Subunit III (Psa-F) of photosystem I reaction center of the C4 dicotyledon Flaveria trinervia

An immunological survey of C3, C4 and C3-C4-intermediate Flaveria species showed that subunit III (PsaF) of the photosystem I reaction center (PSI-RC) is present in all these species. This was confirmed by the isolation of the gene encoding the PSI-RC subunit III (PsaF) from Flaveria trinervia, the first psaF gene to be isolated from a C4 plant. The deduced amino acid sequence showed a high degree of similarity to the corresponding protein of spinach which is a C3 species. A region of 17 hydrophobic amino acids in the C-terminal part of the F. trinervia protein was found to be especially conserved in all PsaF proteins studied so far (cyanobacteria and Chlamydomonas).Abbreviations PSI-RC Photosystem I reaction center - cTPs chloroplast-targeted-proteins - chl chlorophyll - SDS sodium dodecyl sulfate     

Morphological differences between larvae and in vitro-cultured adults of Anisakis simplex (sensu stricto) and Anisakis pegreffii (Nematoda: Anisakidae)

Proper identification of Anisakis species infecting host fishes is very important to both human health and fish disease diagnosis. The foremost problem in the identification of Anisakis larvae in fishes is that L3 larvae cannot be easily differentiated morphologically, especially between A. simplex (sensu stricto) (s.s.) (Rudolphi, 1809) and A. pegreffii Campana-Rouget et Biocca, 1955. Instead, molecular means such as allozyme, mitochondrial DNA (mtDNA) cox2 region and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analyses had been successfully used. In this study, morphological differences of L3 larvae collected from fishes and in vitro-cultured L4 larvae and adult A. simplex (s.s.) and A. pegreffii were evaluated. Anisakis larvae were collected from 7 different host fishes within Japan. Undamaged A. simplex (s.s.) and A. pegreffii collected from Oncorhynchus keta (Walbaum) and Scomber japonicus Houttuyn, respectively, were used for in vitro-culture in order to obtain L4 and adult stages. Species identification was confirmed by PCR-RFLP analysis of the ITS region (ITS1-5.8S-ITS2) of ribosomal DNA and by mtDNA cox2 gene sequencing. Results revealed that L3, L4 and adult stages of A. simplex (s.s.) and A. pegreffii are morphologically distinguishable based on ventriculus length, wherein the former has longer ventriculus (0.90–1.50 mm) than the latter (0.50–0.78 mm). For oesophagus/ventriculus ratio, these two species are distinguishable only during L4 and adult stages. Also, adult male A. simplex (s.s.) and A. pegreffii were found to be distinguishable by differences in the distribution pattern of the caudal papillae, particularly the 3rd pair of distal papillae.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

The effects of ghrelin on the in vitro spontaneous and sGnRH-A stimulated luteinizing hormone (LH) release from the pituitary cells of common carp (Cyprinus carpio L.)

In fish, like in mammals, ghrelin affects gonadotropin release acting at the level of the hypothalamus as well as directly on the pituitary gland. In the present study, enzymatically dispersed pituitary cells obtained from sexually mature male and female carp (Cyprinus carpio L.) were incubated in the presence of human ghrelin at the concentration of 10^− 7 or 10^− 6 M, salmon GnRH analogue (Des-Gly¹⁰, D-Arg⁶, Trp⁷, Leu⁸, Pro⁹)-LHRH (sGnRH-A) at the concentration of 10^− 8 M or the combination of ghrelin (both concentrations) and sGnRH-A. ELISA method was used for carp LH levels determination in the media collected after 10 or 24 h of incubation. Ghrelin at the concentration of 10^− 6 M caused the increase of the spontaneous LH secretion from female pituitary cells only. The combination of ghrelin (both concentrations) with sGnRH-A resulted in the significant elevation of LH levels in the incubations of both male and female pituitary cells in comparison with control incubations as well as with sGnRH-A alone treated cells. The results obtained in this study show that ghrelin functions as LH-stimulating hormone in common carp and that it acts directly on gonadotrophic cells, potentiating also the action of GnRH.     

Transport of Poly(n-butylcyano-acrylate) nanoparticles across the blood-brain barrier in vitro and their influence on barrier integrity

In previous studies it was shown that polysorbate 80(PS80)-coated poly(n-butylcyano-acrylate) nanoparticles (PBCA-NP) are able to cross the blood–brain barrier (BBB) in vitro and in vivo. In order to explore and extend the potential applications of PBCA-NP as drug carriers, it is important to ascertain their effect on the BBB. The objective of the present study was to determine the effect of PS80-coated PBCA-NP on the BBB integrity of a porcine in vitro model. This has been investigated by monitoring the development of the transendothelial electrical resistance (TEER) after the addition of PBCA-NP employing impedance spectroscopy. Additionally, the integrity of the BBB in vitro was verified by measuring the passage of the reference substances ¹⁴C-sucrose and FITC-BSA after addition of PBCA-NP. In this study we will show that the application of PS80-coated PBCA-NP leads to a reversible disruption of the barrier after 4 h. The observed disruption of the barrier could also be confirmed by ¹⁴C-sucrose and FITC-BSA permeability studies. Comparing the TEER and permeability studies the lowest resistances and maximal values for permeabilities were both observed after 4 h. These results indicate that PS80-coated PBCA-NP might be suitable for the use as drug carriers. The reversible disruption also offers the possibility to use these particles as specific opener of the BBB. Instead of incorporating the therapeutic agents into the NP, the drugs may cross the BBB after being applied simultaneously with the PBCA-NP.     

Schistosoma mansoni: Schistosomicidal effect of mefloquine and primaquine in vitro

We investigated the effects of the anti-malarials mefloquine and primaquine against the juvenile and adult life stages of Schistosoma mansoniin vitro. Cercariae were incubated with 0.5 μg/ml, 1 μg/ml and 2 μg/ml mefloquine or primaquine and with 1 μg/ml praziquantel for 12 h. Schistosomula, pre-adults and adults were incubated with 0.5 μg/ml, 1 μg/ml and 2 μg/ml mefloquine or primaquine and with 1 μg/ml praziquantel for 7 days. The viability status was classified as viable, damaged or dead and was checked every 3 h for cercariae and every 12 h for schistosomula, pre-adults and adults. Both, mefloquine and primaquine show time and dose-dependent schistosomicidal effects on the four life stages of S. mansoni. The promising in vitro effects on all stages of the blood fluke S. mansoni warrants further evaluation of both anti-malarials and their derivatives for their prophylactic and therapeutic values in early and late schistosomiasis in field trials.     

Comparative Development and Performance of Artificially Reared versus Host-RearedDiapetimorpha introita(Cresson) (Hymenoptera: Ichneumonidae) Wasps

Diapetimorpha introita(Cresson) (Hymenoptera: Ichneumonidae), a native ectoparasitoid ofSpodopteraspp. pupae, was reared in the laboratory on an artificial diet devoid of any insect host components. Diet-reared wasps demonstrated a propensity to search for and parasitize natural hosts in a field cage trial. Longevity of the diet-reared wasps was comparable with the longevity of wasps reared on host pupae. Survival rate ofD. introitawas 61.3% when reared on diet and 76.3% when reared on host pupae. Wasps reared on the artificial diet had longer developmental times, reduced fecundity, and reduced adult weights compared to wasps reared on host pupae. These studies suggest that future research efforts should focus on increasing fecundity and weight of diet-reared wasps and decreasing the mortality and developmental time of wasps reared on the artificial diet. The ability to rearD. introitaon an inexpensive, artificial diet significantly enhances the potential of mass rearing this parasitoid for inundative releases againstSpodopteraspp.     

Differential effects of muscle fibre length and insulin on muscle-specific mRNA content in isolated mature muscle fibres during long-term culture

The aims of this study were (1) to determine the relationship between muscle fibre cross-sectional area and cytoplasmic density of myonuclei in high- and low-oxidative Xenopus muscle fibres and (2) to test whether insulin and long-term high fibre length caused an increase in the number of myonuclei and in the expression of α-skeletal actin and of myogenic regulatory factors (myogenin and MyoD) in these muscle fibres. In high- and low-oxidative muscle fibres from freshly frozen iliofibularis muscles, the number of myonuclei per millimetre fibre length was proportional to muscle fibre cross-sectional area. The in vivo myonuclear density thus seemed to be strictly regulated, suggesting that the induction of hypertrophy required the activation of satellite cells. The effects of muscle fibre length and insulin on myonuclear density and myonuclear mRNA content were investigated on high-oxidative single muscle fibres cultured for 4–5 days. Muscle fibres were kept at a low length (~15% below passive slack length) in culture medium with a high insulin concentration (~6 nmol/l: “high insulin medium”) or without insulin, and at a high length (~5% above passive slack length) in high insulin medium. High fibre length and high insulin medium did not change the myonuclear density of isolated muscle fibres during culture. High insulin increased the myonuclear α-skeletal actin mRNA content, whereas fibre length had no effect on α-skeletal actin mRNA content. After culture at high fibre length in high insulin medium, the myonuclear myogenin mRNA content was 2.5-fold higher than that of fibres cultured at low length in high insulin medium or in medium without insulin. Myonuclear MyoD mRNA content was not affected by fibre length or insulin. These in vitro experiments indicate that high muscle fibre length and insulin enhance muscle gene expression but that other critical factors are required to induce adaptation of muscle fibre size and performance.This work was partially supported by a research grant from the Haak Bastiaanse Kuneman Stichting.     

A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)n block of the frataxin gene

We analyzed HeLa nuclear proteins binding to the (gaa)_n harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)₉ form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)_n block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)_n fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)₁₂₁ binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.     

Preliminary assessment of somatic cell nuclear transfer in the dromedary (Camelus dromedarius)

Somatic cloning may enable the maintenance/expansion of the population of camels with the highest potential for milk production or the best racing performances. However, there have been no reports of embryonic or somatic nuclear transfer in camels. The aim of this study was to produce dromedary embryos by nuclear transfer using in vitro matured oocytes and two somatic cells from two sources (adult fibroblasts or granulosa cells). A total of 58 adult females were superstimulated by a single dose of eCG (3500 IU). Ten days later, their ovaries were collected postmortem. Cumulus–oocytes-complexes (COCs) were aspirated from stimulated follicles and were matured in vitro for 30 h. Fibroblasts (from live adult male) and granulosa cells (from slaughtered adult females) were used as donor karyoplasts and injected into mature enucleated dromedary oocytes.The cleavage rate was significantly higher (P < 0.05) for embryos reconstructed with fibroblasts (59%) versus those with granulosa cells (45%). However, there was no difference between the two groups in the proportion of cloned embryos reaching the blastocyst stage (fibroblasts: 14% vs. granulosa cells: 15%) or those that hatched (fibroblasts: 10% vs. granulosa cells: 12%). The viability of reconstructed dromedary embryos from the two sources of donor cells (fibroblasts; n = 5 vs. granulosa cells; n = 7) was examined by transferring them to synchronized recipients. Two females (fibroblasts: 1/5; 20%, granulosa cells: 1/7; 14%) were confirmed pregnant by ultrasonography at 15 and 25 days following transfer. Later, the pregnancies were followed by pregnancy empirical-symptoms. These two pregnancies were lost between 25 and 60 days following transfer, respectively.In conclusion, the present study shows for the first time that the development of dromedary NT embryos derived from either adult fibroblasts or granulosa cells can occur in vitro and the transfer of these cloned embryos to recipients can result in pregnancies.     

温度对荔枝异形小卷蛾发育和繁殖的影响

荔枝异形小卷蛾的蛀梢为害是珍贵树种格木人工林健康发展的主要限制因子。依据其天然分布和潜在推广区的温度范围设置系列温度梯度,探讨温度对荔枝异形小卷蛾发育和繁殖的综合影响。结果显示,温度对荔枝异形小卷蛾各阶段的发育历期具有显著影响,在研究温度范围内,发育历期随温度升高呈显著地下降趋势,世代历期在18℃时为66.87 d,30℃时降至35.77 d。预蛹和蛹的存活率对温度的响应不敏感,而卵、幼虫、成虫的存活率和世代存活率以及产卵量均随温度升高表现为先上升后下降的变化趋势,且繁殖力对温度的反映较存活率敏感,其存活率和繁殖力在18℃时均最低,分别为41.20%和13.90粒,在27℃时发育最适,分别为83.80%和45.40粒,在30℃时虽有下降,分别为66.00%和32.40粒,但仍高于18℃时,即其对低温较高温敏感。荔枝异形小卷蛾完整世代发育起点温度为5.77℃,所需有效积温为876.76 d℃,其中幼虫发育所需有效积温最高,占整个历期的45.23%,发育速率与温度显著正相关。根据荔枝异形小卷蛾为害方式、发育和繁殖特征分析可知,在低温地区其幼虫期长,但世代数少、存活率和繁殖力低,对寄主植物受害部位的单次为害程度严重;在高温地区则幼虫期短,但世代数多、存活率和繁殖力高,对寄主植物受害部位的单次为害程度低,但更为频繁,持续为害程度高。研究结果对于不同地区选择荔枝异形小卷蛾的防治具有借鉴意义,同时有助于指导格木人工林的合理推广和健康发展。     

Synthesis and intracellular transport of proteins in the exocrine pancreas of the frog (Rana esculenta)

Summary Frog pancreatic tissue was pulse-labelled in vitro with ³H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions.Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae.Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues.The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30° C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.     

Agrin binds to the N-terminal region of Lrp4 protein and stimulates association between Lrp4 and the first immunoglobulin-like domain in muscle-specific kinase (MuSK)

Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four β-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.     

The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm inArabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm inA. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.Abbreviations DIC differential interference contrast optics - MT microtubule - PPB preprophase band of microtubule We thank Ms. Margaret Travers for her helpful English translation of Yakovlev and Alimova (1976) and Mr. James Whitehead for preparation of Fig. 11. M.C.W. was supported by an Australian Postgraduate Research Award.     

Micropropagation and genetic stability of a Dendrobium hybrid (Orchidaceae)

Summary Dendrobium hybrids have great economic importance in a number of countries. Asymbiotic seed germination and the conventional vegetative method have been commonly used by growers to propagate these plants. To overcome somaclonal variation, which is commonly exhibited by Dendrobium (Nobile group) when micropropagated from protocorm-like bodies, a protocol for propagating Dendrobium Second Love in vitro using axillary buds in the presence of thidiazuron was developed. Random amplified polymorphic DNA analysis was also carried out to check for possible genetic alterations in plants originating from six consecutive subcultures. The results revealed that the established protocol was efficient for the in vitro cloning of this orchid hybrid and the plants obtained from the six subcultures did not exhibit any type of polymorphism.     

Cryopreservation of in vitro-cultured multiple bud clusters of asparagus (Asparagus officinalis L. cv Hiroshimagreen (2n=30) by the techniques of vitrification

Summary A culture line of asparagus forming green bulbous structures consisting of numerous multiple bud clusters designated bud clusters was induced from a meristem culture of asparagus (Asparagus officinalis L.cv. Hiroshimagreen, 2n=30). Small cubic segments (2 mm3) cut from bud clusters were cryopreserved using three different cryogenic protocols. Only vitrification produced very high levels of shoot formation after cooling to –196°C. Segments were treated with a vitrification solution (PVS2) at 25°C for 45 min or at 0°C for 120 min prior to a direct plunge into liquid nitrogen. After rapid warming, the segments were expelled into Murashige and Skoog medium containing 1.2 M sucrose for 10 min and then plated on agar shoot outgrowth medium. The average rate of shoot formation of vitrified segments producing normal shoots was near 90% without any preculture and/or cold-acclimation treatment. Revived segments resumed growth within 3 days and developed about three shoots per segment. In vitro-cultured bud clusters appear promising as material for cryopreserving asparagus germplasm.Abbreviations DMSO dimethyl sulfoxide - PVS 2-vitrification solution - LN liquid nitrogen - IBA 3-indolbutyric acid - BA 6-benzylaminopurine - FDA fluorescein diacetate - DSC differential scanning calorimeter