Chronic Exposure of NG108-15 Cells to Amyloid β Peptide (Aβ1–42) Abolishes Calcium Influx via N-Type Calcium Channels

We investigated whether amyloid--peptide (A_1–42) has an effect on the elevations of the intracellular concentration of Ca²⁺ ions ([Ca²⁺]_i) induced by depolarizations of NG108-15 cells and on related Ca²⁺ channels. A_1–42 (10-1000 nM) had no immediate effect on depolarization-induced [Ca²⁺]_i elevations. [Ca²⁺]_i increases were slightly diminished in cells grown in the presence of 100 or 1000 nM A_1–42. Nifedipine (1 M) reduced these elevations equally in cells grown in the absence or presence of A_1–42. In contrast, the ability of -conotoxin GVIA to diminish the depolarization-induced [Ca²⁺]_i responses became lost in cells grown in the presence of 100 nM A_1–42. This indicates that the influx of calcium through the N-type Ca²⁺ channels was compromised by the chronic exposure of cells to a submicromolar concentration of A_1–42, presumably because of impairement of their function or diminished expression. This may be important in the pathogeny of Alzheimer's dementia in view of the pivotal role of N-type Ca²⁺ channels in neurotransmitter release.     

A selDABC cluster for selenocysteine incorporation in Eubacterium acidaminophilum

The four genes required for selenocysteine incorporation were isolated from the gram-positive, amino acid-fermenting anaerobe Eubacterium acidaminophilum, which expresses various selenoproteins of different functions. The sel genes were located in an unique organization on a continuous fragment of genomic DNA in the order selD1 (selenophosphate synthetase 1), selA (selenocysteine synthase), selB (selenocysteine-specific elongation factor), and selC (selenocysteine-specific tRNA). A second gene copy, encoding selenophosphate synthetase 2 (selD2), was present on a separate fragment of genomic DNA. SelD1 and SelD2 were only 62.9% identical, but the two encoding genes, selD1 and selD2, contained an in-frame UGA codon encoding selenocysteine, which corresponds to Cys-17 of Escherichia coli SelD. The function of selA, selB, and selC from E. acidaminophilum was investigated by complementation of the respective E. coli deletion mutant strains and determined as the benzyl viologen-dependent formate dehydrogenase activity in these strains after anaerobic growth in the presence of formate. selA and selC from E. acidaminophilum were functional and complemented the respective mutant strains to 83% (selA) and 57% (selC) compared to a wild-type strain harboring the same plasmid. Complementation of the E. coli selB mutant was only observed when both selB and selC from E. acidaminophilum were present. Under these conditions, the specific activity of formate dehydrogenase was 55% of that of the wild type. Transformation of this selB mutant with selB alone was not sufficient to restore formate dehydrogenase activity.     

Morphology-related effects on gene expression and protein accumulation of the yeast Arxula adeninivorans LS3

The dimorphism of the yeast Arxula adeninivorans LS3 is regulated by cultivation temperatures. Up to 42 degrees C the yeast grows as budding cells, which turn to mycelia at higher temperatures. To test whether the dimorphism is exclusively induced by high temperatures or also by other conditions, mutants were selected with an altered behaviour with respect to dimorphism. After mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, five of 25,000 colonies formed a very rough surface consisting of mycelia at 30 degrees C, in contrast to the wild-type. These mutants allow temperature-mediated and morphology-related effects on gene expression and protein accumulation to be distinguished. Budding cells and mycelia showed different expression of genes encoding secretory proteins at the same temperature. Mycelia secreted two-fold more protein than budding cells, including the enzymes glucoamylase and invertase. This indicated that morphology, rather than temperature, is the decisive factor in the analysed processes.     

Establishment of introduced antagonistic bacteria in the rhizosphere of transgenic potatoes and their effect on the bacterial community

In a field release experiment, rifampicin resistant mutants of two antagonistic plant-associated bacteria were used for seed tuber inoculation of transgenic T4 lysozyme expressing potatoes, transgenic control potatoes and non-transgenic parental potatoes. The T4 lysozyme tolerant Pseudomonas putida QC14-3-8 was originally isolated from the tuber surface (geocaulosphere) of T4 lysozyme producing plants and showed in vitro antibacterial activity to the bacterial pathogen Erwinia carotovora ssp. atroseptica. The T4 lysozyme sensitive Serratia grimesii L16-3-3 was originally isolated from the rhizosphere of parental potatoes and showed in vitro antagonism toward the plant pathogenic fungus Verticillium dahliae. The establishment of the inoculated bacteria in the rhizosphere and geocaulosphere of the different plant lines was monitored over one growing season to assess the effect of T4 lysozyme produced by transgenic potato plants on the survival of both inoculants. Both introduced isolates were able to colonize the rhizo- and geocaulosphere of transgenic plants and non-transgenic parental plants, and established in the rhizosphere at levels of ca. log(10) 5 colony forming units g(-1) fresh weight of root. During flowering of plants, significantly more colony counts of the T4 lysozyme tolerant P. putida were recovered from transgenic T4 lysozyme plants than from the transgenic control and the parental line. At this time, the highest level of T4 lysozyme (% of total soluble protein) was detected. Effects of the inoculants on the indigenous microbial community were monitored by analysis of PCR-amplified fragments of the 16S rRNA genes of the whole bacterial community after separation by denaturing gradient gel electrophoresis (DGGE). At any sampling time, the DGGE pattern of rhizosphere and geocaulosphere communities did not show differences between the inoculated and non-inoculated potatoes. Neither of the introduced strains became a dominant member of the bacterial community. This work was the first approach to assess the establishment of plant growth promoting rhizobacteria and potential biocontrol agents on transgenic plants.     

Sewage-fed aquaculture: The Calcutta model

Wastes have been rightly referred to as resources out of place. Since household wastewater often intermixes with effluents from industries and agricultural runoff, multidimensional approaches have been made towards maximizing protein production through rational exploitation of available resources. Sewage-fed aquaculture is a unique system and has manifold advantages in developing tropical countries acting as a major source of nutrients for crop farming and aquaculture, economical for sustainable production and helps to combat environmental pollution. The use of municipal wastewater fed to fertilize ponds began in Calcutta in the 1930s; the city now has perhaps the largest wastewater-fed aquaculture system in the world. A large number of people derive their livelihood from the sewage-fed aquaculture using the principles of systems ecology and applying it through ecological engineering. The subject of sewage-fed aquaculture is reviewed in terms of source, chemical nature, diversity pattern, recycling practices, production potential of aquaculture, environmental issues and safety measures for ecofriendly sustainable environmental management strategies.     

Interaction between embryogenic cultures of Scots pine and ectomycorrhizal fungi

 Embryogenic cell masses of three Scots pine (Pinus sylvestris) cell lines K779, K884 and K1009 were cultivated with the ectomycorrhizal (ECM) fungi Laccaria bicolor, L. proxima, Pisolithus tinctorius, Paxillus involutus and two strains of Suillus variegatus. The average growth ratio of the slowly proliferating cell line K1009 was improved by L. proxima and S. variegatus strain H, while of the rapidly proliferating lines K779 and K884 the non-mycorrhizal controls grew best. The fungi caused two distinct reactions in embryogenic cultures. In the positive reaction, the shape and light yellow colour of the cultures resembled the controls, while in the negative reaction the embryogenic cells became brown and necrotic and the fungi grew aggressively over them. These reactions to the fungi did not correlate completely with effects on the growth ratio. All the cell lines enhanced the radial growth of S. variegatus H and of P. tinctorius, while the Laccaria species and S. variegatus strain 1 thrived better alone. This study shows that early-stage embryogenic cells of Scots pine and ECM fungi are able to interact. As some fungi produced a positive reaction or even increased proliferation, they could be used to enhance somatic embryogenesis of Scots pine. Specific fungi might be used to induce the growth of slowly proliferating cell lines, and knowledge of positive cell line-fungus interactions could be useful in work with later stages of somatic embryogenesis, such as rooting. Accepted: 16 July 1998     

Cryopreservation of embryogenic cultures of Scots pine

The aim of the study was to develop an effective cryopreservation method for Scots pine (Pinus sylvestris L.) embryogenic cultures. Altogether nine cell lines derived from three mother trees were cryopreserved after cold hardening using dimethylsulfoxide or two different mixtures of polyethyleneglycol 6000, glucose and dimethylsulfoxide as cryoprotectants. Seventy-eight percent of the cell lines remained viable after cryostorage, the best cryoprotectant treatment being 10% polyethyleneglycol 6000, 10% glucose, and 10% dimethylsulfoxide in water. This treatment resulted in significantly better regrowth of the embryogenic cultures than with the other cryoprotectants or with the controls. According to microscopical observations, the cells that retained their viability and regrowth ability after cryopreservation were the embryonal head cells, as well as some elliptic suspensor cells close to the embryonal head cell area. When proliferation growth of the frozen cultures had started, their morphological appearance was the same as the non-frozen cultures. In addition, the RAPD assays suggested that the cryostorage treatment used here preserved the genetic fidelity of the Scots pine embryogenic cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Signal transduction in isolated fat body from the cockroach Blaptica dubia exposed to hypertrehalosaemic neuropeptide

Hypertrehalosaemic hormones stimulate trehalogenesis while inhibiting glycolysis in cockroach fat body. Signal transduction of the hypertrehalosaemic peptide Bld HrTH was examined in isolated fat body of the Argentine cockroach Blaptica dubia with respect to its effects on the increase in trehalose production and decrease in the content of the glycolytic activator fructose 2,6-bisphosphate in the tissue. Cyclic AMP does not seem to be involved in these processes as the cAMP analogue cpt-cAMP and the phosphodiesterase inhibitor IBMX, which both permeate cell membranes, had no effect on either parameter. Octopamine at physiological concentrations (10⁻⁷ mol · l⁻¹) was also ineffective, but at 10⁻⁵ mol · l⁻¹ or above, octopamine stimulated trehalose production although the content of fructose 2,6-bisphosphate in fat body was not affected. Both calcium entry and the release of Ca²⁺ from intracellular stores seem to be involved in the action of the hormone. If Ca²⁺ was omitted from the incubation medium, the hormone stimulated trehalose production less, though still significantly, whereas the hormone effect on fructose 2,6-bisphosphate was completely abolished in the absence of extracellular Ca²⁺. With Ca²⁺ present in the medium, the effect of the hormone on fructose 2,6-bisphosphate could be fully mimicked by the calcium ionophore A23187, suggesting that calcium entry is a␣decisive step in this signalling pathway. Trehalose production, on the other hand, was increased by thimerosal and thapsigargin which increase cytosolic Ca²⁺ from intracellular stores, whereas thimerosal in the absence of extracellular Ca²⁺ increased rather than decreased the content of fructose 2,6-bisphosphate, thus dissociating the two effects, which are normally coordinated by the hormone. Trehalose production and the content of fructose 2,6-bisphosphate were not significantly affected by mepacrine and mellitin, which are known to inhibit, respectively stimulate, phospholipase A₂. Our data suggest that the effects of Bld HrTH on the stimulation of trehalose production and reduction of fructose 2,6-bisphosphate content in fat body are mediated by Ca²⁺, but that different signalling pathways are involved, suggesting that the two processes, although they are functionally linked, could be regulated separately. Accepted: 10 November 1997     

The Inhibitory Effects of Anacardic Acid on Hepatitis C Virus Life Cycle

Hepatitis C virus (HCV) is a small positive-sense single-stranded RNA virus that causes severe liver diseases. Current anti-HCV therapies involving direct-acting antivirals have significantly enhanced efficacy in comparison to traditional interferon and ribavirin combination. However, further improvement is needed to eradicate HCV. Anacardic acid (AnA) is a phytochemical compound that can inhibit the activity of various cellular enzymes including histone acetyltransferases (HATs). In this study, we investigated the effects of AnA on different phases of HCV life cycle. Our data showed that AnA can inhibit HCV entry, replication, translation, and virion secretion in a dose-dependent manner with no measurable effects on cell viability. In addition, we showed that two HAT inhibitors and knocking down HAT (PCAF) by RNAi can reduce HCV replication, suggesting a mechanism of AnA’s inhibitory effects on HCV. Elucidation of the AnA-mediated inhibitory mechanism should facilitate the development of new drug candidates for HCV infection.     

Effects of an energy-dense diet and nicotinic acid supplementation on production and metabolic variables of primiparous or multiparous cows in periparturient period

It is well observed that feeding energy-dense diets in dairy cows during the dry period can cause metabolic imbalances after parturition. Especially dairy cows with high body condition score (BCS) and fed an energy-dense diet were prone to develop production diseases due to metabolic disturbances postpartum. An experiment was conducted to determine the effects of an energy-dense diet and nicotinic acid (NA) on production and metabolic variables of primiparous and multiparous cows in late pregnancy and early lactation which were not pre-selected for high BCS. Thirty-six multiparous and 20 primiparous German Holstein cows with equal body conditions were fed with energy-dense (60% concentrate/40% roughage mixture; HC group) or adequate (30% concentrate/70% roughage mixture; LC group) diets prepartum. After parturition, concentrate proportion was dropped to 30% for all HC and LC groups and was increased to 50% within 16 days for LC and within 24 days for HC cows. In addition, half of the cows per group received 24 g NA supplement per day and cow aimed to attenuate the lipid mobilisation postpartum. Feeding energy-dense diets to late-pregnant dairy cows elevated the dry matter (p < 0.001) and energy intake (p < 0.001) as well as the energy balance (p < 0.001) without affecting the BCS (p = 0.265) during this period. However, this did not result in any metabolic deviation postpartum as the effects of prepartum concentrate feeding were not carried over into postpartum period. Multiparous cows responded more profoundly to energy-dense feeding prepartum compared with primiparous cows, and parity-related differences in the transition from late pregnancy to lactation were obvious pre- and postpartum. The supplementation with 24 g NA did not reveal any effect on energy metabolism. This study clearly showed that energy-dense feeding prepartum did not result in metabolic imbalances postpartum in multiparous and primiparous cows not selected for high BCS. A genetic predisposition for an anabolic metabolic status as indicated by high BCS may be crucial for developing production diseases at the onset of lactation.