The Naturally Occurring PKC Inhibitor Sphingosine and Tumor Promoter Phorbol Ester Potentially Induce Tyrosine Phosphorylation/Activation of Oncogenic Proline-Directed Protein Kinase FA/GSK-3α in a Common Signalling Pathway

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase F_A/glycogen synthase kinase-3 (kinase F_A/GSK-3) could be decreased to ~25% of control. Conversely, when treated with 1 M TPA for 24 hr, the activity could be reversibly increased to ~200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 M could also induce activation of kinase F_A/GSK-3 to ~200% of control within 60 min. Further, when cells were chronically treated with 1 M TPA for 24 hr and then with 27 M sphingosine for 60 min, the activity of kinase F_A/GSK-3 could only be increased to ~200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase F_A/GSK-3 activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase F_A/GSK-3 in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase F_A/GSK-3. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKC in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.     

Effects of prolonged oral administration of fumonisin B1 and aflatoxin B1 in rats

The effects of prolonged oral administration (21 days) of fumonisin B₁ (FB₁) and aflatoxin B₁ (AFB₁) were evaluated on male Wistar rats. The animals were housed in individual metabolic cages and submitted to the following treatments: 1-0 g AFB₁ + 0 mg FB₁/100g bw.; 2-72 g AFB₁+ 0 mg FB₁/100 g bw; 3-0 g AFB₁ + 0.5 mg FB₁ g bw; 4-0 g AFB₁ + 1.5 mg FB₁/100 g bw; 5-72 g AFB₁ + 0.5 mg FB₁/100g bw; 6-72 gAFB₁ + 1.5 mg FB₁/100g bw. On day 21, the rats were sacrificed for evaluation. The results showed that treated animals presented differences in body weight and absolute/relative weights of liver and kidney as well as altered hepatic function and cholesterol blood levels. Rats fed with the greatest doses of AFB₁ and FB₁ gained less weight (2.79 g/day) at the end of the experimental period; their blood concentrations of liver enzymes aspartate aminotransferase (AST) and alkaline phosphatase (AP) were above control levels (130.35 /l and 471.00 /l, respectively). Blood cholesterol increased in the groups treated with the highest dose ofFB₁ or FB₁ associated with AFB₁. Histopathology revealed the occurrence of apoptosis in the liver of rats exposed to FB₁. The association of aflatoxin B₁ with fumonisin B₁ at higher dose probably potentiated the effects of the higher dose of fumonisin B₁acting singly.This revised version was published online in October 2005 with corrections to the Cover Date.     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

The inheritance of radiation induced semi-sterility in Rhodnius prolixus

Eggs from crosses of 40 adult male R. prolixus irradiated with 6K rad -rays with normal females had a mean fertility of 23.9%, only 2 crosses being completely sterile. The 86 F₁ progeny of both sexes, when outcrossed with normal mates, had a mean egg fertility of 12.6%, and 43 of these matings were completely sterile. Twenty-eight F₂ bugs reared from F₁ x normal crosses were mated with normal partners and had a mean fertility of 44.6%, 6 of them being fully fertile, a reversal towards normal fertility. Cytogenetic examination of F₁ F₂ and F₃ males showed that these changes in fertility correlated well with the degree of chromosomal abnormality found. The very high recovery rate of translocations in F₁ generation males can be related to the holocentric chromosomes of these bugs which precludes the formation of dicentric chromosomes which are inviable in monocentric species. In F₁ and F₂ males the majority of translocations were associated as chains of III+I or as chains of IV. Only one bug was found with a ring of IV chromosome association and it is suggested that chromosome morphology, combined with a low chiasma frequency, favours chain association. Most chain multivalents showed linear orientation which may lead to duplication deficiencies and zygotic death. However parallel, indifferent and the more stable convergent modes of chain orientation were also all observed indicating that survival of some translocations in this species may be possible. The survival to the F₂ generation of chromosomal fragments confirmed the holocentric nature of triatomine chromosomes. It is suggested that semi-sterile males would prove more effective than releases of completely sterile males for reducing wild populations of R. prolixus, because of the delayed effects of sterilizing radiation consequent upon the holocentric structure of triatomine chromosomes.     

Identification of subunits required for the catalytic activity of the F1-ATPase

F₁() complexes containing equimolar ratios of the and subunits have been shown to function as active ATPases, whereas individually isolated and subunits show no real ATPase activity. These results indicate that the single-copy subunits are not required for F₁-ATPase activity. The minimal F₁()-core complexes exhibit, however, lower rates and some different properties from those of their parent whole F₁ or ₃₃ complexes. It is therefore concluded that for obtaining a full spectrum of the characteristic functional properties of an F₁-ATPase the presence of the F₁- subunit is also required. The implications of these findings on the subunit location of both catalytic and noncatalytic nucleotide binding sites is discussed.     

Confidence interval estimators for heritability for several mating and experiment designs

Summary Confidence interval estimators have not been described for several heritability (H) estimators relevant to recurrent family selection. Previously described H interval estimators do not apply to onefactor mating designs in split-plot in time experiment designs in one or more locations, one-factor mating designs for several experiment designs in two or more locations and years, and two-factor mating designs for several experiment designs in two or more locations or years. Our objective was to derive H interval estimators for these cases. H reduced to a function of constants and a single expected mean square ratio in every case; H=1–E(M)/E(M) where E(M) is a linear function of expected mean squares and E(M) is a single expected mean square. It was shown that F=[M/E(M)]/[M/E(M)] has an approximate F-distribution with df and df degrees of freedom, respectively, where M and M are mean squares corresponding to E(M) and E(M), respectively. H is a function of F, therefore, we used F to define an approximate (1–) interval estimator for H.Oregon Agricultural Experiment Station Technical Paper No. 7923     

A light-enhanced metabolism of sulfite in cells of Cucumis sativus L. cotyledons

The effects of light and several photosynthetic inhibitors on the rate of sulfite metabolism in cells obtained from Cucumis sativus L. cotyledons was studied. The cells were treated with 200 M Na₂SO₃ and the disappearance of sulfite was monitored using either dithiobisnitrobenzoic acid or fuchsin. The rate of sulfite disappearance in light was double the dark rate. Disalicylidene propanediamine at 1 mM increased this light-enhanced metabolism approx. 50%; neither 1 M 3,4-dichlorophenyl-N,N-dimethylurea nor 0.1 mM cyanazine, which completely inhibited CO₂-dependent oxygen evolution, affected the rate of sulfite metabolism. Addition of 200 M Na₂SO₃ to the cells partially inhibited ¹⁴CO₂ fixation. The rate of sulfite consumption by the cells did not affect this inhibition. We conclude that light-dependent sulfite metabolism is cucumber cells may utilize reduced ferredoxin generated as a result of photosynthetic electron transport. An injurious interaction between CO₂ fixation and sulfite appears to occur independently of the sulfite-metabolism process.Abbreviations DCMU 3,4-dichlorophenyl-N,N-dimethylurea - DSPD disalicylidene propanediamine - DTNB 5,5-dithiobis-(2-nitrobenzoic acid)     

Chemical modification of active sites in relation to the catalytic mechanism of F1

Recent studies of chemically modified F₁-ATPases have provided new information that requires a revision of our thinking on their catalytic mechanism. One of the subunits in F₁-ATPase is distinguishable from the other two both structurally and functionally. The catalytic site and regulatory site of the same subunit are probably sufficiently close to each other, and the interaction between the various catalytic and regulatory sites are probably sufficiently strong to raise the uni-site rate of ATP hydrolysis by several orders of magnitude to that of promoted (multi-site) ATP hydrolysis. Although all three subunits in F₁ possess weak uni-site ATPase activity, only one of them () catalyzes promoted ATP hydrolysis. But all three subunits catalyze ATP synthesis driven by the proton flux. Internal rotation of the ₃₃ or ₃ moiety relative to the remainder of the F₀F₁ complex did not occur during oxidative phosphorylation by reconstituted submitochondrial particles.     

N-Carbamoyl-α-Amino Acids Rather than Free α-Amino Acids Formation in the Primitive Hydrosphere: A Novel Proposal for the Emergence of Prebiotic Peptides

Our previous kinetic and thermodynamic studies upon the reactional system HCHO/HCN/ NH₃ in aqueous solutions are completed. In the assumed prebiotic conditions of the primitive earth ([HCHO] and [HCN] near 1 g L^–1, T = 25 °C, pH = 8, [NH₃] very low), this system leads to 99.9% of -hydroxyacetonitrile and 0.1% of -aminoacetonitrile (precursor of the -amino acid). The classical base-catalyzed hydration of nitriles, slow and not selective, can not modify significantly this proportion. On the contrary, we found two specific and efficient reactions of -aminonitriles which shift the initial equilibrium in favor of the -aminonitrile pathway. The first reaction catalyzed by formaldehyde generates -aminoamides, precursors of -aminoacids. The second reaction catalyzed by carbon dioxide affords hydantoins, precursors of N-carbamoyl--aminoacids. In the primitive hydrosphere, where the concentration in carbon dioxide was estimated to be higher than that of formaldehyde, the formation of hydantoins was consequently more efficient. The rates of hydrolysis of the -aminoacetamide and of the hydantoin at pH 8 being very similar, the synthesis of the N-carbamoyl--amino acid seems then to be the fatal issue of the HCHO/HCN/NH₃ system that nature used to perform its evolution. These N-protected -amino acids offer new perspectives in prebiotic chemistry, in particular for the emergence of peptides on the prebiotic earth.     

Potential for use of a cyanobacterium Synechocystis sp. immobilized in poly(vinylalcohol): Application to the detection of pollutants

A cyanobacterium, Synechocystis sp. PCC 6803, was immobilized by entrapment in poly(vinylalcohol) bearing styrylpyridinium groups. Its properties in a single-compartment micro-photoelectrochemical cell using platinum electrodes in potentiosatic mode were compared with the native material. The operational activity was measured in the presence of an electrolytic solution containing 20 mM sodium phosphate, 0.15 mM NaCl and 1 mM MgCl₂. The best conditions of use are pH 7.0, 38 °C and a 2,5-dichlorobenzoquinone concentration equal to 350 M with native cyanobacteria or pH 6.5, 25 °C and 500 M 2,5-dichlorobenzoquinone after entrapment. Using this procedure, the photocurrent could be inhibited by pollutants such as Diuron or HgCl₂. After entrapment, the detection limits (corresponding to a 10% inhibition) were respectively 0.5 M and 50 M for Diuron and HgCl₂ after five minutes of incubation. A permeabilization technique was used to increase sensitivity of the procedure to the detection of HgCl₂ (25% inhibition with 50 M after five minutes of incubation).     

The energy transmission in ATP synthase: From the γ-c rotor to the α3β3 oligomer fixed by OSCP-b stator via the βDELSEED sequence

ATP synthase (F₀F₁) is driven by an electrochemical potential of H⁺ (H⁺). F₀F₁ is composed of an ion-conducting portion (F₀) and a catalytic portion (F₁). The subunit composition of F₁ is ₃₃. The active ₃₃ oligomer, characterized by X-ray crystallography, has been obtained only from thermnophilic F₁ (TF₁). We proposed in 1984 that ATP is released from the catalytic site (C site) by a conformational change induced by the DELSEED sequence via -F₀. In fact, cross-linking of DELSEED to stopped the ATP-driven rotation of in the center of ₃₃. The torque of the rotation is estimated to be 420 pN·å from the H⁺ and H⁺-current through F₀F₁. The angular velocity () of is the rate-limiting step, because H⁺ increased theV _max of H⁺ current through F₀, but not theK _m (ATP). The rotational unit of F₀ (=ab₂c₁₀) is /5, while that in ₃₃ is 2/3. This difference is overcome by an analog-digital conversion via elasticity around DELSEED with a threshold to release ATP. The distance at the C site is about 9.6 å (2,8-diN₃-ATP), and tight Mg-ATP binding in ₃₃ was shown by ESR. The rotational relaxation of TF₁ is too rapid (=100 nsec), but the rate of AT(D)P-induced conformational change of ₃₃ measured with a synchrotron is close to . The ATP bound between the P-loop and E188 is released by the shift of DELSEED from RGL. Considering the viscosity resistance and inertia of the free rotor (-c), there may be a stator containing OSCP (= of TF₁) and F₀-d to hold free rotation of ₃₃.     

Prolongation of MGE 412 Transposition Induction after γ-Irradiation in an Isogenic Line of Drosophila melanogaster

The dose dependence of the rate of -induced transpositions and consequent dynamics of the MGE 412pattern after -irradiation were investigated in isogenic line 49 in generations F₁, F₁₂, F₁₄₀, and F₁₇₀. It was shown that the results on dose dependence of transpositions was very similar with the corresponding results of the classic works by Timofeeff-Ressovsky et al.(1935). It is suggested that the transcribed copies of retrotransposon 412cure -radiation-induced double-strand DNA breaks. The phenomenon of prolongation of MGE transposition induction during early generations after treatment was shown. In this period (F₁–F₁₂), the maximum transposition rate ( 2 × 10^–2events per MGE copy, per haploid genome, per generation) and the maximum number of heterozygous MGE copies were achieved. In the late generations (F₁₄₀–F₁₇₀), the reduced induction level ( 10^–3) was established. In the population of effective size N _e= 2000 individuals, this corresponds to the state when 1/4N _e, i.e., when the transposition flow prevails over the MGE copy loss by genetic drift. These data together with some indirect evidence argue for the hypothesis that the spontaneous transposition rate is proportional to the average number of heterozygous MGE copies per diploid genome.     

Structure and function of H+-ATPase

(1) Extensive studies on proton-translocating ATPase (H⁺-ATPase) revealed that H⁺-ATPase is an energy transforming device universally distributed in membranes of almost all kinds of cells. (2) Crystallization of the catalytic portion (F₁) of H⁺-ATPase showed that F₁ is a hexagonal molecule with a central hole. The diameter of F₁ is about 90 Å and its molecular weight is about 380,000. (3) Use of thermophilic F₁ permits the complete reconstitution of F₁ from its five subunits (, , , , and ) and demonstration of the gate function of the -complex, the catalytic function of (supported by and ), and the H⁺-translocating functions of all five subunits. (4) Studies using purified thermostable F₀ showed that F₀ is an H⁺-channel portion of H⁺-ATPase. The direct measurement of H⁺-flux through F₀, sequencing of DCCD-binding protein, and isolation of F₁-binding protein are described. (5) The subunit stoichiometry of F₁ may be ₃₃. (6) Reconstitution of stable H⁺-ATPase-liposomes revealed that ATP is directly synthesized by the flow of H⁺ driven by an electrochemical potential gradient and that H⁺ is translocated by ATP hydrolysis. This rules out functions for all the hypothetical components that do not belong to H⁺-ATPase in H⁺-driven ATP synthesis. The roles of conformation change and other phenomena in ATP synthesis are also discussed.     

Weitere Untersuchungen an Nachkommen von Kürbis-Artbastarden

Zusammenfassung Aus 1950 durchgeführten Artkreuzungen zwischenCucurbita maxima Duch. und weichschaligemC. pepo L. wurden mittels Embryonenkultur F₁-Bastarde gewonnen. An Hand zweimaliger Rückkreuzung mitC. maxima und anschließender Selbstung gelang es, zum Teil wiederum mit Hilfe der Embryonenkultur, 4 verschiedene Bastardkombinationen bis in die F₄ zu verfolgen. In dieser Generation spalteten (1954 und 1955) bei Auswertung von 72 Familien mit insgesamt 733, davon 600 fertilen Pflanzen in 2 Bastardlinien resp. 3 Familien, insgesamt 4 weichschalige Pflanzen heraus. Da diese Pflanzen frei abblühten und das gewonnene Saatgut heterozygot war, mußten erneut zwei Selbstungsgenerationen aufgezogen werden, die in den Jahren 1956–58 mehrere erbreine Stämme weichschaliger Pflanzen lieferten.Nach Kurzer Einführung in die Anatomie der Samenschale und ihre verschiedenen Typen sowie in die bisherigen Ergebnisse genetischer Untersuchungen zur Frage der Testaausbildung wird an Hand der Spaltungsergebnisse beim erstmaligen Auftreten weichschaliger Pflanzen in der F₄ sowie der nach erneuter Selbstung in der F₆ erzielten Spaltungsverhältnisse aufgezeigt, daß das Merkmal weichschalig durchzwei Gene bedingt ist. Nach kurzer Besprechung von Spaltungen pachyspermer und leptospermer Pflanzen in der F₃ wird die Variabilität der Testaausbildung bis zum Auftreten weichschaliger Pflanzen beschrieben.In einem weiteren Kapitel wird die Variabilität in der Ausbildung der weichschaligen Testa der Bastardnachkommen, das Ergebnis der Selektion auf rein weichschalige Typen im Verlauf der 7. bis 11. Generation (1957–1964) und die Größenvariabilität der Samen der weichschaligen Typen im Vergleich zu den Ausgangsarten sowie einem Weltsortiment vonC. maxima erörtert. Zur Veranschaulichung dieser Verhältnisse wird die erstmalig vonF. Galton benutzte Methode der Contourellipsen herangezogen, deren Berechnung und zeichnerische Darstellung mit Hilfe der Großrechenanlage IBM 7090 erfolgte.Abschließend wurden die beobachtetenMendel-Spaltungen unter dem Gesichtspunkt der die Pollenmeiose sowie die Pollenentwicklung der F₁ charakterisierenden Störungen, ferner die Bedeutung des erzielten Ergebnisses für die Züchtung diskutiert. Es wird eine Formel für die Berechnung der Wahrscheinlichkeit mitgeteilt, mit der unter ähnlichen Bedingungen rezessive Pflanzen erwartet werden können, wenn das rezessive Merkmal durch n Faktoren bedingt ist und im Anschluß an die Artkreuzungj Rückkreuzungen mit der dominanten Art erforderlich sind. Auch auf die Bedeutung von Untersuchungen der vorliegenden Art für Fragen der Genlokalisation und Genanalyse wird hingewiesen.

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Further investigations on progeny of pumpkin species hybridsI. Seed formation, especially in progeny with soft seed coat, and the regularity in transmission of a recessive trait from one parental species to fertile progeny with characteristics of the other parental species

Summary Using embryo culture, we obtainedF ₁-hybrids from interspecific crosses realized in 1950 betweenCucurbita maxima Duch. and a form ofC. pepo L. with soft shell seed or weichschaligen Samen (a so called naked seed type). These and their descendants, the latter in part also obtained by embryo culture, were back-crossed toC. maxima, and theF ₃ selfed. In this way we obtained descendants from 4 different crosses between the two species.Studying (in 1954 and 1955) 72F ₄-families with a total of 733 (600 fertile) plants, we found 4 plants with soft shell seeds. They were descended from 3F ₄-families or from 2 crosses respectively between the above named species.Since these plants flowered in the presence of other progeny from our species crosses, their seed was heterozygous, and two generations of selfing were necessary to obtain (in 1956–1958) different true breeding families, whose plants furnished homozygous soft shell seed.The anatomy of seed coats and results of studies by other authors on the genetic control of different seed coat types are discussed briefly. Given the segregation ratios in theF ₄ of our soft shell seeds, as well as those obtained inF ₆ after renewed selfing, it is shown that the trait soft shell is controlled by two genes. After a short discussion of segregation in theF ₃ of pachyspermic and leptospermic plants, the variability of seed coat formation until the appearance of soft shell seed is described.In a special chapter we discuss the variability of the soft-shell seed character in hybrid progeny, the result of our selection experiments from the 7^th up to the 12^th generation (1957–1964), and the variability in the seed size of our types compared with that of pure species and of a collection ofC. maxima. To illustrate these relationships we used the method of contour-ellipses introduced byF. Galton. These ellipses were computed and designed by means of the computer IBM 7090.Finally we discuss Mendelian segregation of our plants in the light of abnormalities characteristic for meiosis of pollen-mother-cells and pollen development inF ₁, and the significance of the results to plant breeding. A formula is given for calculating the probability of obtaining recessive plants when the recessive trait depends onn factors, and whenj backcrosses to the dominant variety are necessary to establish a stable strain. The importance of experiments of this kind to the problem of gene localization and gene analysis is also pointed out.

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Herrn Professor Dr. Dr. Dr. h. c. O.Heinisch, geb. 23. 4. 1896, gest. 3. 5. 1966, zum Gedächtnis.     

Subunit movement during catalysis by F1-F0-ATP synthases

The catalytic portion (F₁) of ATP synthases have the subunit composition ₃, ₃, , , . This composition imparts structural asymmetry to the entire complex that results in differences in nucleotide binding affinity among the six binding sites. Evidence that two or more sites participate in catalysis, alternating their properties, led to the notion that the interactions of individual pairs with the small subunits must change as binding site properties alternate. A rotation of the subunit within the ₃₃ hexamer has been proposed as a means of alternating the properties of catalytic sites. Evidence argues that the rotation of the complete subunit during ATP hydrolysis is not mandatory for activity. The subunit of chloroplast F₁ may be cleaved into three large fragments that remain bound to F₁. This cleavage enhances ATPase activity without loss of evidence of site-site interactions. Complexes of ₃₃ have been shown to have significant ATPase activity in the absence of . Mg²⁺ATP affects the interaction of with the different subunits, and induces other changes in F₁, but whether these changes are induced by catalysis, or are fast enough to be involved in the catalytic turnover of the enzyme has not been established. Likewise, changes in structure and in binding site properties induced in thylakoid membrane bound CF₁ by formation of an electrochemical proton gradient may activate the enzyme rather than be apart of catalysis. Mechanisms other than rotary catalysis should be considered.     

Quaternary structure of ATP synthases: Symmetry and asymmetry in the F1 moiety

It has been proposed that during ATP synthesis/hydrolysis F₁ ATPases experience a complex pattern of nucleotide binding and release during the catalytic cycle (binding change mechanism). This type of mechanism has implications that can be correlated with the structure of the enzyme. F₁-ATPases (stoichiometry ₃₃) are essentially a symmetrical trimer of pairs of the major subunits ( and ); the minor subunits (, and ) are in single copies and interact with the trimer in an asymmetrical fashion. The asymmetry introduced by the minor subunits has important structural and functional consequences: (1) it introduces differences between the potentially equivalent binding and catalytic sites in the major subunits, (2) it restricts the ways in which a binding change mechanism can occur, and (3) it governs the way in which the F₁ interacts with the (asymmetrical) F₀ sector.     

Structure of F1-ATPases

F₁-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F₁-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type ₃₃ and ₂₂₂₂₂ were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F₁-ATPases.     

Functional sites in F1-ATPases: Location and interactions

This review focuses on the location and interaction of three functional sites in F₁-ATPases. These are catalytic sites which are located in subunits, noncatalytic nucleotide-binding sites which are located at interfaces of and subunits and modulate the hydrolytic activity of the enzyme, and a site that binds inhibitory amphipathic cations which is at an interface of and subunits. The latter site may participate in transmission of conformational signals between catalytic sites in F₁ and the proton-conducting apparatus of F₀ in the intact ATP synthases.     

The Structural and Functional Connection between the Catalytic and Proton Translocating Sectors of the Mitochondrial F 1 F 0 -ATP Synthase

The structural and functional connection between the peripheral catalytic F₁ sector and theproton-translocating membrane sector F₀ of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F₀I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F₁ to F₀ and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF₁) binds at one sideof the F₁F₀ connection. The carboxy-terminal segment of IF₁ apparently binds to OSCP. The42L-58K segment of IF₁, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F₁, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis.     

Differential sensitivity of astrocyte primary cultures and derived spontaneous transformed cell lines to 70-hydroxycholesterol: effect on plasma membrane lipid composition and fluidity,and on cell surface protein expression

Summary The cytotoxicity of 7-hydroxycholesterol (7-OHC) was investigated on rat astrocyte primary cultures and spontaneously transformed cell lines derived from them. Confluent astrocyte primary cultures (normal cells) were unaffected by 20 µM 7(3-OHC over a period of 72 h whereas 30 µM markedly affected the viability of the transformed cells within the first 72 h. Both cell types incorporated 18% of the total amount of 7-OHC added to the cultures at concentrations of 20 µM or 30 µM. Cellular fractionation after incubation with 20 µM or 30 µM 7-OHC indicated that the plasma membrane incorporated 2 or 6 fold more 7-OHC than the intracellular one's respectively. Plasma membrane cholesterol (CH) and phospholipid (PL) analysis showed that 20 µM 7-OHC did not affect CH/PL in normal cells; in contrast, plasma membranes of transformed cells displayed a significant CH/PL decrease, which was more pronounced with 30 µM 7-OHC treatment. Fluorescence anisotropy measurements indicated that 20 µM 7-OHC slightly fluidified the plasma membrane of normal cells whereas it has not effect on that of the transformed cells one; however, an increase in plasma membrane fluidity was observed when the transformed cells were treated with 30 µM 7-OHC. Lactoperoxidase catalyzed radioiodination of cell surface proteins and subsequent autoradioelectrophoretic analysis demonstrated that the labelled protein pattern was unchanged when both cell types were incubated with 30 µM 7-OHC.