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1

Biotechnology of insect cell tissue culture

100%

Olejnik A. , Grajek W.

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2003

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nr 1

176-192

EN

The commercial exploitation of the baculovirus expression system for heterologous protein or biopesticides production requires an efficient large-scale cultivation method. This review summarized recent developments concerning the scale-up of insect cell culture and baculovirus gene expression. We described novel bioreactor systems (stirred tank bioreactor, bioreactor airlift and cell-lift, membrane bioreactor), culture modes (batch, fed-batch, continuous) and different strategies used for cell cultivation and baculovirus replication.

2

Exoproteinases of the type A in pathogenesis of insect bacterial diseases

88%

Andrejko M.

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nr 3-4

135-141

EN

Immune inhibitors produced in infected larvae of Galleria mellonella by such entomopathogens as Pseudomonas aeruginosa, Serratia marcescens and Heterorhabditis bacteriophora effectively blocked in vitro bactericidal activity of insect haemolymph against Escherichia coli D31, both in Galleria mellonella and Pieris brassicae pupae previously vaccinated with Enterobacter cloacae. Even at a trace concentration, the extracellular proteinases, by proteolytic degradation, totally destroyed the activity of cecropin peptides from Galleria and cecropin-like and attacin-family proteins from Pieris, but no ability to destroy antibacterial activity was shown by extracts obtained from Galleria larvae killed by massive doses of bacterial saprophytes. It is suggested that by blocking antibacterial immune response of the host, the proteinases help the bacteria to multiply in the haemolymph, thus they could be considered an important factor in the pathogenesis of bacterial diseases of insects.

3

Structure and development of ovaries in the weevil, Anthonomus pomorum (Coleoptera, Polyphaga). II. Germ cells of the trophic chamber

88%

Swiatek P.

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tom 50

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nr 3-4

153-163

EN

The analysis of the germ cell cluster formation in Anthonomus pomorum (Coleoptera, Polyphaga, Curculionidae) has revealed that both linear and branched clones of cystocytes occur in the pupa stage. In the branched clones a poorly developed polyfusome is formed and cystocytes with maximally 3 intercellular bridges were found. In the linear clones the polyfusomes are absent. Further divisions of cystocytes produce exclusively linearly arranged cells. Just after metamorphosis (Imago-A stage), the process of the germ cell membrane reduction starts. Only 2 groups of cells retain cell membranes: i.e the most anteriorly localized group of cystocytes and the posteriorly located presumptive oocytes. The former cells divide mitotically during the summer. As a result an anterior-posterior gradient of the syncytialization process arises in the Imago-B stage (females preparing for hibernation). In the sexually mature females (Imago-C) the trophic chamber consists of a huge syncytial area with numerous nurse cell nuclei embedded in a common cytoplasm, and posteriorly located young oocytes surrounded by prefollicular cells. In the light of recent hypothesis concerning the germ cell cluster formation and telotrophy anagenesis in Polyphaga the significance of the presented results is discussed.

4

Structure and development of ovaries in the weevil, Anthonomus pomorum (Coleoptera, Polyphaga). I. Somatic tissues of the trophic chamber

88%

Swiatek P.

Folia Biologica

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2001

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tom 49

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nr 3-4

215-224

EN

In developing ovarioles of Anthonomus pomorum (Coleoptera, Polyphaga, Curculionidae) the trophic chambers (tropharia) are relatively large and consist of clusters (clones) of germ cells and various somatic tissues. Each ovariole is enclosed within an outer epithelial sheath (tunica externa). Throughout the pupal phase, the growth of this sheath is accelerated and precedes the development of the rest of the ovariole. As a result, the epithelial sheath proliferates anteriorly and forms an elongated ?sleeve? that during the later stages of development becomes gradually filled by the growing tropharium. In the early pupal stage, a few terminal filament cells are observed in contact with the anterior end of the tropharium. These cells are separated from the rest of the trophic chamber by a transverse septum, which maintains continuity with the basal lamina. Beneath the basal lamina there is a layer of inner sheath cells, whereas inside the tropharium there are interstitial cells. These two types of cell differ morphologically in a mature ovary but they retain, until the end of the imago-B stage, a similar ultrastructure testifying to their common origin. At the posterior end of the tropharium, from the imago-B stage on, many young oocytes, surrounded by prefollicular cells, are observed. This is the so-called neck region of the tropharium. Extraction with Triton X-100 detergent showed that in a mature trophic chamber there are only individual microtubules arranged along the projections of interstitial cells. This indicates that the cytoskeleton elements (microfilaments and microtubules) participate only to a very limited extent in the spatial organisation of the tropharium in A. pomorum.

5

Beauvericin cytotoxicity to the invertebrate cell line SF-9

75%

Calo L. , Fornelli F. , Nenna S. , Tursi A. , Caiaffa M. F. , Macchia L.

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nr 4

515-520

EN

The cyclic hexadepsipeptide beauvericin, initially known as a secondary metabolite produced by the entomopathogenic fungus Beauveria bassiana and toxic to Artemia salina larvae, has been more recently recognized as an important mycotoxin synthesized by a number of Fusarium strains, which parasite maize, wheat and rice. Therefore, this mycotoxin may enter the food chain, causing yet unknown effects to the health of both domestic animals and humans. The cytotoxic effects of beauvericin on mammalian cells have been studied. We investigated the cytotoxicity of this compound in an in vitro invertebrate model, viz. the insect cell line SF-9 (immortalized pupal ovarian cells of the lepidopter Spodoptera frugiperda). Cultures of SF-9 cells in the stationary phase were exposed to beauvericin at concentrations ranging from 100 nM to 300 M, for different periods of time (from 30? to 120 h). The effects on cell viability were assessed by the trypan blue exclusion method. After 4 h of incubation no significant decrease in cell viability was recorded in SF-9 cell cultures exposed to low concentrations of beauvericin, i.e. 100 nM and 300 nM. However, a slight decrease in viability (3.9%) was seen already in cells exposed to the mycotoxin at the 1 M concentration. This effect became gradually more evident at higher concentrations ( 28% at 30 M, 50% at 100 M, 68% at 300 M). An even more pronounced reduction in cell viability was observed after a 24 h exposure. Under these conditions, 1 M beauvericin caused an approx. 10% decrease in the number of viable cells, which became more significant at higher concentrations 23% at 3 M, 47% at 10 M, 65% at 30 M, 90% at 100 M, 99% at 300 M). Therefore, the 50% cytotoxic concentrations (CC50) at 4 h and 24 h could be estimated as 85 M and 10 M, respectively. In time-course experiments, no effect of beauvericin (30 M) on cell viability could be seen after exposure for periods of time as long as 30?, 1 h and 2 h, respectively. In contrast, when SF-9 cells were exposed to the mycotoxin for longer periods of time, from 8 h to 120 h, we recorded a strong cytotoxic effect already in the low micromolar concentration range. Thus, the CC50 after both 72 h and 120 h exposure times was assessed as 2.5 M. Higher concentrations caused a virtually 100% cell death. The data collected suggest that beauvericin exerts a substantial dose- and time-dependent cytotoxic effect on invertebrate cells, comparable to the effects described in mammalian cells.