Phosphorylation of F1F0 ATPase δ-Subunit Is Regulated by Platelet-Derived Growth Factor in Mouse Cortical Neurons In Vitro

Abstract: The δ subunit of F₁F₀ ATPase (ATP synthase complex) is part of the stalk connecting the F₁ and F₀ moieties. Studies in Escherichia coli suggest that the analogous bacterial subunit, called ε, is essential for the ATPase assembly energy coupling. Platelet-derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS. Using two-dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the δ subunit or a closely related peptide in cultured mouse cortical neurons.     

Functional asymmetry of the F0 motor in bacterial ATP synthases

F₁F₀ ATP synthases use the electrochemical potential of H⁺ or Na⁺ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F₀ parts of a Na⁺- and H⁺-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of ∼100 times higher Na⁺ or H⁺ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F₀ parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo . In Escherichia coli , we observed respiratory chain-driven ATP production at pH 7–8, while P -site pH values < 6.5 were required for ATP synthesis in vitro . This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.     

Tributyltin-resistant Methanothermobacter thermautotrophicus mutant with mutational substitutions in the A1A0-ATP synthase operon

A spontaneous mutant of Methanothermobacter thermautotrophicus resistant to tributyltin chloride (TBT) was isolated. TBT, the inhibitor of the A₀ domain of A₁A₀-ATP synthase, inhibits methanogenesis in the wild-type cells; however, the TBT-resistant mutant exhibited methanogenesis even in the presence of 800 μM TBT. ATP synthesis driven by methanogenic electron transport was markedly diminished in the mutant strain. While TBT profoundly inhibited ATP synthesis driven by methanogenic electron transport in the wild type, only a slight inhibition was observed in the mutant strain. These results suggested a modification in the ATP-synthesizing system of the mutant strain. The sequence of the complete A₁A₀-ATP synthase operon ( Mth952 – Mth961 ) in the wild-type and mutant strains was determined and compared. Three mutations leading to amino acid substitutions in two A₁A₀-ATP synthase subunits were identified – Val₃₃₈Ala in subunit A and Leu₂₅₂Ile and Ser₂₉₃Ala in subunit B. Moreover, this study revealed the differential expression of several proteins that may contribute to TBT resistance. The results imply that change of TBT sensitivities of TBT-resistant mutant is due to mutational substitutions in the A₁A₀-ATP synthase operon.     

Can Extensive Reticulation and Concerted Evolution Result in a Cladistically Structured Molecular Data Set?

Hierarchy is the main criterion for informativeness in a data set, even if no explicit reference to evolution as a causal process is provided. Sequence data (nuclear ribosomal DNA ITS) from Armeria (Plumbaginaceae) contains a certain amount of hierarchical structure as suggested by data decisiveness and distribution of tree lengths. However, ancillary evidence suggests that extensive gene flow and biased concerted evolution in these multicopy regions have significantly shaped the ITS data set. This argument is discussed using parsimony analysis of four data sets, constructed by combining wild sequences with those from different generations of artificial hybrids (wild + F₁, F₂, and backcrosses; wild + backcrosses; wild + F₁; wild + F₂). Compared to the F₁ hybrids, F₂ show a certain degree of homogenization in polymorphic sites. This effect reduces topological disruption caused by F₁ and is considered to be illustrative of how extensive gene flow and biased concerted evolution may have modeled the wild ITS data. The possibility that hierarchy has arisen as a result of—or despite a significant contribution from—those two such potentially perturbing forces raises the question of what kind of signal are we recovering from this molecular data set.     

Depletion of stromal Pi induces high 'energy-dependent' antenna exciton quenching (qE) by decreasing proton conductivity at CFO-CF1 ATP synthase

This work tests two models to account for the effects of depletion of stromal inorganic phosphate (P_i), which results in down-regulation of light capture via the exciton quenching (q_E) mechanism and has been proposed to act in feedback regulation of the light reactions. In both models, antenna down-regulation is activated by acidification of the lumen, despite the fact that linear electron flow (LEF) (and associated proton flux) is decreased upon P_i depletion. In one model, an imbalance of ATP or NADPH activates cyclic electron transfer around photosystem I (CEF1), increasing proton influx to the lumen. In the second, the effective conductivity of the CF_O-CF₁ ATP synthase to protons ( g _H⁺) is decreased, retarding proton efflux from the lumen. Sequestering of P_i by mannose infiltration increased sensitivities of q_E and pmf to LEF. The effects were attributable to decreases in g _H⁺, but not to CEF1 and were largely reversed by subsequent P_i feeding. Rapid recovery of g _H⁺ in the dark suggested that dark-labile metabolic pools are responsible for regulation of the ATP synthase. Overall, these results support models where accumulation of Benson–Calvin cycle intermediates or lowering of stromal P_i below its K _Mat the ATP synthase, retards proton efflux from the lumen, leading to build-up of pmf and subsequent down-regulation of photosynthetic light capture.     

Time-resolved microfluorescence for measuring coenzyme F420 in Methanobacterium thermoautotrophicum

Abstract The time course of photobleaching and the nanosecond fluorescence decay have been measured from microscopic samples of methanogenic bacteria, to our knowledge the first application of these methods in this field. Decay times of about 1 ns and 3 ns were obtained for the specific coenzymes F₄₂₀ and 7-methylpterin, respectively. In contrast to methylpterin and other fluorescent compounds the intensity of F₄₂₀ fluorescence was reduced selectively due to photobleaching. This effect, as well as the different decay time constants could be used to discriminate F₄₂₀ from other fluorescent components. In addition, active and inactive bacterial cells could be differentiated following the course of photobleaching.     

Sex ratio bias in the F1 adult progeny of African rice gall midge, Orseolia oryzivora H. and G. (Dipt., Cecidomyiidae)

Abstract:  Against the background of conflicting reports of variable sex ratio distribution in different populations of the African rice gall midge (AfRGM), Orseolia oryzivora Harris and Gagne, the number and sex ratios of F₁ adult progeny produced per female from wild and screen house populations were investigated in the screen house at 27 ± 3°C, 60–70% relative humidity and 12 : 12 h (light : dark) photoperiod. Results indicated the occurrence of a single sex-biased ratio in all the F₁ adult progeny produced per female of this pest. Regardless of the source, whether from wild or screen house population, each mated female AfRGM produced a full complement of either sex only but never both. The difference between the number of eggs laid and F₁ adult progeny that emerged per female from wild and screen house populations was not significant (P > 0.05), indicating consistency in the occurrence of this phenomenon among AfRGM populations. The reason for this could not be ascertained in this study but AfRGM eggs must be fertilized for development and eclosion to occur. No F₂ adults emerged when a cohort of emergents from the same female were allowed to re-infest susceptible seedlings, indicating absence of parthenogenesis.     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.     

Chloroplast ultrastructure in two Crassulacean species and an F1 hybrid with differing biomass δ13C values

Abstract. The chloroplasts of two species of the Crassulaceae and their F₁ hybrid were compared by electron microscopy. The two species had contrasting leaf tissue δ¹³C values of −25°/_∞ ( Sedum greggii ) and −13°/_∞ ( Cremnophila linguifolia ), and the F₁ hybrid had a value of − 18°/_∞. S. greggii had a mean of 8.9 thylakoids per granum in contrast to C. linguifolia which had a mean of only 3.8 thylakoids per granum. The F₁ hybrid had a mean of 6.4 thylakoids per granum. Crystaloids were observed in S. greggii and the hybrid but not in C. linguifolia     

Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase

A mutagenesis programme using ethyl methanesulphonate (EMS) was carried out on Lotus japonicus (Regel) Larsen cv. Gifu in order to isolate photorespiratory mutants in this model legume. These mutants were able to grow in a CO₂-enriched atmosphere [0.7% (v/v) CO₂] but showed stress symptoms when transferred to air. Among them, three mutants displayed low levels of glutamine synthetase (GS; EC 6.3.1.2) activity in leaves. The mutants accumulated ammonium in leaves upon transfer from 0.7% (v/v) CO₂ to air. F₁ plants of back crosses to wild type were viable in air and F₂ populations segregated 3 : 1 (viable in air : air-sensitive) indicative of a single Mendelian recessive trait. Complementation tests showed that the three mutants obtained were allelic. Chromatography on DEAE-Sephacel used to separate the cytosolic and plastidic GS isoenzymes together with immunological data showed that: (1) mutants were specifically affected in the plastidic GS isoform, and (2) in L. japonicus the plastidic GS isoform eluted at lower ionic strength than the cytosolic isoform, contrary to what happens in most plants. The plastidic GS isoform present in roots of wild type L. japonicus was also absent in roots of the mutants, indicating that this plastidic isoform from roots was encoded by the same gene than the GS isoform expressed in leaf tissue. Viability of mutant plants in high-CO₂ conditions indicates that plastidic GS is not essentially required for primary ammonium assimilation. Nevertheless, mutant plants did not grow as well as wild type plants in high-CO₂ conditions.     

Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae

Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO₂ with H₂ or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F₄₂₀-nonreactive hydrogenase, NADP⁺: F₄₂₀ oxidoreductase, NADP⁺-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F₄₂₀ dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F₄₂₀-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F₄₂₀ to provide electrons for biosynthesis, was formulated.     

New results about structure, function and regulation of the chloroplast ATP synthase (CF0CF1)

The chloroplast ATP synthase utilises the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. This multi-subunit thylakoid membrane-bound enzyme consists of a proton channel, CF₀, and an extrinsic catalytic sector, CF₁. Stimulated by the elucidation of a three-dimensional partial structure of the mitochondrial enzyme, substantial progress has been made to understand the catalytic mechanism and interesting hypotheses have been proposed about the molecular mechanism of energy coupling. The review discusses the present state of knowledge concerning the structure, molecular genetics, catalytic mechanism, energy coupling and regulation of this important enzyme involved in photophosphorylation.     

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose

Sal1p, a novel Ca²⁺-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K _m of 0.20 ± 0.03 mM and 0.28 ± 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca²⁺ dependent. Ca²⁺ increases the V _max with a S _0.5 of 15 μM, and no changes in the K _m for ATP-Mg. Glucose sensing in yeast generates Ca²⁺ transients involving Ca²⁺ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H⁺-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca²⁺-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.     

The ATP-dependent synthesis of factor 390 by cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH)

Abstract Cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH) converted the 8-OH-5-deazaflavin coenzyme F₄₂₀ to factor 390, a 8-adenylyl derivative (F₄₂₀-AMP). Activity was only observed upon exposure of the crude cell-free extract to oxygen. The ability to synthesize F₃₉₀ was lost when crude cell-free extract was subsequently brought to an anaerobic reducing environment. The enzymatic reaction used ATP and oxidized coenzyme F₄₂₀ as substrates and inorganic pyrophosphate was formed next to F₃₉₀. GTP could be used instead of ATP resulting in a guanylylated derivative. The crude cell-free extract showed K _m values of 154 μM for coenzyme F₄₂₀ and 2.4 mM for ATP. A partially purified enzyme preparation exhibited a K _eq of 0.32. In accordance, coenzyme F₄₂₀ and ATP could be synthesized from F₃₉₀ and PPi by the reverse reaction.     

Growth of methanogens on cyclopentanol/CO2 and specificity of alcohol dehydrogenase

Abstract Growth of 5 strains of secondary alcohol-utilizing methanogens on cyclopentanol/CO₂ was demonstrated. Cultures reached only low optical densities, comparable to those observed during growth on 2-propanol/CO₂ or 2-butanol/CO₂. The conversion of cyclopentanol to cyclopentanone was reversible in the presence of hydrogen. The specificity of ADHs of nine secondary alcohol-utilizing methanogens for coenzyme requirement and substrates was tested using crude enzyme preparations. The ADH of Methanobacterium bryantii M.o.H.G, Methabobacterium palustre, Methanocorpusculum parvum and Methanocorpusculum bavaricum was only active with NADP⁺, the ADH of Methanomicrobium paynteri, Methanogenium marisnigri, Methanogenium bourgense , strain GKZPZ and strain INSLUZ with F₄₂₀ as electron carrier. FAD, FMN, methylene blue, benzyl viologen and tetrazolium, chloride could be used as electron carriers by the F₄₂₀-dependent ADHs if endogenous F₄₂₀ was introduced with the crude ADH preparation. No reaction with these electron carriers occurred if dialyzed extracts were used as a source of ADH. The ADH of all tested strains oxidized 2-propanol, 2-butanol, 2-pentanol and cyclopentanol, while the ADH of Mg. marisnigri and Mm. paynteri oxidized cyclohexanol, of Mb. palustre and Mcp. bavaricum cyclopentanol and 2,3-butanediol,and of Mcp. parvum cyclopentanol, 2,3-butanediol, ethanol and 1-propanol in addition.     

The induction of the CO2 concentrating mechanism in a starch-less mutant of Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii the formation of a starch sheath surrounding the pyrenoid is observed when cells grown under high CO₂ (5% CO₂ in air) are transferred to low CO₂ (0.03%) conditions. Formation of the starch sheath occurs coincidentally with induction of the CO₂ concentrating mechanism and with de novo synthesis of 5 polypeptides with molecular masses of 21, 36, 37, 42–44 kDa. We studied the effect of CO₂ concentrations on photosynthesis, ultrastructure and protein synthesis in Chlamydomonas reinhardtii cw-15 (wild phenotype for photosynthesis) and in the starch-less mutant BAFJ -6, with the aim to clarify the role of the pyrenoid starch sheath in the operation of the CO₂ concentrating mechanism and whether these low CO₂-inducible polypeptides are involved in the formation of starch sheath. When wild type and starch-less mutant cells were transferred from high to low CO₂, the CO₂ requirement for half-maximal rates of photosynthesis decreased from 40 μM to 2 μM CO₂. ³⁵SO₄^2- labeling analyses showed that the starch-less mutant induced the 5 low CO₂-inducible polypeptides. These observations suggest that the starch-less mutant was able to induce a fully active CO₂ concentrating mechanism. Since the starch-less mutant did not form a pyrenoid starch sheath, we suggest that the starch sheath is not involved in the operation of the CO₂ concentrating mechanism and that none of these 5 low CO₂-inducible proteins is involved in the formation of the starch sheath in Chlamydomonas .     

Mapping of QTL on chromosome X for fat deposition, muscling and growth traits in a wild boar x Meishan F2 family using a high-density gene map

Quantitative trait loci (QTL) for fat deposition, growth and muscling traits have been previously mapped on the basis of low-density linkage maps in a wild boar × Meishan F₂ family to the chromosome X region flanked by SW2456 and SW1943 . Improved QTL resolution was possible using data for F₂ animals with a marker density of 2.7 cM distance in the SW2456 to SW1943 region, including AR , SERPINA7 and ACSL4 as candidate genes. The resolution of the QTL scan was increased substantially, as evidenced by the higher F -ratio values for all QTL. Maxima of F -ratio values for fat deposition, muscling and growth traits were 28.6, 18.2 and 16.5 respectively, and those QTL positions accounted for 7.9%, 5.0% and 4.5% of the F₂ phenotypic variance (VF₂) respectively. QTL for fatness and growth and for most muscling traits mapped near ACSL4 , with the exception of the QTL for ham traits that mapped proximally, in the vicinity of AR . An analysis performed separately for F₂ male animals showed the predominant QTL affecting fat deposition traits (up to 13.6% VF₂) near AR and two QTL for muscling traits (up to 9.9% VF₂) mapped close to ACSL4 . In the F₂ female animals, QTL affecting muscling (up to 12.1% VF₂) mapped at ACSL4 and SW2456 , and QTL for fat deposition (10% VF₂) and growth (up to 10.5% VF₂) mapped at ACSL4 .     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

A one-year investigation of the relationship between serum GH levels and the growth of F4 transgenic and non-transgenic common carp Cyprinus carpio

It has been demonstrated that growth hormone (GH) transgenic fish often posses a trait for fast growth. Here, we investigated the growth of F₄'all-fish' GH transgenic carp Cyprinus carpio and their serum GH levels for a year. The results showed that F₄ all-fish GH transgenic carp were significantly larger in body mass ( c . two-fold, P < 0·001) and body length ( c . 1·3 fold, P < 0·001), compared with the non-transgenic group. The discrepancy of serum GH levels between the transgenic carp group and control group is 54 fold, when the water temperature was 12–34° C. When the water temperature decreased to 3·5° C in January, the discrepancy was 256 fold. The serum GH level of the transgenic group was relatively constant, while that of control varied greatly based on month and water temperature. The changes of growth rates between the transgenic group and the control group were similar for a year. Taken together, the results indicated that F₄ all-fish GH transgenic carp had not only higher and constant serum GH levels but also a significant fast-growing effect, compared with the control. To our knowledge, this is the first report on a one-year investigation of growth trait and serum growth hormone level in F₄ all-fish GH transgenic carp.