Maintenance of redox balance by antioxidants in hemolymph of the greater wax moth Galleria mellonella larvae during encapsulation response

The lipid peroxidation process in hemocytes, activities of phenoloxidase and key enzymatic antioxidants (superoxide dismutase, glutathione‐S‐transferase, catalase) and nonenzymatic antioxidants (thiols, ascorbate) in hemolymph of the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) were studied during the encapsulation process of nylon implants. It has been established that as soon as 15 min after piercing a cuticle with the implant, a capsule is formed on its surface. Active melanization of the capsule has been shown to last for 4 h. During the first hours after incorporating the implant, an increase in phenoloxidase activity and lipid peroxidation in the insect hemocytes has been revealed. Adhesion and degranulation on the surface of foreign object lead to the depletion of total hemocytes count (THC). Our results indicated that thiols and ascorbate molecules take part in the immediate antioxidant response, during later stages of encapsulation process hemolymph glutathione‐S‐transferase detoxifies and protects insect organism thereby restoring the internal redox balance. We suggest that nonenzymatic and enzymatic antioxidants of hemolymph plasma play a key role in the maintenance of redox balance during encapsulation of foreign targets.     

Atypical Micromorphology and Uncommon Location of Cryptococcosis: A Histopathologic Study Using Special Histochemical Techniques (One Case Report)

Here we report an unusual case of disseminated cryptococcosis in a patient with AIDS. Although typical Cryptococcus neoformans micromorphology was observed in tongue biopsy, cervical lymph node examination revealed atypical histopathologic findings. These included pseudohyphae, chains of budding yeasts and structures resembling germ tubes. Cryptococcus neoformans infection in supraclavicular lymph nodes was also confirmed by culture. The importance of using special histochemical techniques—Mayer’s mucicarmine stain for mucicarminophilic capsule and Grocott’s silver stain—in the diagnosis of cryptococcosis is reinforced.     

Hemocytic encapsulation of implants in the tickDermacentor variabilis

Implants of Epon, inserted inDermacentor variabilis (Say) through incisions in the cuticle, were encapsulated by hemocytes. We followed this process at intervals of 1, 3, 6, 12 and 24 h, and every 24 h thereafter up to 120 h. Degranulation of Type 1 granulocytes and coagulation of hemolymph were first seen at 1 h after implantation and were the earliest evidence of encapsulation. By 3 h after implantation, the degranulation and disintegration of granulocytes had formed a matrix at the Epon surface. From 6 h until encapsulation was completed, plasmatocytes and granulocytes continued to respond to degranulation and formed multiple cell layers around the Epon implant. The capsule was complete at 72 h after implantation. Completion was marked by decreasing degranulation, migration of hemocytes from the outermost layers of the capsule, and by the appearance of loosely attached hemocytes on the outer surface of the capsule. The most common junctional complex observed was gap junctions.     

Production of reactive oxygen species by the mitochondrial electron transport chain in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Mitochondrial free radicals and in particular mitochondrial Reactive Oxygen Species (mtROS) are considered to be totally or partially responsible for several different diseases including Parkinson, diabetes or cancer. Even more importantly, mtROS have also been proposed as the main driving force behind the aging process. Thus, in the last decade, there has been a growing interest in the role of free radicals as signalling molecules. Collectively this makes understanding mechanisms controlling free radical production extremely important. There is extensive published literature on mammalian models (essentially rat, mouse and guinea pig) however; this is not the case in Drosophila melanogaster. Drosophila is an excellent model to study different physiological and pathological processes. Additionally a robust method to study mtROS is extremely useful. In the present article, we describe a simple—but extremely sensitive—method to study mtROS production in Drosophila. We have performed various experiments to determine which specific respiratory complexes produce free radicals in the electron transport chain of Drosophila melanogaster. Complex I is the main generator of ROS in Drosophila mitochondria, leaking electrons either in the forward or reverse direction. The production of ROS during reverse electron transport can be prevented either by rotenone or by the oxidation of NADH by complex I. These results clearly show that Drosophila mitochondria function in a very similar way to mammalian mitochondria, and therefore are a very relevant experimental model for biochemical studies related to ageing.     

The role of oxidative stress in lung injury induced by cigarette smoke

Oxidative stress is a damaging process resulting from an imbalance between excessive generation of oxidant compounds and insufficient antioxidant defence mechanisms. Oxidative stress plays a crucial role in the initiation and progression of cigarette smoke-induced lung injury, deterioration in lung functions, and development of chronic obstructive pulmonary disease (COPD). In smokers and in patients with COPD, the increased oxidant burden derives from cigarette smoke per se, and from activated inflammatory cells releasing enhanced amounts of reactive oxygen and nitrogen species (ROS, RNS, respectively). Although mild oxidative stress resulting from cigarette smoking leads to the upregulation of the antioxidative enzymes synthesis in the lungs, high levels of ROS and RNS observed in patients with COPD overwhelm the antioxidant enzymes capacities, resulting in oxidant-mediated lung injury and cell death. In addition, depletion of antioxidative systems in the systemic circulation was consistently observed in such patients. The imbalance between the generation of ROS/RNS and antioxidant capacities — the state of “oxidative stress” — is one of the major pathophysiologic hallmarks in the development of COPD. Detrimental effects of oxidative stress include impairment of membrane functions, inactivation of membrane-bound receptors and enzymes, and increased tissue permeability. In addition, oxidative stress aggravates the inflammatory processes in the lungs, and contributes to the worsening of the protease-antiprotease imbalance. Several markers of oxidative stress, such as increases in lipid peroxidation products and reductions in glutathione peroxidase activity, have been shown to be related to the reductions in pulmonary functions. In the present article we review the current knowledge about the vicious cycle of cigarette smoking, oxidative stress, and inflammation in the pathogenesis of COPD.     

Antioxidant system in programmed cell death of sycamore (<Emphasis Type="Italic">Acer pseudoplatanus</Emphasis> L.) cultured cells

Reactive oxygen species (ROS) have pleiotropic effects in plants. ROS can lead to cellular damage and death or play key roles in control and regulation of biological processes, such as programmed cell death (PCD). This dual role of ROS, as toxic or signalling molecules, is possible because plant antioxidant system (AS) is able to achieve a tight control over ROS cellular levels, balancing properly their production and scavenging. AS response in plant PCD has been clearly described only in the hypersensitive response in incompatible plant–pathogen interactions and in the senescence process and has not been completely unravelled. In sycamore (Acer pseudoplatanus L.) cultured cells PCD can be induced by Fusicoccin (Fc), Tunicamycin (Tu), and Brefeldin A (Ba). These chemicals induce comparable PCD time course and extent, while H₂O₂ production is detectable only in Fc- and, to a lesser extent, in Ba-treated cells. In this paper the AS has been investigated during PCD of sycamore cells, measuring the effects of the three inducers on the cellular levels of non-enzymatic and enzymatic antioxidants. Results show that the AS behaviour is different in the PCD induced by the three chemicals. In Fc-treated cells AS is mainly devoted to decrease the concentration of toxic intracellular H₂O₂ levels. On the contrary, in cells treated with Tu and Ba, the cell redox state is shifted to a more reduced state and the enzymatic AS is partially down-regulated, allowing ROS to act as signalling molecules.     

Effect of seed-specific expression of the <Emphasis Type="Italic">ipt</Emphasis> Gene on <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. seed composition

A transgenic approach to manipulation of endosperm development has been investigated. Nicotiana tabacum cv. Xanthi, an endosperm-containing dicotyledon, has been used as a model plant and the 2.6 kb wheat high molecular weight (HMW) glutenin subunit 12 promoter has been used fused either to the gus reporter gene (HMWgus construct)—to study promoter characteristics—or to the Agrobacterium ipt gene—to study the effect of cytokinin (CK) over-expression on assimilate accumulation in the seed. In transgenic tobacco the promoter:gus fusion showed that HMW is an endosperm-specific promoter with maximum expression 20 days after anthesis (DAA), corresponding to the mid to late stages of seed development. Transgenic plants containing the HMWipt construct showed no morphological abnormalities but they had an average increase in seed weight and total ethanol-insoluble carbohydrates and protein content of 8.1%, 7.0% and 8.3%, respectively. SDS PAGE analysis demonstrated that the effect on protein accumulation was non-specific. The highest values of the parameters analysed correlated with moderate increases in the levels of biologically active CKs. These results suggest that ectopic expression of small amounts of CKs can be used to increase storage assimilate accumulation without a detrimental effect on development.     

The viability of the first generation roach (<Emphasis Type="Italic">Rutilus rutilus</Emphasis> L.), bream (<Emphasis Type="Italic">Abramis brama</Emphasis> L.), and blue bream (<Emphasis Type="Italic">Abramis ballerus</Emphasis> L.) hybrids at early stages of development

The viability of bream, roach, and blue bream F ₁hybrids at early stages of their development is analyzed. Viability is controlled according to parameters of the survival rate at stages from fertilized eggs to yearlings. During embryogenesis, significant stages (blastula—gastrula and hatching) are revealed by the amount of losses. The viability of hybrids of the first generation (compared to pure species) from the hatching stage and in the subsequent development constantly increases. At the stage of fingerlings, the viability of F ₁hybrids significantly exceeds that of pure species, which points to the heterozygous effect according to the parameters of hybrid survival for the first generation, which is absent in embryogenesis.     

Characterisation of the initial degradation stage of Scots pine (<Emphasis Type="Italic">Pinus sylvestris</Emphasis> L.) sapwood after attack by brown-rot fungus <Emphasis Type="Italic">Coniophora puteana</Emphasis>

In our study, early period degradation (10 days) of Scots pine (Pinus sylvestris L.) sapwood by the brown-rot fungus Coniophora puteana (Schum.: Fr.) Karst. (BAM Ebw.15) was followed at the wood chemical composition and ultrastructurelevel, and highlighted the generation of reactive oxygen species (ROS). An advanced decay period of 50 days was chosen for comparison of the degradation dynamics. Scanning UV microspectrophotometry (UMSP) analyses of lignin distribution in wood cells revealed that the linkages of lignin and polysaccharides were already disrupted in the early period of fungal attack. An increase in the lignin absorption A₂₈₀ value from 0.24 (control) to 0.44 in decayed wood was attributed to its oxidative modification which has been proposed to be generated by Fenton reaction derived ROS. The wood weight loss in the initial degradation period was 2%, whilst cellulose and lignin content decreased by 6.7% and 1%, respectively. Lignin methoxyl (–OCH₃) content decreased from 15.1% (control) to 14.2% in decayed wood. Diffuse reflectance Fourier-transform infrared (DRIFT) spectroscopy corroborated the moderate loss in the hemicellulose and lignin degradation accompanying degradation. Electron paramagnetic resonance spectra and spin trapping confirmed the generation of ROS, such as hydroxyl radicals (HO^∙), in the early wood degradation period. Our results showed that irreversible changes in wood structure started immediately after wood colonisation by fungal hyphae and the results generated here will assist in the understanding of the biochemical mechanisms of wood biodegradation by brown-rot fungi with the ultimate aim of developing novel wood protection methods.     

Biotechnological production of itaconic acid and its biosynthesis in <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

More than 80,000 tons of itaconic acid (IA) is produced worldwide each year and is sold at a price of around US$ 2/kg. The IA production yield from sugar is higher than 80 g/l. The widespread use of IA in synthetic resins, synthetic fibers, plastics, rubbers, surfactants, and oil additives has resulted in an increased demand for this product. However, at present, the IA production capacity exceeds the demand because this product has a restricted range of applications. Studies have been actively conducted in different biomedical fields—dental, ophthalmic, and drug delivery—to extend the range of applications of IA. Recently, many researchers have attempted to replace the carbon source used for microbial production of IA with cheaper alternative substrates. However, there is still a need for new biotechnology innovations that would help to reduce the production costs, such as innovative process development and strain improvement to allow the use of a low-quality carbon source. In this short review, we discuss the following aspects of IA production: strain improvement, process development, identification of the key enzyme cis-aconitic acid decarboxylase (CAD) in the IA metabolic pathway, metabolic importance of CAD, and new applications of IA.     

Redescription of large-scale eelpout <Emphasis Type="Italic">Lycodes macrolepis</Emphasis> Taranetz et Andriashev, 1935 (Perciformes: Zoarcidae)

Morphology of a rare species—large-scale eelpout Lycodes macrolepis Taranetz et Andriashev, 1935—has been studied. It was shown that the main lateral line of this species has a ventrolateral configuration rather than medial, as was considered previously. Large-scale eelpout is an endemic species of the Sea of Okhotsk; evidence of its finding in the Sea of Japan is based on the erroneous classification of another eelpout species.     

The consequences of predator control for brown hares (<Emphasis Type="Italic">Lepus europaeus</Emphasis>) on UK farmland

The brown hare Lepus europaeus is a valued game species but also a species of conservation concern owing to its severe decline in abundance on farmland throughout Europe during the twentieth century. Changes in the farmland habitat and predation have both been cited as causative factors. Their relative roles have been unclear, but most conservation action has focused on improving habitat. We analyse data from a sequence of three unique studies (one experiment and two demonstrations) covering the period 1985–2006 in which control of several common predator species was undertaken to increase densities of wild game on farmland in England. Across the three studies, regression modelling of the proportional change in hare numbers between successive years showed that—after site, year differences and harvesting were accounted for—predator control was a significant determinant of hare population change. Where habitat improvement also took place, hares reached autumn densities that were exceptional for the UK and which could sustain substantial harvests. When predation control was stopped, hare densities fell, even where habitat improvements remained in place. This analysis demonstrates that even where farmland habitat is greatly improved, uncontrolled predation prevents hares making full use of its carrying capacity. This helps explain the mixed—and at best modest—success of agri-environment schemes in the UK and elsewhere in Europe to increase hare densities. Game-shooting estates, on which effective predator control takes place, probably have a special significance within the landscape as source areas for brown hares.     

Dependence of size of the great ramshorn (<Emphasis Type="Italic">Planorbarius corneus</Emphasis> L., Gastropoda,Pulmonata) on population density

The dependence of the mean mass (M) of great ramshorn (Planorbarius corneus) individuals on the number of individuals (N) that reached 82-days age in culture with constant conditions—water volume 50 ml, temperature 25°C, and redundant food (dandelion leaves)—has been studied. The relationship between these parameters has been shown to be approximated by the equation M = 139/N mg. Consequently, at least in these conditions the total biomass of same-aged ramshorn individuals in the culture is relatively constant and does not depend on the number of individuals in the population.     

Mitochondrial generation of reactive oxygen species is enhanced at the Q<Subscript>o</Subscript> site of the complex III in the myocardium of <Emphasis Type="Italic">Trypanosoma cruzi-</Emphasis>infected mice: beneficial effects of an antioxidant

In this study, we have characterized the cellular source and mechanism for the enhanced generation of reactive oxygen species (ROS) in the myocardium during Trypanosoma cruzi infection. Cardiac mitochondria of infected mice, as compared to normal controls, exhibited 63.3% and 30.8% increase in ROS-specific fluorescence of dihydroethidium (detects O₂ ^•−) and amplex red (detects H₂O₂), respectively. This increase in ROS level in cardiac mitochondria of infected mice was associated with a 59% and 114% increase in the rate of glutamate/malate- (complex I substrates) and succinate- (complex II substrate) supported ROS release, respectively, and up to a 74.9% increase in the rate of electron leakage from the respiratory chain when compared to normal controls. Inhibition studies with normal cardiac mitochondria showed that rotenone induced ROS generation at the Q_Nf-ubisemiquinone site in complex I. In complex III, myxothiazol induced ROS generation from a site located at the Q_o center that was different from the Q_i center of O₂ ^•− generation by antimycin. In cardiac mitochondria of infected mice, the rate of electron leakage at complex I during forward (complex I-to-complex III) and reverse (complex II-to-complex I) electron flow was not enhanced, and complex I was not the main site of increased ROS production in infected myocardium. Instead, defects of complex III proximal to the Q_o site resulted in enhanced electron leakage and ROS formation in cardiac mitochondria of infected mice. Treatment of infected mice with phenyl-α-tert-butyl-nitrone (PBN) improved the respiratory chain function, and, subsequently, decreased the extent of electron leakage and ROS release. In conclusion, we show that impairment of the Q_o site of complex III resulted in increased electron leakage and O₂ ^•− formation in infected myocardium, and was controlled by PBN.     

Spawning and early ontogenesis of the littoral polychaete <Emphasis Type="Italic">Namanereis littoralis</Emphasis> (Grube, 1876) (Nereididae,Namanereidinae)

For the first time, under laboratory conditions, development of the polychaete Namanereis littoralis (Grube, 1876) is investigated. Under conditions of the Sea of Japan, its reproduction occurs in July and is confined to the season of monsoon rains. Fertilization is external. Spawning manifests no epitocous transformations. Fecundity is low, ovicells are rich in yolk, and development is nonpelagic, lecithotrophic, embryonized, characterized by a high rate—5–8 days—and occurs in mucous clutches up to hatching of benthic juveniles. Temperature and salinity optima of development are 22–27°C and 16–21‰, respectively, characterizing the species as subtropical brackish-water by its origin. Archaic and specialized traits are noted in the early ontogenesis of N. littoralis.     

<Emphasis Type="Italic">Hox</Emphasis> cluster duplication in the basal teleost <Emphasis Type="Italic">Hiodon alosoides</Emphasis> (Osteoglossomorpha)

Large-scale—even genome-wide—duplications have repeatedly been invoked as an explanation for major radiations. Teleosts, the most species-rich vertebrate clade, underwent a “fish-specific genome duplication” (FSGD) that is shared by most ray-finned fish lineages. We investigate here the Hox complement of the goldeye (Hiodon alosoides), a representative of Osteoglossomorpha, the most basal teleostean clade. An extensive PCR survey reveals that goldeye has at least eight Hox clusters, indicating a duplicated genome compared to basal actinopterygians. The possession of duplicated Hox clusters is uncoupled to species richness. The Hox system of the goldeye is substantially different from that of other teleost lineages, having retained several duplicates of Hox genes for which crown teleosts have lost at least one copy. A detailed analysis of the PCR fragments as well as full length sequences of two HoxA13 paralogs, and HoxA10 and HoxC4 genes places the duplication event close in time to the divergence of Osteoglossomorpha and crown teleosts. The data are consistent with—but do not conclusively prove—that Osteoglossomorpha shares the FSGD. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Investigation of the influence of temperature on polyelectrolyte microcapsules containing and not containing proteins

Using the methods of light scattering and optical microscopy, data have been obtained on the thermosensitivity of polyelectrolyte microcapsules formed of alternating layers of polyallylamine and polystyrenesulfonate, hollow and with included polyelectrolyte complexes and proteins. It is shown that all three types of capsule shrink with increasing temperature and time interval of thermal influence, and their diameter decreases. It is proposed that the thermosensitivity of microcapsules be estimated by the temperature factor of the rate of their shrinkage (E _s). For all three types of microcapsule containing from 6 to 10 layers in the shell, the phenomenon of alternant thermosensitivity depending on the number of shell layers is revealed—with an odd number of layers the shrinkage is stronger than with an even one. Using the transport proteins of blood—hemoglobin and bovine serum albumin—as an example, the dependence of the thermosensitivity of microcapsules on the quantity, the degree of ionization, and the conformational state of the encapsulated protein has been investigated.     

Gene frequency-based estimation of natural outcrossing in opium poppy (<Emphasis Type="Italic">Papaver somniferum</Emphasis> L.)

Mating systems are generally thought to be important factors in determining the amount and nature of genetic variability in a population. Nearly 1,000 individuals at a single location (Lucknow) and over two years were crossed and subsequently scored for selfing versus outcrossing in 9–10 monohybrid populations of opium poppy (Papaver somniferum). Three different alleles of two marker loci (two—R/r and P/p—for anthocyanin locus and B/b for capsule size locus) were used to determine the male gametes that had effected fertilizations in F₂ recessives (rr, pp and bb). The estimates of the gene frequency-based outcrossing parameter (α) were found to vary with year, cross and marker locus used (α range: 7.21–71.03%). Study of the two dihybrid crosses concerning the two marker loci simultaneously, further confirmed that outcrossing at the R/r or P/p locus was significantly greater than that at the B/b locus. The nature of the outcrossing was, in general, nonrandom. In this species, in general, selfing predominated, with one exception in respect of monohybrid crosses involving the purple form of anthocyanin locus, in which outcrossing predominated.     

Ontogenesis of the cricket Gryllus argentinus Sauss. (Orthoptera, Gryllidae)

The development of Gryllus argentinus Sauss. was studied under stable laboratory conditions: the temperature of 26°C, the air humidity of 60%, and the photoperiod of 12h light: 12 h dark. The life cycle of Gryllus argentinus includes four stages: egg, pronymph, nymph, and adult. The duration of embryonic development is 18 days. The depth of egg bedding in the peat is 9.63 ± 0.12 mm (n =145), the clutch containing 2–4 eggs. A female can lay over 1100 viable eggs during the entire oviposition period. Nymphal development includes 9 instars and lasts 97 days. The duration of nymphal instars (days) is: I—5; II—6; III—6; IV—6; V—8; VI—10; VII—13; VIII—14; IX—29. The duration of the adult life is 51 days in males and 69 days, in females. In the imaginal ontogenesis of males and females, three periods can be distinguished: pre-reproductive, reproductive, and postreproductive. Males start to emit the aggressive signal on the 6th (5–8th) day (the pre-reproductive period). They enter the reproductive period (start to emit the calling song) on the 9th (8–13th) day. Females enter the reproductive period (become capable of responding to the calling song and of copulation) on the 9th (8–10th) day. Oviposition starts on the day after the first copulation. The reproductive period lasts about 40 (15–59) days in males and 58 (21–70) days in females. The post-reproductive period starts in females at the moment of finishing the egg laying period and in males, with disappearance of reproductive behavior. The period ends in the animal’s death.