphosphatase Activity from Nicotiana tabacum L. Leaves with Alkyltrimethylammonium Bromide

Cetyltrimethylammonium bromide (CTAB), a cationic detergent,more effectively inhibited the activity of membrane-bound epidermaladenosine triphosphatase (ATPase) of tobacco (Nicotiana tabacumL. cv. Samsun) leaves than anionic or non-ionic detergents.The inhibition of ATPase activity was highly dependent on thelength of the alkyl chain of alkyltrimethylammonium: CTAB >dodecyltrimethylammonium bromide > n-octyltrimethylammoniumbromide trimethylammonium chloride cetyl bromide, comparedat 10^–4 M. The last three derivatives hardly inhibitedthe activity. CTAB inhibition was equivalent to that due toother cationic detergents, cetylpyridinium bromide and cetylamine, but less than that by gramicidin S and tyrocidine andstronger than that by N,N'-dicyclohexylcarbodiimide and vanadate. These results show that a certain length of the alkyl chain(C_n>12) and the combination of both hydrophobic and chargedgroups of a detergent moiety are indispensable for inhibitingthe membrane-bound epidermal ATPase activity. (Received January 26, 1982; Accepted April 10, 1982)     

Characterization of Maize Chlorotic Mottle Virus Associated with Maize Lethal Necrosis Disease in China

A maize lethal necrosis disease was observed in Yunnan province of China. Isometric virus particles 30 nm in diameter were found in infected maize leaf tissues. Using DAS‐ELISA, diseased maize plant samples reacted positively with the antiserum of Maize chlorotic mottle virus (MCMV). The complete nucleotide sequence (4436 nt) of a Yunnan isolate of MCMV was determined; it shares 97% nucleotide sequence identity with previously reported MCMV isolates. This is the first report of MCMV occurring in China.     

Preparation of Corn Root Plasmalemma with Low Mg-ATPase Latency and High Electrogenic H Pumping Activity after Phase Partitioning

Crude plasma membranes of corn (Zea mays L.) roots were obtained according to MI De Michelis and RM Spanswick (1986 Plant Physiol 81: 542-547). This preparation, which contained tightly sealed vesicles displaying Mg-ATP dependent H⁺-transport, was purified by phase partitioning. The percentage of inside-out vesicles (10%) was determined from the Mg-ATPase latency, revealed with lysophosphatidylcholine. A Triton X-100 treatment described previously (JP Grouzis, R Gibrat, J Rigaud, C Grignon 1987 Biochim Biophys Acta 903: 449-464) was applied to phase-partitioned plasma membranes. The percentage of catalytic sites freely accessible to Mg-ATP increased to 50% after Triton X-100 treatment. Treated vesicles remained capable of electrogenic H⁺-pumping, as demonstrated by Mg:ATP-dependent quinacrine fluorescence quenching and oxonol absorbance shift. As expected from the large increase of the catalytic sites accessibility, increases of the dye responses were observed. Concanavalin A binding was estimated from microelectrophoretic measurements of individual vesicles. Statistical analysis of concanavalin A binding and Mg-ATPase latency suggest that treated membranes have lost their asymmetric structure.     

Diazotrophic Burkholderia Species Associated with Field-Grown Maize and Sugarcane

Until recently, diazotrophy was known in only one of the 30 formally described species of Burkholderia. Novel N₂-fixing plant-associated Burkholderia species such as B. unamae, B. tropica, and B. xenovorans have been described, but their environmental distribution is scarcely known. In the present study, the occurrence of N₂-fixing Burkholderia species associated with different varieties of sugarcane and maize growing in regions of Mexico and Brazil was analyzed. Only 111 out of more than 900 isolates recovered had N₂-fixing ability as demonstrated by the acetylene reduction assay. All 111 isolates also yielded a PCR product with primers targeting the nifH gene, which encodes a key enzyme in the process of nitrogen fixation. These 111 isolates were confirmed as belonging to the genus Burkholderia by using a new 16S rRNA-specific primer pair for diazotrophic species (except B. vietnamiensis) and closely related nondiazotrophic Burkholderia. In Mexico, many isolates of B. unamae (predominantly associated with sugarcane) and B. tropica (more often associated with maize) were recovered. However, in Brazil B. tropica was not identified among the isolates analyzed, and only a few B. unamae isolates were recovered from one sugarcane variety. Most Brazilian diazotrophic Burkholderia isolates (associated with both sugarcane and maize plants) belonged to a novel species, as revealed by amplified 16S rRNA gene restriction profiles, 16S rRNA gene sequencing, and protein electrophoresis. In addition, transmissibility factors such as the cblA and esmR genes, identified among clinical and environmental isolates of opportunistic pathogens of B. cenocepacia and other species of the B. cepacia complex, were not detected in any of the plant-associated diazotrophic Burkholderia isolates analyzed.     

Reduced Soluble Proteins Associated with Maize Endosperm Protein Bodies

Endosperm protein bodies from developing maize were purifiedby discontinuous sucrose gradient centrifugation and the proteincontent analysed by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAE). Major proteins detected were zeinpolypeptides plus a component with Mr 28 000 and a doublet aroundMr 58 000. These proteins were present only in the protein bodyfraction of the sucrose gradient. Treatment of protein bodieswith the reducing agent dithiothreitol (DTT) in aqueous bufferdissolved the components with Mr 28 000 and 58 000, plus minorones, but not zein. The reduced soluble proteins were separatedby DEAE-Sephacel chromatography into three fractions: two ofthese contained the component with Mr 28 000, and the thirdthe components around Mr 58 000 plus minor ones. Proteins fromthe three fractions had characteristic amino acid compositions,markedly different from those of zein polypeptides. Chymotrypticdigestion experiments performed on protein bodies under variousconditions, and two-dimensional electrophoresis of proteinsfrom protein bodies suggested that the major zein polypeptides,the protein with Mr 28 000 and the other reduced soluble proteinshave different native organizations.     

Nitrogenase Activity Associated with Halodule wrightii Roots

Nitrogen fixation (acetylene reduction) associated with roots of the seagrass Halodule wrightii was measured offshore near Beaufort and Moorhead City, N.C. Rates of acetylene reduction were higher in aerobic than in anaerobic assays and were linear for up to 5 days. The temperature range for acetylene reduction was 15 to 35°C with a maximum activity at 35°C. Nitrogenase activity was shown to vary seasonally with highest activities occurring during warmer summer months (23 μg of N₂ fixed per m² per day). At in situ temperature, nitrogenase activities associated with surface-sterilized and non-surface-sterilized roots were similar. One morphological bacterial type was isolated from surface-sterilized roots and identified as Klebsiella pneumoniae type 4B.