Coordination between the electrical activity of developing indirect flight muscles and the firing activity of a population of neurosecretory cells in the silkmoth, Bombyx mori

The developing indirect flight muscles of pharate moths are characterized by a rhythmic discharge of a long bout of flight-pattern-like muscle potentials in the absence of contractions. The electrical activity of the dorsal longitudinal flight muscles (DLMs) in the silkmoth, Bombyx mori, was discernible as a cluster of many series of muscle potentials that last for several minutes on day 4 of the pupal period. The duration of the active phases and the period of rhythmic activity gradually increased to a peak value on day 7 or 8 and then declined until the end of the pupal period. Mean duration of the active phases (+/-SD) and the mean period of the rhythmic activity (+/-SD) at the peak were 38.7+/-8.7 min and 74.5+/-7.3 min, respectively. The rhythmic electrical activity of immature DLMs was closely coordinated with the rhythmic (bursting) activity of a population of neurosecretory cells that are known to produce pheromone-biosynthesis activating neuropeptide (PBAN) and its related peptides, which belong to the multifunctional peptide family, pyrokinin/PBAN. The DLMs always became active a few minutes after the neurosecretory cells, and the timing of onset of these two activities appeared to be strictly regulated by a neural mechanism. The implication of the coordinated activity for development and maturation of imaginal tissues, including the flight motor system, and possible functions of the neuropeptides in this development are discussed.     

Co-ordination of firing activity of neurosecretory cells with cardiac activity in the silkmoth Bombyx mori

Electrocardiograms and electrical action potentials of cerebral neurosecretory cells producing bombyxin (an insulin-related neuropeptide) were simultaneously recorded from male pupae of the silkmoth Bombyx mori. A pupa showed alternations in the flow of haemolymph due to a rhythmic heartbeat reversal: a train of retrograde heartbeat with a slow pulse rate followed a train of anterograde heartbeat with a higher pulse rate. Intervals of heartbeat reversals changed throughout the pupal period. At any stage of the pupal period, firing activity of a population of bombyxin-producing (BP) cells rapidly declined after the start of anterograde heartbeat and an inactive state of cells continued during an anterograde heartbeat period. Analyses of ultradian bursting rhythmicity of a single BP cell revealed that a bursting phase of the cell often delayed at a time when the anterograde cardiac activity occurred at the preceding inter-burst period of firing rhythm. The results support the postulation that firing (secretory) activity of an insect neurosecretory cell system may be co-ordinated with circulation of haemolymph for rapid and pulsatile delivery of the peptides released to target organs.     

Periodic abdominal pumping supports leg development during metamorphosis in tenebrionid beetle Zophobas atratus

Rhythmic abdominal pumping movements in a pupa of giant mealworm beetle Zophobas atratus caused large hemolymph pressure pulses of approximately 20 mmHg. The abdominal pumping movements were completely blocked by transecting the ventral nerve cord (VNC) between the first and second abdominal ganglia. Transection of the VNC until 2 days after pupation caused a developmental defect of adult legs: morphogenesis of the tibial and tarsal segments was severely retarded, and the segments remained covered with a thick pupal cuticle. The developmental defect was rescued by artificially inducing rhythmic abdominal bending for 3 days after transection of VNC. Blocking of the abdominal pump did not increase the amount of water loss during the pupal period. The transplanted tibial segments lacking active tracheal ventilation could form a thick adult cuticle. The results suggest that abdominal pumping movements during the pupal period support the development of adult legs by facilitating hemolymph circulation.     

Alachalasins A-G, new cytochalasins from the fungus Stachybotrys Podospora vesticola [corrected

Alachalasins A-G (1-7), seven new cytochalasin-type of metabolites, have been isolated from cultures of an isolate of Stachybotrys Podospora vesticola [corrected] The structures of these compounds were determined mainly by analysis of their NMR spectroscopic data, and the absolute configuration of 1 was assigned by application of the modified Mosher method. Alachalasin A (1) displayed inhibitory effect on HIV-1LAI replication in C8166 cells with an EC50 of 8.01 microM; alachalasins D (4) and G (7) showed modest antimicrobial activity.     

Firing activity of "diapause hormone" producing cells in the male silkmoth,Bombyx mori

Diapause hormone (DH) originally identified to be a factor originating from neurosecretory cells in the suboesophageal ganglion acts on developing ovaries to produce diapause eggs in a female silkmoth, Bombyx mori. A male silkmoth has homologous neurosecretory cells, but little is known of the physiological nature of the cells and actions of their products. We examined the long-term firing activity of putative DH-producing neurosecretory cells and hormonal activity of their products in male pupae that had been experienced different environmental regimens for diapause induction. Firing activity patterns of male labial cells strongly depended on diapause types of pupae: cells in a diapause-type male were active throughout the pupal period, whereas the same cells in a non-diapause-type male were usually inactive during the early two-thirds of the pupal period. A male pupa with electrically active labial cells could induce diapause eggs in a female pupa connected parabiotically to that male. The firing activity of male neurosecretory cells and hormonal action of their products are qualitatively the same as in the female previously examined. We suggest that there is no evident sexual dimorphism in the physiological and biochemical nature of neurosecretory cells producing DH and the amidated peptide DH has different functions in a male.     

Molecular identification and functional characterisation of uncoupling protein 4 in larva and pupa fat body mitochondria from the beetle Zophobas atratus

Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis.     

Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora

The incidence of diapause was shown to be determined humorally during the larval-pupal ecdysis by means of brain extirpation experiments.On the basis of this observation, light and electron microscopic changes in the neurosecretory type II cells in the pars intercerebralis-corpus cardiacum system during pharate pupal and early pupal stages were examined in insects reared under long day-length (non-diapause individuals) and in insects reared under short day-length (diapause individuals). In the diapause individuals, neurosecretory granules in NS-II cells increased during the pupal instar and large aggregates of granules packed the cytoplasm. Thereafter, inclusion bodies showing cytoplasmic breakdown of the granules appeared.In the non-diapause individuals, on the contrary, electron micrographs suggesting the release of neurosecretory material from axon terminals were obtained just after the pupal ecdysis. There were very few granules, with many Golgi bodies and much rough ER 8 to 12 hr after the ecdysis.It is concluded that adult development is determined by the release of neurosecretory material from the axon terminals of NS-II cells at the larval-pupal ecdysis. If release does not occur, the pupae enter diapause. It is also thought that differences in day-length during the larval stages influence the activities of NS-II cells before pupation.     

Ultracytochemical characteristics of cultured goat brain microvascular endothelial cells [corrected]

This ultrastructural study was undertaken to determine the localization of cytochemically demonstrable blood-brain barrier (BBB)-associated enzymatic activities and of some nonenzymatic constituents in goat [corrected] brain microvascular endothelial cells (ECs) growing in vitro. Positive reactions for alkaline phosphatase (AP), 5'-nucleotidase (5'N), transport ATPase (Na+,K(+)-ATPase), and adenosine diphosphatase (ADPase) were present on both apical and basolateral plasma membranes (PMs) of the ECs. The reaction for calcium-dependent ATPase (Ca(2+)-ATPase) was less intense and was restricted to basolateral PM and associated plasmalemmal pits. These cells also revealed an abundance of anionic sites labeled with cationic colloidal gold (CCG) and Ricinus communis agglutinin 120 (RCA)-binding sites, specific for beta-D-galactosyl residues, on the apical PM. The labeling of the apical PM with Ulex europaeus agglutinin (UEA)-gold complex, specific for alpha-L-fucosyl residues, was negligible. When compared with results of cytochemical examination of the ECs of goat [corrected] brain capillary in vivo, these observations indicate that although cells cultivated in vitro retain at confluence the enzymatic activities typical for BBB-type ECS, they lose their characteristic (polar) localization. This loss is interpreted as a reflection of lost functional polarity of the microvascular endothelium in vitro resulting from deprivation of the normal influence of the components of brain parenchyma.     

Firing activities of neurosecretory cells producing diapause hormone and its related peptides in the female silkmoth,Bombyx mori. II. mandibular and maxillary cells

A gene encoding a precursor polyprotein of diapause hormone (DH) and four related peptides is expressed by three groups of neurosecretory cells in the suboesophageal ganglion (SOG) of Bombyx mori. Long-term chronic recordings of firing activities were made from a common axonal tract (the maxillary nerve) of two groups of cells localized in the mandibular and maxillary neuromeres of SOG during pupal-adult development. Mandibular and maxillary cells usually produced a cluster of action potentials at an interval of 30-60 min during pupal-adult development and there was no significant difference in the firing activity profile between diapause-egg and non-diapause-egg producers. We suggest that rather than DH secretion, pupal mandibular and maxillary cells are involved in the secretion of DH-related neuropeptides. DH secretion seems to be assigned to the third group of cells (labial cells); hence, there may be different posttranslational processing of the precursor polyprotein in different neurosecretory cell groups.     

Amides from Streptomyces hygroscopicus and their antimicrobial activity [corrected

Three amides, N-salicyloyl-2-aminopropan-1,3-diol (1) and 1-acetyl-N-salicyloyl-2-aminopropan-3-ol (2) including a natural product, N-salicyloyl-2-aminopropan-1-ol (3) were isolated from an ethyl acetate extract of the culture filtrate of Streptomyces hygroscopicus [corrected] The structures of these compounds were unambiguously established by interpretation of their spectral data including, a series of 1D and 2D-NMR and MS analyses. Compounds 1-3 showed significant antibacterial activity against a wide range of Gram positive and Gram negative bacteria.     

Cloning and sequencing of LlaDCHI [corrected] restriction/modification genes from Lactococcus lactis and relatedness of this system to the Streptococcus pneumoniae DpnII system.

The natural 7.8-kb plasmid pSRQ700 was isolated from Lactococcus lactis subsp. cremoris DCH-4. It encodes a restriction/modification system named LlaDCHI [corrected]. When introduced into a phage-sensitive L. lactis strain, pSRQ700 confers strong phage resistance against the three most common lactococcal phage species, namely, 936, c2, and P335. The LlaDCHI [corrected] endonuclease was purified and found to cleave the palindromic sequence 5'-GATC-3'. It is an isoschizomer of Streptococcus pneumoniae DpnII. The plasmid pSRQ700 was mapped, and the genetic organization of LlaDCHI [corrected] was localized. Cloning and sequencing of the entire LlaDCHI [corrected] system allowed the identification of three open reading frames. The three genes (llaIIA, llaIIB, and llaIIC) overlapped and are under one putative promoter. A putative terminator was found at the end of llaIIC. The genes llaIIA and llaIIB coded for m6A methyltransferases, and llaIIC coded for an endonuclease. The LlaDCHI [corrected] system shares strong genetic similarities with the DpnII system. The deduced amino acid sequence of M.LlaIIA was 75% identical with that of M.DpnII, whereas M.LlaIIB was 88% identical with M.DpnA. However, R.LlalII shared only 31% identity with R.DpnII.     

Rhythmicity of the activity of the supraoptic and paraventricular nuclei of the hypothalamus of the C57Bl mouse

Rhythmical changes in the activity of the neurosecretory processes have been compared in the supraoptic (SON) and paraventricular (PVN) nuclei in the C57Bl mice hypothalamus during vernal equinox. Parts of the neurosecretory cells, being at stages of synthesis, excretion and accumulation of secrete, volumes of the cell nuclei and nucleoli survey as criteria of their activity. Similar feature for the rhythmic of both nuclei studied is the highest activation of the processes during day time, when mice are resting; this is demonstrated as the maximal amount of actively synthesizing cells, maximal volumes of the cell nuclei and nucleoli. The peculiarities of the rhythmic display in the activity is manifested as a greater ability of the SON cells to accumulate neurosecrete. The accumulation of the secretory material in the SON cells precedes to the period of its maximal activity (1-7 PM) characterized: by making the cells free from the secrete and by a maximal increasing the volume of the nucleoli. In the PVN intensified display of the activity is noted at early hours of the day, and the amount of the cells not containing the secrete--at 6 PM. Lack of the neurosecrete accumulation in the PVN cells speaks in favour of more steady than in the SON cells excretion of the secrete. This demonstrates a more even maintenance of neurohormones concentration in the organism.     

Inhibition by [corrected] ursolic acid of [corrected] calcium-induced mitochondrial permeability transition and release of two proapoptotic proteins

The possible inhibition by [corrected] ursolic acid (UA) of [corrected] mitochondrial permeability transition (MPT) in mouse liver was investigated to identify the mechanisms underlying the hepatoprotective effect of UA. The effect of UA on liver MPT induced by Ca2+ was assessed by measuring changes in mitochondrial volume, mitochondrial membrane potential (MMP), release of matrix Ca2+, and transfer of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from the intermembrane space to the cytoplasm. The results showed that obvious mitochondrial swelling, loss of MMP, and release of matrix Ca2+ occurred after the addition of 50 microM Ca2+. However, preincubation with 20, 50 or 100 microg ml(-1) UA significantly blocked the above changes. Addition of 100 microg ml(-1) UA inhibited on mitochondrial swelling by 73.2% after 5 min, while the MMP dissipating and Ca2+ releasing were, respectively, suppressed by 59.3% and 54.1% after 3 min. In addition, Western blot analysis showed Cyt c and AIF transferred from mitochondrial pellet to the supernatant after the addition of 50 microM Ca2+, but the process was significantly inhibited by various concentrations of UA. The results suggest that the mechanisms underlying the hepatoprotection of UA may be related to its direct inhibitory action on MPT.     

Cardioacceleratory action of tachykinin-related neuropeptides and proctolin in two coleopteran insect species

Several cardioactive peptides have been identified in insects and most of them are likely to act on the heart as neurohormones. Here we have investigated the cardioactive properties of members of a family of insect tachykinin-related peptides (TRPs) in heterologous bioassays with two coleopteran insects, Tenebrio molitor and Zophobas atratus. Their effects were compared with the action of the pentapeptide proctolin. We tested the cardiotropic activity of LemTRP-4 isolated from the midgut of the cockroach Leucophaea maderae, CavTK-I and CavTK-II isolated from the blowfly Calliphora vomitoria. The semi-isolated hearts of the two coleopteran species were strongly stimulated by proctolin. We observed a dose dependent increase in heartbeat frequency (a positive chronotropic effect) and a decrease in amplitude of contractions (a negative inotropic effect). In both beetles the TRPs are less potent cardiostimulators and exert lower maximal frequency responses than proctolin. LemTRP-4 applied at 10(-9)-10(-6) M was cardiostimulatory in both species inducing an increase of heart beat frequency. The amplitude of contractions was stimulated only in Z. atratus. CavTK-I and CavTK-II also exerted cardiostimulatory effects in Z. atratus at 10(-9)-10(-6) M. Both peptides stimulated the frequency, but only CavTK-II increased the amplitude of the heart beat. In T. molitor, however, the CavTKs induced no significant effect on the heart.Immunocytochemistry with antisera to the locust TRPs LomTK-I and LomTK-II was employed to identify the source of TRPs acting on the heart. No innervation of the heart by TRP immunoreactive axons could detected, instead it is possible that TRPs reach the heart by route of the circulation. The likely sources of circulating TRPs in these insects are TRP-immunoreactive neurosecretory cells of the median neurosecretory cell group in the brain with terminations in the corpora cardiaca and endocrine cells in the midgut.In conclusion, LemTRP-4, CavTK-I and CavTK-II are less potent cardiostimulators than proctolin and also exert stimulatory rather than inhibitory action on amplitude of contractions. The differences in the responses to proctolin and TRPs suggest that the peptides regulate heart activity by different mechanisms.     

Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.     

Prolactin-cytokine network in the defence against Acanthamoeba castellanii in murine microglia [corrected

In the central nervous system, cytokine-primed microglia play a pivotal role in host defence against parasite infections. In this study, the effect of recombinant (r) prolactin (rPRL) and rIFN-gamma on A. castellanii infection in murine microglia was examined. Priming of microglia with rPRL and rIFN-gamma synergistically triggered, in a dose-dependent manner, amebastatic activity and the release of endogenous IL-1alpha, IL-1beta, IL-6 and TNF-alpha. More than 53, 57, 49, 89 or 96 % of amebastatic activity was abrogated by anti-PRLR, IFN-gammaR, IL-1beta (but not IL-1alpha), IL-6 or TNF-alpha antibodies, suggesting that these two receptors and proinflammatory cytokines participate in the anti-microbial function. Inasmuch as DPI and AET, both inhibitors of NO synthase, blocked amebastatic activity only during the priming process, the NO-dependent pathway itself appears not to be directly involved in the anti-parasitic capacity. These data suggest that the PRL/PRLR and IFN-gamma/IFN-gamma/R complexes, by the induction of the IFN-gammaR on microglia, up-regulate the release of endogenous TNF-alpha, IL-6 and IL-1beta by these cells, which could trigger anti-microbial activity against A. castellanii infection in the brain [corrected].     

The heterotrimeric [corrected] G protein subunit G alpha i is present on mitochondria

Receptors that signal through heterotrimeric [corrected] GTP binding (G) proteins mediate the majority of intercellular communication. Recent evidence suggests that receptors acting through G proteins also transfer signals across the nuclear membrane. Here we present cell fractionation and immunolabeling data showing that the heterotrimeric [corrected] G protein subunit Galphai is associated with mitochondria. This finding suggests that G protein receptor signaling may be a feature common to all membranes.     

Distribution of aspartate aminotransferase activity in yeasts, and purification and characterization of mitochondrial and cytosolic isoenzymes from Rhodotorula minuta [corrected

The distribution of aspartate aminotransferase activity in yeasts was determined. The number of species of the enzyme in each yeast was determined by zymogram analysis. All the yeasts, except for the genus Saccharomyces, showed two or three activity bands on a zymogram. From among the strains, Rhodotorula minuta [corrected] and Torulopsis candida were selected for examination of the existence of yeast mitochondrial isoenzymes, because these strains showed two clear activity bands on the zymogram and contained a high amount of the enzyme. Only one aspartate aminotransferase was purified from T. candida: the component in the minor band on the zymogram was not an isoenzyme of aspartate aminotransferase. On the other hand, two aspartate aminotransferases were purified to homogeneity from R. minuta [corrected]. The components in the main and minor activity bands on the zymogram were identified as the mitochondrial and cytosolic isoenzymes, respectively, in a cell-fractionation experiment. The enzymatic properties of these isoenzymes were determined. The yeast mitochondrial isoenzyme resembled the animal mitochondrial isoenzymes in molecular weight (subunits and native form), absorption spectrum, and substrate specificity. The amino acid composition was closely similar to that of pig mitochondrial isoenzyme. Rabbit antibody against the yeast mitochondrial isoenzyme, however, did not form a precipitin band with the pig mitochondrial isoenzyme.