Proton pumping pyrophosphatase from higher plant mitochondria

In higher plant cells, there are some enzymes capable of utilizing pyrophosphate (PP_i) as an energy donor. Among these, membrane-bound proton pumping pyrophosphatases (H⁺-PP_iase) have been identified. In addition to the well-known vacuolar H⁺-PP_iase (V-PP_iase), there is evidence for the presence of a mitochondrial H⁺-PP_iase. This enzyme is localized on the inner surface of the inner membrane and catalyzes the specific hydrolysis of PP_i, coupled to proton transport, with a H⁺/PP_i stoichiometry of ca 2. This activity is Mg²⁺-requiring, is stimulated by monovalent cations, and is inhibited by Ca²⁺, F⁻ and diphosphonates. The H⁺-PP_iase contains a catalytic head which is constituted by a 35-kDa protein which is loosely bound to the inner membrane. This protein exhibits a PP_iase activity, stimulated by phospholipids, with characteristics very similar to the membrane-bound enzyme. The mitochondrial PP_iase is distinct from the V-PP_iase, because an antibody raised against the 35-kDa protein does not react with tonoplast membranes. The mitochondrial H⁺-PP_iase seems to have an F-type structure, similar to the F-ATP synthase and the membrane-bound PP_iases from mammalian and yeast mitochondria. It is suggested that, beside synthesizing PP_i, this enzyme may act as a buffer for the electrochemical proton gradient, by hydrolyzing PP_i, during conditions of oxygen deprivation.     

Temperature-dependent regulation of d-cis-[H]diltiazem binding to Ca channels by 1,4-dihydropyridine channel agonists and antagonists

The binding of the Ca²⁺-channel blocker d-cis-[³H]diltiazem to guinea pig skeletal muscle microsomes is temperature-dependent. At 2°C the K_D is 39 nM and B_max is 11 pmol/mg protein. The binding is fully reversible (K₋₁ = 0.02 min⁻¹). The binding sites discriminate between the diastereoisomers 1- and d-cis-diltiazem, recognize verapamil, gallopamil and tiapamil, and are sensitive to La³⁺-inhibition. At 30°C the K_D is 37 nM and the B_max is 2.9 pmol/mg protein. D-cis-diltiazem-labelling is regulated by the 1,4-dihydropyridine Ca²⁺-channel blockers and a novel Ca²⁺-channel activator in a temperature-dependent manner. At 30°C an enhancement of d-cis-diltiazem binding by the channel blockers is observed. This is attributed to a B_max increase. EC₅₀-values for enhancement and the maximal enhancement differ for the individual 1,4-dihydropyridines. At 2°C 1,4-dihydropyridines inhibit d-cis-[³H]diltiazem binding. This is attributed to a B_max decrease. We have directly labelled one of the drug receptor sites within the Ca²⁺-channel which can allosterically interact with the 1,4-dihydropyridine binding sites.     

Swelling-activated chloride channels in cardiac physiology and pathophysiology

Characteristics and functions of the cardiac swelling-activated Cl current (I_Cl,swell) are considered in physiologic and pathophysiologic settings. I_Cl,swell is broadly distributed throughout the heart and is stimulated not only by osmotic and hydrostatic increases in cell volume, but also by agents that alter membrane tension and direct mechanical stretch. The current is outwardly rectifying, reverses between the plateau and resting potentials (E_m), and is time-independent over the physiologic voltage range. Consequently, I_Cl,swell shortens action potential duration, depolarizes E_m, and acts to decrease cell volume. Because it is activated by stimuli that also activate cation stretch-activated channels, I_Cl,swell should be considered as a potential effector of mechanoelectrical feedback. I_Cl,swell is activated in ischemic and non-ischemic dilated cardiomyopathies and perhaps during ischemia and reperfusion. I_Cl,swell plays a role in arrhythmogenesis, myocardial injury, preconditioning, and apoptosis of myocytes. As a result, I_Cl,swell potentially is a novel therapeutic target.     

Determination of (-)-threo-chlorocitric acid in human plasma by gas chromatography—positive chemical-ionization mass spectrometry

A method is described for measuring (-)-threo-chlorocitric acid in human plasma. Plasma is acidified to pH 1 to minimize lactonization and a¹³C analogue of (-)-threo-chlorocitric acid is added as internal standard. The acidified plasma is then extracted with ethyl acetate containing 10% methanol. The ethyl acetate—methanol extract is back-extracted with acetate buffer (pH 5). This extract, following adjustment to pH 1, is reextracted with ethyl acetate. The residue after removal of the ethyl acetate is treated with ethereal diazomethane. The wet residue is reconstituted in ethyl acetate and a portion of this solution is analyzed by gas chromatography—chemical ionization mass spectrometry. The mass spectrometer is set to monitorm/z269 [MH⁺ of trimethylated (-)-threo-chlorocitric acid] andm/z270 [MH⁺ of trimethylated (-)-threo-[¹³C]chlorocitric acid] in the gas chromatographic effluent. Them/z/269 tom/z270 ion ratio in a sample containing an unknown amount of (-)-threo-chlorocitric acid is converted to an amount of compound using a calibration curve. The calibration curve is generated by analyzing control plasma spiked with various known amounts of (-)-threo-chlorocitric acid and a fixed amount of (-)-threo-[¹³C]chlorocitric acid. The limit of quantitation is 0.1–0.6 μg ml⁻¹, depending on the characteristics of the calibration curve generated with each set of samples. The precision (relative standard deviation) at a concentration of 2 μg ml⁻¹ is 3.3%.     

Spectral study of b cytochromes in yeast mitochondria and intact cells

Three types of b cytochromes are demonstrated in Candida utilis mitochondria. One of these b cytochromes has a symmetrical -band at 561.5 nm at room temperature. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate both in coupled and uncoupled mitochondria. The second b cytochrome has a double -band at 565 nm and 558 nm. This b cytochrome is readily reduced either by anaerobiosis or by cyanide treatment in the presence of glycerol 1-phosphate or succinate in coupled mitochondria, but in uncoupled mitochondria it is slowly reduced after anaerobiosis and this reduction rate is enhanced by antimycin A addition. Thus the oxidation-reduction state of this cytochrome is energy dependent. The first cytochrome is spectroscopically identified as cytochrome b_K and the second as cytochrome b_T. The third b cytochrome has an -band around 563 nm (b₅₆₃) and is reduced slowly after anaerobiosis in uncoupled mitochondria but faster than the b_T. Further properties of this component are not known. Midpoint potentials of cytochromes b_T, b₅₆₃ and b_K are approximately −50 mV, +5 mV, and +65 mV, respectively.

In intact cells, cytochrome b_T is reduced immediately after anaerobiosis or cyanide treatment, and rapidly oxidized when uncoupler is added. Addition of antimycin A instead of uncoupler to the anaerobic cells causes oxidation of mainly cytochrome b_T while addition of antimycin A to the aerobic cells results in a reduction of the cytochrome b_T.     

Characterization of Ligand Binding to the Cannabinoid Receptor of Rat Brain Membranes Using a Novel Method: Application to Anandamide

Abstract: Ligand binding to the cannabinoid receptor of brain membranes has been characterized using [³H]CP 55,940 and the Multiscreen Filtration System. Binding of [³H]CP 55,940 is saturable and reaches equilibrium by 45 min at room temperature. At a concentration of 10 µg of membrane protein/well, the K _D for [³H]CP 55,940 is 461 p M and the B _max is 860 fmol/mg of protein. The apparent K _D of [³H]CP 55,940 is dependent upon tissue protein concentration, increasing to 2,450 p M at 100 µg of membrane protein. Binding of [³H]CP 55,940 is dependent upon the concentration of bovine serum albumin in the buffer; the highest ratio of specific to nonspecific binding occurs between 0.5 and 1.0 mg/ml. The K _i of anandamide, a putative endogenous ligand of the cannabinoid receptor, is 1.3 µ M in buffer alone and 143 n M in the presence of 0.15 m M phenylmethylsulfonyl fluoride. When [¹⁴C]anandamide is incubated with rat forebrain membranes at room temperature, it is degraded to arachidonic acid; the hydrolysis is inhibited by 0.15 m M phenylmethylsulfonyl fluoride. These results support the hypothesis that anandamide is a high-affinity ligand of the cannabinoid receptor and that it is rapidly degraded by membrane fractions.     

Breeding systems and phylogeny in Poa,with special attention to Northeast Asia: The problem of Poa shumushuensis and sect. Nivicolae (Poaceae)

Poa sect. Poa subsect. Nivicolae (Prob.) Tzvelev was circumscribed to include four species of the Soviet Union: Poa shumushuensis, Poa caucasica, Poa irkutica, and Poa veresczaginii. Bayesian phylogenetic analyses of plastid and nuclear‐ribosomal DNA revealed that it is polyphyletic, none of these species are closely related. Poa shumushuensis, type of sect. Nivicolae, or its ancestor, likely displayed the nrDNA genotype characteristic of the higher polyploid P. sect. Malacanthae. Genotype codes are designated for each species: Hx, C c, Php, and Shp. Poa sect. Nivicolae s.s. is restricted to P. shumushuensis; P. sect. Irkuticae is restricted to P. irkutica; P. caucasica is moved to P. subg. & sect. Caucasicae nov.; and P. sect. Dschungaricae is resurrected for P. veresczaginii and two other species. Although diclinous breeding systems are known in many western hemisphere species of Poa, dicliny is infrequent and little studied in Asian Poa. Poa shumushuensis is judged to be either sequentially gynomonoecious or gynodioecious. A ratio of 2 perfect‐, to 2 mixed‐, to 1 pistillate‐flowered inflorescences from different plants in P. shumushuensis is suggestive of a recessive allele for stamen suppression, and this is associated with subtle sexual‐dimorphism. Poa irkutica is diclinous with a breeding system between simple gynomonoecy and sequential gynomonoecy; P. caucasica is perfectly flowered; and P. veresczaginii has infrequent abortive anthers, indicative of limited dicliny, or sterility for other reasons possibly related to its reticulate origin. In total, 23 Asian species are here reported to be diclinous and their breeding systems are characterized. A lectotype is designated for P. fauriei.     

Nitrogen fixation and nitrate reduction in the root nodules of legumes

Published data on, and hypotheses regarding the effect of NO⁻₃ on functioning of legume root nodules are reviewed. It is concluded that a short-term reversible effect of NO⁻₃ may act via an increased resistance to O₂ diffusion in nodules; this is coupled to decreased bacteroid respiration. For longer exposures to NO⁻₃ nodule activity is irreversibly lost, but how this relates to carbohydrate deprivation or NO-₂ accumulation is unclear. Complicating factors include denitrification reactions and the interaction of NO⁻₂ with leghaemoglobin.     

Specific effect of manganese on the allantoinase of Vigna Radiata

Vigna radiata seedlings germinated in the presence of Mn²⁺ show an unusual increase in allantoinase activity which is proportional to Mn²⁺ concentration up to 5 mM. Though Mn²⁺ is not an activator for V. radiata allantoinase, it specifically protects allantoinase against thermal as well as papain-catalysed inactivation. Evidence is presented to show that the primary effect of Mn²⁺ is a protective one, both in vitro and in vivo, and that this is reflected in the observed enhancement of allantoinase activity in Mn²⁺ grown seedlings. That this unusual effect of Mn²⁺ is a specific one is indicated by the lack of a similar effect with Mg²⁺. Cu²⁺ is shown to destabilize V. radiata allantoinase in vitro as well as in vivo.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.     

The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434

A new plasmid vector, pNSI, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. It is a derivative of pBR327, and it includes a regulatory region from the lambdoid phage 434. The expression of the Tc^R gene of pNS1 is under the control of the o_Rp_R operator-promoter of phage 434, which is regulated by the represser gene c1. The cloning sites of pNSI (StuI, NdeI, HpaI, HindIII, AsuII and EcoRI) are situated within cI; hence insertion of foreign DNA into these sites causes derepressed expression of the Tc^R gene from p_R thus conferring the Tc^R phenotype on the harboring Escherichia coli strain. The use ofpNS1 is facilitated by the presence of another selectable marker, Ap^R its small size, and its known nucleotide sequence; no special host strain is required.     

The intrinsic rate of natural increase and reproductive effort in primates

The intrinsic rate of natural increase (r_max) is calculated for 58 primate species. It is found that this parameter is negatively correlated with body weight, so that larger primate species consistently have a lower r_max than do smaller species. Although there is no apparent link between the raw value of r_max and environmental predictability, a relationship between a high r_max, relative to body weight, and an unpredictable environment is found to exist. However, there is no relationship between a predictable environment and either the raw value of r_max, or the relative r_max. After body size effects are removed, r_max is not correlated with basal metabolic rate (BMR). Pre-natal maternal investment (MI) is found to correlate highly with BMR, even after the removal of body weight effects. MI does not correlate either with r_max or with environmental predictability. Diet appears to have little influence either on r_max on MI, but there is some indication that folivores have both a high relative r_max and a high relative MI. These results are discussed in the light of theories of life-history strategy evolution.     

根虫瘟霉和相关种的分类地位研究及其ITS核苷酸序列分析

根虫瘟霉是最常见的一种虫霉,寄主广泛,世界广布。目前有学者认为该种是个复合种。本研究对世界不同地区和不同寄主的根虫瘟霉及其近缘类群总计19个菌株,进行3个靶位点（ITS、LSU rDNA、RPB2）的分子系统发育学分析。结果显示,根虫瘟霉ITS长度较为保守性,介于1 321-1 324bp之间,而所研究的虫霉亚门的其他类群的长度范围较大,为556-1 654bp。本研究确认根虫瘟霉是单系种,同时西虫瘟霉、矛孢虫瘟霉和英吉利虫瘟霉具有明确种的分类地位。鬼笔状虫瘟霉种应该被视为西虫瘟霉的异名。     

BIOCHEMICAL CHARACTERIZATION OF THE NEURON: RIBONUCLEIC ACID COMPOSITION OF THE NEURONAL CELL BODIES

Abstract— The fluorescence of chlorotetracycline (CTC) in the presence of synaptosomes isolated from sheep brain is selectively increased by Ca²⁺ under conditions in which Mg²⁺, Na⁺, K⁺, Li⁺ or choline have only a small effect. The monovalent cations release bound Ca²⁺ from synaptosomes, and this effect is reflected by a decrease in the CTC fluorescence. Under optimal conditions there is a near parallelism between Ca²⁺ and CTC binding to the synaptosomes membranes, and Li⁺ is the monovalent cation tested which interferes the most with the binding of both substances. These results obtained in a predominantly sucrose medium become less distinct when media simulating physiological composition are utilized, which limits the usefulness of the method. Brain mitochondria and myelin also bind Ca²⁺ and CTC. The ratio of the fluorescence signal (or CTC bound) to Ca²⁺ bound is highest of all for mitochondrial membranes, and the apparent fluorescence quantum yield of CTC is also the highest in these membranes, which suggests that the Ca²⁺ in these membranes is localized in a more apolar region than is the case for synaptosomes and myelin.     

Characterization of purified cytochrome c1 from Rhodobacter sphaeroides R-26

Cytochrome c₁ from a photosynthetic bacterium Rhodobacter sphaeroides R-26 has been purified to homogeneity. The purified protein contains 30 nmol heme per mg protein, has an isoelectric point of 5.7, and is soluble in aqueous solution in the absence of detergents. The apparent molecular weight of this protein is about 150 000, determined by Bio Gel A-0.5 m column chromatography; a minimum molecular weight of 30 000 is obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The absorption spectrum of this cytochrome is similar to that of mammalian cytochrome c₁, but the amino acid composition and circular dichroism spectral characteristics are different. The heme moiety of cytochrome c₁ is more exposed than is that of mammalian cytochrome c₁, but less exposed than that of cytochrome c₂. Ferricytochrome c₁ undergoes photoreduction upon illumination with light under anaerobic conditions. Such photoreduction is completely abolished when p-chloromercuriphenylsulfonate is added to ferricytochrome c₁, suggesting that the sulfhydryl groups of cytochrome c₁ are the electron donors for photoreduction. Purified cytochrome c₁ contains 3 ± 0.1 mol of the p-chloromercuriphenylsulfonate titratable sulfhydryl groups per mol of protein. In contrast to mammalian cytochrome c₁, the bacterial protein does not form a stable complex with cytochrome c₂ or with mammalian cytochrome c at low ionic strength. Electron transfer between bacterial ferrocytochrome c₁ and bacterial ferricytochrome c₂, and between bacterial ferrocytochrome c₁ and mammalian ferricytochrome c proceeds rapidly with equilibrium constants of 49 and 3.5, respectively. The midpoint potential of purified cytochrome c₁ is calculated to be 228 mV, which is identical to that of mammalian cytochrome c₁.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

The preparation and properties of the membranal DPNH dehydrogenase from Escherichia coli

1. The membrane bound DPNH oxidase of Escherichia coli can reduce the artificial electron acceptors: ferricyanide, dichlorophenolindophenol (DCIP) and menadione. All three are reduced by the flavoprotein of DPNH oxidase, but at different sites of the enzyme.

2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of K_m(K₃Fe(CN)₆) from 2.0 to 0.25 mM, while no change is detected in K_m(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.

3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.

4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher K_m(DPNH) and slower v_max., while the K_m(K₃Fe(CN)₆) remains unchanged.

5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed.     

不同天然居群小蓬竹的遗传多样性

从分子水平探讨不同居群小蓬竹的遗传多样性以及与环境因子的相关性,揭示其濒危原因,为小蓬竹的保护和后续开发利用提供理论支撑,助力实施极危物种最佳保护策略。运用RAPD标记技术和POPGENE32对16个小蓬竹天然居群进行遗传多样性研究和遗传变异分析。结果表明,8个RAPD随机引物共扩增出105条清晰、重复性高的条带,其中多态性条带有98条,分子量300~2000bp;物种水平多态性位点百分率PPL=93.33%,有效等位基因数Ne=1.4942,Nei’s基因多样性H=0.3005,Shannon多样性指数I=0.4586;落湾(ZY1)居群的遗传多样性水平最高(PPL=60.95%,H=0.2329,I=0.3451),[JP3]桃坡(PT1)居群的最低(PPL=44.76%,H=0.1700,[JP]I=0.2523);16个天然居群的遗传分化系数Gst=0.3231,基因流Nm=1.0478,基于Shannon’s多样性指数的分化系数[(HSP-HPOP)/HSP]为0.3429。小蓬竹居群内存在丰富的遗传多样性,各个天然居群间具有一定的遗传分化但分化水平并不高,主要的遗传变异存在于居群内部。