Spiroplasma infection in Drosophila melanogaster: What is the advantage of killing males?

Male-killing bacteria are maternally inherited agents that cause death of sons of infected females. Their transmission rate is commonly high but imperfect and also sensitive to different environmental factors. Therefore, the proportion of infected females should be reduced in each generation. In order to explain male-killers spread and persistence in host population, a mechanism resulting in the relative increase of infected females must outweigh the losses caused by the imperfect transmission. The resource release hypothesis states that the males’ death results in increased resources available to sibling females which would otherwise be used by their male siblings. Infected females are then expected: to be larger than uninfected females in natural populations; or to have higher viability; or to have shorter development times; or any combination of these outcomes. Here, we tested the resource release hypothesis by measuring body size of infected and uninfected wild-caught Drosophila melanogaster females and carried out other fitness related measures in the laboratory. Wild-caught infected females produced more daughters than uninfected females in their first days in the laboratory. However, although no significant difference in viability was found in a controlled experiment with infected and uninfected flies from a standard laboratory strain, there was a decrease in development time probably mediated by reduced competition. Fitness effects conditioned by the host genetic background are pointed out as a possible explanation for this difference between wild and laboratory flies. Our findings are discussed in the context of the resource advantage hypothesis.     

Precocene I and II inhibition of vitellogenic oöcyte development in Drosophila melanogaster

Adult female Drosophila melanogaster were exposed to precocene I and II, antiallatropin compounds which result in juvenile hormone deficiency in many insects. The presence of juvenile hormone in Drosophila adults was evaluated by examining vitellogenic oöcyte development, a process regulated by juvenile hormone in these flies. Both precocenes reduced the number of vitellogenic oöcytes present 43 hr after exposure in a dose-dependent manner. Precocene I was effective when applied to either newly eclosed females prior to vitellogenic oöcyte development or to gravid females. Precocene I was also effective in decapitated females, indicating that the action of the compound is not mediated by the brain. Corpus allatum volume, presumably a reflection of secretory activity, increased between 0 and 24 hr after eclosion in control females but not in precocene-treated females even after 48 hr. However, when females were removed from precocene medium, gland volumes increased within 48 hr to approximately those of control flies. This result is consistent with the reversibility of the precocene effect on Drosophila adults. These results suggest that precocene acts on the corpus allatum of Drosophila adult females to produce juvenile hormone deficiency.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

A simple method for preparation of valency hybrid hemoglobins, (α3+β2+)2 and (α2+β3+)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

cDNA cloning and bacterial expression of phospholipase A2 inhibitor PLIα from the serum of the Chinese mamushi, Agkistrodon blomhoffii siniticus1

The cDNA encoding of a phospholipase A₂ inhibitor (PLIα) of the Chinese mamushi, Agkistrodon blomhoffii siniticus, was identified from a liver cDNA library by use of a probe prepared by polymerase chain reaction (PCR) on the basis of the amino acid sequence of PLIα. It encoded a polypeptide of 166 amino acid residues, including 19 residues of the signal sequence and 147 residues of the complete mature sequence of PLIα. The PLIα cDNA was subcloned into the expression vector pET-16b and used to transform Escherichia coli strain BL21(DE3)pLysS. The recombinant PLIα expressed as a fusion protein was solubilized and purified to homogeneity by use of a metal affinity resin. The purified PLIα fusion protein underwent folding to form a trimeric structure like the intact PLIα, and showed inhibitory activity against the group II acidic PLA₂ from A. blomhoffii siniticus venom; although its binding constant (1/K_i) value was 30-fold lower than that of the natural PLIα. The elimination of the N-terminal additional peptide from the fusion protein resulted in a marked increase in the inhibition activity with a binding constant comparable to that of the natural PLIα against the acidic PLA₂. Furthermore, the carbohydrate chains of the natural PLIα were found to play an important role in the inhibitory activity against the basic PLA₂.     

Phytochrome-controlled phototropism of protonemata of the moss Ceratodon purpureus: physiology of the wild type and class 2 ptr–mutants

Phototropism and polarotropism in protonemata of the moss Ceratodon purpureus are controlled by the photoreceptor phytochrome. One class of phototropism mutants is characterised by growing randomly when kept for a prolonged time (5 d or longer) in unilateral red light. It was found that a subclass of these mutants grows faster than the wild type, the rate of cell division and the length of the cells being increased. This difference is found for light-grown and dark-grown filaments. It is therefore suggested that the mutant phenotype neither results from a defect in phytochrome photoconversion nor from a defect in phytochrome-gradient formation. Instead, it is possible that a factor which is involved in both signal transduction of phototropism and regulation of cell size and cell division is deregulated. If dark-grown mutant filaments are phototropically stimulated for 24 h, they show a weak phototropic response. Phototropism and polarotropism fluence-rate effect curves for mutants were flattened and shifted to higher fluence rates compared with those for the wild type. With wild-type filaments, a previously unreported response was observed. At a low fluence rate, half of the filaments grew positively phototropically, while the other half grew negatively phototropically. It seems that under these conditions, a phytochrome gradient with two maxima for the far-red-absorbing form of phytochrome (Pfr) within the cross-section of the cell is displayed by the response of the filaments. At higher fluence rates, all filaments of the wild type grew towards the light. These data and results from microbeam irradiation experiments and from phototropism studies with filaments growing within agar, indicate that light refraction plays an important role in the formation of the Pfr gradient in phototropism of Ceratodon. Received: 10 September 1998 / Accepted: 30 December 1998     

Seasonal changes of thermogenic capacity in Melano-bellied oriental voles (Eothenomys melanogaster)☆

Seasonal changes of thermogenic capacity in Melano-bellied oriental voles (Eothenomysmelanogaster) were studied by measurements of body mass, basal metabolic rate (BMR), non-shivering thermogenesis (NST), thermogenic properties of brown adipose tissue (BAT), relative weight of liver mass, mitochondrial protein (MP) content, and activities of mitochondrial cytochrome C oxidase (COX) in autumn (October), winter (December), spring (April) and summer (July), respectively.
Results showed that: (1) Body mass of E.melanogaster was the highest in autumn, and the lowest in summer. The seasonal change in body mass was subject not only by environment temperature but also to other factors such as reproduction, feeding habit and climate in the habitat. Reproductive periods of E.melanogaster were in early spring (February–March) and late autumn (September–October), and therefore, the body mass was higher in spring and autumn. In addition, E.melanogaster feeds on leaves and stems of plants in spring and summer, whereas mainly on fruits in autumn and winter as they contain more energy. The difference in the feeding habit could result in higher body mass in the late half of the year, but lower body mass in winter. The lower body mass in winter provides E.melanogaster with an advantage in motility. (2) The BMR and NST of E.melanogaster was higher in winter than in other seasons, which suggests that the regulating thermogenic capacity is an important adaptive strategy for E.melanogaster. However, there was no difference in relative oxygen consumption (NST–BMR/BMR), suggesting that the regulative capacity of BMR is approximately equal to NST in E.melanogaster. This is different from some small mammal species in north China, which have higher regulative capacity in NST than E.melanogaster. Melano-bellied oriental voles live in mountainous area with subtropical zone monsoon climate and abundant food resource, and therefore have lower regulative capacity in NST, which can reduce an absolute request for energy. The low regulative capacity in NST can also help E.melanogaster to maintain energy balance and stable body temperature. (3) The relative mass of BAT in winter was significantly higher than that in summer. Different seasons significantly affected the MP content of BAT. The MP content of BAT was in the following order: winter > fall > spring > summer. The trend as following was observed for the mitochondrial COX activity of BAT: winter > spring > fall > summer. In experimental conditions, the thermogenic rate of many small mammal species increased under cold acclimation. Short photoperiod alone could also induce E.melanogaster to increase NST and mitochondrial COX activity of BAT. Therefore, ambient temperature and photoperiod are important environmental cues to induce seasonal changes in thermogenic capacity in E.melanogaster. (4) The relative mass of liver in summer was higher than that in other seasons, but no seasonal changes were found among other seasons. The MP contents of liver showed highest level in winter and lowest in spring and summer. Significant seasonal changes were also observed in the mitochondrial COX activity of liver. It increased significantly in winter, but not in other seasons. This indicates that liver participate in adjustment of thermogenesis. These data indicate that both increasing whole-animal thermogenesis rate and thermogenic capacity at the cells of BAT and liver play an important role in the adaption of E.melanogaster to seasonal changes, and they are also closely correlated with the habitat condition and their life habit.     

Purification and some properties of Δ2-isopentenylpyrophosphate:5′AMP Δ2-isopentenyltransferase from the cellular slime mold Dictyostelium discoideum

Δ²-Isopentenylpyrophosphate:5′AMP Δ²-isopentenyltransferase, which catalyzes the formation of isopentenyl-AMP from Δ²-isopentenylpyrophosphate and 5′AMP, was purified 6800-fold from the fruiting body of the cellular slime mold Dictyostelium discoideum using several separation procedures including 5′AMP^ox-redAH-Sepharose 4B affinity column chromatography. The final preparation was very unstable and lost its activity in a day. Various properties of the 1000-fold-purified enzyme preparation were examined. The molecular mass was 40,000 ± 2000 Da, as determined by Sephadex G-100 superfine gel filtration. The divalent metal ions Mn²⁺, Zn²⁺, and Mg²⁺ profoundly affected the enzymatic activity depending on their concentration, and also altered the optimum pH and temperature. Of the compounds tested, 5′AMP was the best acceptor of the isopentenyl group and, interestingly, ADP also served as a substrate, being 60–80% as effective as 5′AMP. Adenine, adenosine, and ATP were not substrates for this enzyme. Under the optimum assay conditions (pH 7.0, 1 mm Zn²⁺, and 25 °C) the K_m values for 5′AMP and Δ²-isopentenylpyrophosphate were 1.0 × 10^?7m and 2.2 × 10^?6m, respectively.     

Metabolomics of Ndufs4−/− skeletal muscle: Adaptive mechanisms converge at the ubiquinone-cycle

Leigh syndrome is one of the most common childhood-onset neurometabolic disorders resulting from a primary oxidative phosphorylation dysfunction and affecting mostly brain tissues. Ndufs4^?/? mice have been widely used to study the neurological responses in this syndrome, however the reason why these animals do not display strong muscle involvement remains elusive. We combined biochemical strategies and multi-platform metabolomics to gain insight into the metabolism of both glycolytic (white quadriceps) and oxidative (soleus) skeletal muscles from Ndufs4^?/? mice. Enzyme assays confirmed severely reduced (80%) CI activity in both Ndufs4^?/? muscle types, compared to WTs. No significant alterations were evident in other respiratory chain enzyme activities; however, Ndufs4^?/? solei displayed moderate decreases in citrate synthase (12%) and CIII (18%) activities. Through hypothesis-generating metabolic profiling, we provide the first evidence of adaptive responses to CI dysfunction involving non-classical pathways fueling the ubiquinone (Q) cycle. We report a respective 48 and 34 discriminatory metabolites between Ndufs4^?/? and WT white quadriceps and soleus muscles, among which the most prominent alterations indicate the involvement of the glycerol-3-phosphate shuttle, electron transfer flavoprotein system, CII, and proline cycle in fueling the Q cycle. By restoring the electron flux to CIII via the Q cycle, these adaptive mechanisms could maintain adequate oxidative ATP production, despite CI deficiency. Taken together, our results shed light on the underlying pathogenic mechanisms of CI dysfunction in skeletal muscle. Upon further investigation, these pathways could provide novel targets for therapeutic intervention in CI deficiency and potentially lead to the development of new treatment strategies.     

Analysis of the genetic diversity of Bemisia tabaci populations in Shanxi province, China☆

Five different primer combinations were used for the analysis of 152 B biotype Bemisia tabaci (Gennadius) individuals and five Trialeurodes vaporairiorum individuals collected from 19 counties and seven host plants in Shanxi province in China, respectively. The main objective of the present study was to use AFLP markers to determine the genetic diversity of B. tabaci populations collected from Shanxi Province. The use of these primer combinations allowed the identification of 127 polymorphic bands (52.26%) from 60 to 500 bp. The average number of polymorphic bands per primer was 25.4 while the range for the five primers was 20–32. The average degree of heterozygosity was 0.251, while the range for the five primers was 0.204–0.289. The results suggested definite genetic diversity among different B. tabaci populations. Cluster analysis showed that B. tabaci populations were firstly scattered to three genetic groups according to the regions, then every genetic group was scattered to several subgroups according to the host plants, which revealed the genetic variability of B biotype B. tabaci populations has been not only among different regions, but also among different host plants in Shanxi Province.     

The synthesis of 6,9-deepoxy-6,9-N-phenylimino-Δ6,8-prostaglandin I1 a novel inhibitor of leukotriene C and D synthesis

The pyrrole analog of prostacyclin, 6,9-deepoxy-6,9-N-phenylimino-Δ^6,8-prostaglandin I₁ was synthetized from PGF₂α methyl ester. This pyrroloprostacyclin (U-60, 257) and its methyl ester (U-56, 467) have been shown to inhibit leukotriene C/D biosynthesis and antagonized leukotriene C/D contractions in vitro. Antigen induced bronchopulmonary changes in monkeys and guinea pigs are inhibited by U-60, 257 in vivo.     

Synthesis and characterisation of κ-P and κ-P,N palladium(II) complexes of the open cage water soluble aminophosphine PTN

New palladium(II) complexes containing the water soluble aminophosphine PTN ligand (PTN = 7-phospha-3,7-dimethyl-1,3,5-triazabicyclo[3.3.1]nonane) in 1:1 and 1:2 ratio Pd/PTN ligand, respectively, were prepared and fully characterised by mono and bidimensional ³¹P, ¹H and ¹³C NMR techniques showing that PTN can adopt both κ¹-P and κ²-P,N coordination modes. The complexes with Pd/PTN ratio 1:2 are highly soluble in water at room temperature. Suitable crystals for X-ray structure determination were obtained for the neutral complex κ²-P,N-Pd(PTN)(OAc)₂ (1) and for the monocationic complex [Pd(κ²-P,N-PTN)(κ¹-P-PTN)Cl][PF₆] (5).     

Oligomeric form of the light-harvesting chlorophyll a + b-protein complex CP II,phosphatidyldiacylglycerol, Δ3-trans-hexadecenoic acid and energy transfer in Chlamydomonas reinhardtii,wild type and mutants

Analyses of chlorophyll-protein complexes and of lipids were performed with the wild type of Chlamydomonas reinhardtii and three non-photosynthetic mutants: Fl 39, which was a ‘classical’ high-fluorescent Photosystem II (PS II)-lacking mutant, and mf 1 and mf 2, which lacked also functional PS II but were low-fluorescent and showed an abnormally predominant energy transfer from the main light-harvesting antenna towards Photosystem I. An oligomeric form of the chlorophyll a + b-protein complex CP II was clearly isolated from the wild type and the mutant Fl 39 but it was not detected in the mutants mf 1 and mf 2. The three mutants showed total lipid contents close to or greater than that of the wild type. Their phosphatidyldiacylglycerol (PG) contents, on a chlorophyll basis, were higher (Fl 39) or 1.4- (mf 1) and 2.0- (mf 2) times lower than that of the wild type. The fatty acid compositions of the wild type and of the mutant Fl 39 were comparable, showing about equal amounts of a C18 series and a C16 series which included the Δ3-trans-hexadecenoic acid (C16:1-trans). This C16:1-trans was not detected in the mutants mf 1 and mf 2 which contained the other fatty acids. These results indicate correlations between lack of C16:1-trans-containing PG, lack of an oligomeric form of CP II and an impaired mechanism of the regulation of excitation energy transfer from the main chlorophyll a + b antenna.     

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III_9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities.     

Cell-cell interactions in conjugating Escherichia coli: Con? mutants and stabilization of mating aggregates

Summary Conjugation-deficient (Con^-) mutants of Escherichia coli K-12 have been previously described which were defective in recipient ability. Such Con^- mutants were obtained from several laboratories and retested by a standardized set of procedures. Many of the mutants did not satisfy minimal criteria for conjugation-deficiency and were discarded. The remaining mutants included 11 ConF^- mutants mutated in or near the ompA cistron, 3 ConF^- mutants synthesizing a heptose-deficient lipopolysaccharide and 1 ConI^- mutants synthesizing a defective lipopolysaccharide. This set of mutants was tested for resistance to a variety of bacteriophages and colicins; the only phenotype fully correlated with the ConF^- phenotype was that of resistance to colicin L. No simple correlation existed between the protein profile (on SDS polyacrylamide gel electrophoresis) of cell envelope outer membrane preparations and conjugation deficiency. However, many ConF^- mutants did not synthesize detectable levels of outer membrane protein II^* and protein II^* may have been nonfunctional in the remainder. All the ConF^- mutants were conjugation-deficient when matings were conducted in liquid but (with one exception) were conjugation-proficient on the surfaces of membrane filters. None of the ConF^- mutants formed stable mating aggregates in liquid with (Flac)⁺ donor cells although all bound purified F pili. The ConF^- phenotype associated with a II^*-deficient recipient could be mimicked by the addition of purified protein II^* (solubilized with lipopolysaccharide). In both cases, the formation of stable mating aggregates (analyzed with an improved Coulter counter technique) was inhibited whereas unstable mating aggregates were detected by electron microscopy. F pilus and wall to wall contacts were both observed under these conditions by electron microscopy. These results were used to define a stage in F-promoted conjugation, the stabilization stage, which requires the functional interaction of protein II^* and lipopolysaccharide in the outer membrane of the recipient cell.     

Phospholipase C and sphingomyelinase activities of the Clostridium perfringens α-toxin

α-Toxin is a major pathogenic determinant of Clostridium perfringens, the causative agent of gas gangrene. α-Toxin has been known for long to be a phospholipase C, but up to now its hydrolytic properties have been studied only through indirect methods, e.g. release of cell contents, or under non-physiological conditions, e.g., in micelles, or with soluble substrates. In this report we characterize the phospholipase C and sphingomyelinase activities of α-toxin using a direct assay method (water-soluble phosphorous assay) with phospholipids in bilayer form (large unilamellar vesicles) in the absence of detergents. The simplest bilayer compositions allowing measurable activities under these conditions were DOPC:Chol (2:1 mol ratio) and SM:PE:Chol (2:1:1 mol ratio) for the PLC and SMase activities respectively. PLC activity was five times higher than SMase activity. Both activities gave rise to vesicle aggregation, after a lag time during which ca. 10% of the substrate was hydrolyzed. Vesicle aggregation, measured as an increase in light scattering, was a convenient semi-quantitative method for estimating the enzyme activities. The optimum pH for the combined PLC and SMase activities was in the 5-7 range, in agreement with the proposed role of α-toxin in aiding the bacterium to escape the fagosome and survive within the cytosol.