Molecular cloning, cDNA structure and deduced amino acid sequence for a type I regulatory subunit of cAMP-dependent protein kinase from human testis

A 1.5 kilobase (kb) cDNA clone containing the entire coding region for a regulatory subunit of type I cAMP-dependent protein kinase (RI) was isolated from a human testis cDNA library. The cDNA clone encodes a protein of 381 amino acids that shows 98% and 97% homology to the bovine skeletal muscle RI and rat brain RI, respectively. Northern blot analysis demonstrates two major mRNA-species (1.5 and 3.0 kb) in human testis and one mRNA-species (3.0 kb) in human T-lymphocytes.     

Characterization of eight barley xantha-f mutants deficient in magnesium chelatase.

Magnesium chelatase (EC 6.6.1.1) catalyses the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway. The enzyme is composed of three different subunits of approximately 40, 70 and 140 kDa. In barley (Hordeum vulgare L.) the subunits are encoded by the genes Xantha-h, Xantha-g and Xantha-f. In the 1950s, eight induced xantha-f mutants were isolated. In this work we characterized these mutations at the DNA level and provided explanations for their phenotypes. The xantha-f10 mutation is a 3 bp deletion, resulting in a polypeptide lacking the glutamate residue at position 424. The leaky mutation xantha-f26 has a missense mutation leading to a M632R exchange. The xantha-f27 and -f40 are deletions of 14 and 2 bp, respectively, resulting in truncated polypeptides of 1104 and 899 amino acid residues, respectively. Mutation xantha-f41 is an in-frame deletion that removes A439, L440, Q441 and V442 from the resulting protein. Mutation xantha-f58 is most likely a deletion of the whole Xantha-f gene, as no DNA fragments could be detected by PCR or southern blot experiments. The slightly leaky xantha-f60 and non-leaky -f68 mutations each have a missense mutation causing a P393L and G794E exchange in the polypeptide, respectively.     

Aging bone marrow mesenchymal stromal cells have altered membrane glycerophospholipid composition and functionality

Human mesenchymal stem/stromal cells (hMSC) are increasingly used in advanced cellular therapies. The clinical use of hMSCs demands sequential cell expansions. As it is well established that membrane glycerophospholipids (GPL) provide precursors for signaling lipids that modulate cellular functions, we studied the effect of the donor''s age and cell doublings on the GPL profile of human bone marrow MSC (hBMSC). The hBMSCs, which were harvested from five young and five old adults, showed clear compositional changes during expansion seen at the level of lipid classes, lipid species, and acyl chains. The ratio of phosphatidylinositol to phosphatidylserine increased toward the late-passage samples. Furthermore, 20:4n-6-containing species of phosphatidylcholine and phosphatidylethanolamine accumulated while the species containing monounsaturated fatty acids (FA) decreased during passaging. Additionally, in the total FA pool of the cells, 20:4n-6 increased, which happened at the expense of n-3 polyunsaturated FAs, especially 22:6n-3. The GPL and FA correlated with the decreased immunosuppressive capacity of hBMSCs during expansion. Our observations were further supported by alterations in the gene expression levels of several enzymes involved in lipid metabolism and immunomodulation. The results show that extensive expansion of hBMSCs harmfully modulates membrane GPLs, affecting lipid signaling and eventually impairing functionality.     

Gall midge olfaction: Pheromone sensitive olfactory neurons in Contarinia nasturtii and Mayetiola destructor

This study describes the morphology and function of the antennal sensilla in two gall midge species, Contarinia nasturtii and Mayetiola destructor, where multi-component sex pheromones have been identified. Both species possess sensilla trichodea, s. coeloconica, s. chaetica and s. circumfila. Sensilla circumfila, which consist of several sensilla that bifurcate and fuse into one structure, are unique for the gall midges. In C. nasturtii s. circumfila are sexually dimorphic. In males, they form elongated loops suspended on cuticular spines, whereas in females they run like worm-like structures directly on the antennal surface. Single sensillum recordings demonstrated that olfactory sensory neurons housed in male s. circumfila in C. nasturtii responded to the female sex pheromone. In M. destructor, s. circumfila were attached to the antennal surface in both sexes, and displayed no response to sex pheromone components.A sexual dimorphism was also found in the number of s. trichodea per antennal segment in both C. nasturtii (male 1 vs. female 7) and M. destructor (male 13 vs. female 10). OSNs located in male M. destructor s. trichodea responded to the sex pheromone. This is the first gall midge single sensillum study, and the first demonstration of the functional significance of s. circumfila.     

Pen-2 is sequestered in the endoplasmic reticulum and subjected to ubiquitylation and proteasome-mediated degradation in the absence of presenilin

The gamma-secretase complex catalyzes intramembrane proteolysis of a number of transmembrane proteins, including amyloid precursor protein, Notch, ErbB4, and E-cadherin. gamma-Secretase is known to contain four major protein constituents: presenilin (PS), nicastrin, Aph-1, and Pen-2, all of which are integral membrane proteins. There is increasing evidence that the formation of the complex and the stability of the individual components are tightly controlled in the cell, assuring correct composition of functional complexes. In this report, we investigate the topology, localization, and mechanism for destabilization of Pen-2 in relation to PS function. We show that PS1 regulates the subcellular localization of Pen-2: in the absence of PS, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. PS deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2, which adopts a hairpin structure with the N and C termini facing the luminal space, is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation-proteasome pathway.     

Molecular evidence of a reed warbler × great reed warbler hybrid ( Acrocephalus scirpaceus × A. arundinaceus) in Belgium

We report on the second case of a reed warbler × great reed warbler hybrid (Acrocephalus scirpaceus and A. arundinaceus). The bird was captured during a standardised ringing session in Belgium in autumn 1999, and fell between the parental species in all measurements. Molecular analyses of two microsatellite loci verified that the bird was a female that had a reed warbler father and a great reed warbler mother.     

Response of zooplankton to nutrient enrichment and fish in shallow lakes: a pan-European mesocosm experiment

1. Responses of zooplankton to nutrient enrichment and fish predation were studied in 1998 and 1999 by carrying out parallel mesocosm experiments in six lakes across Europe. 2. Zooplankton community structure, biomass and responses to nutrient and fish manipulation showed geographical and year‐to‐year differences. Fish had a greater influence than nutrients in regulating zooplankton biomass and especially the relative abundances of different functional groups of zooplankton. When fish reduced the biomass of large crustaceans, there was a complementary increase in the biomasses of smaller crustacean species and rotifers. 3. High abundance of submerged macrophytes provided refuge for zooplankton against fish predation but this refuge effect differed notably in magnitude among sites. 4. Large crustacean grazers (Daphnia, Diaphanosoma, Sida and Simocephalus) were crucial in controlling algal biomass, while smaller crustacean grazers and rotifers were of minor importance. Large grazers were able to control phytoplankton biomass even under hypereutrophic conditions (up to 1600 μg TP L^?1) when grazer biomass was high (>80–90 μg dry mass L^?1) or accounted for >30% of the grazer community. 5. The littoral zooplankton community was less resistant to change following nutrient enrichment in southern Spain, at high temperatures (close to 30 °C), than at lower temperatures (17–23 °C) characterising the other sites. This lower resistance was because of a greater importance of nutrients than zooplankton in controlling algal biomass. 6. Apart from the reduced role of large crustacean grazers at the lowest latitude, no consistent geographical patterns were observed in the responses of zooplankton communities to nutrient and fish manipulation.     

Effects of nutrients and fish on periphyton and plant biomass across a European latitudinal gradient

Replicated, factorial mesocosm experiments were conducted across Europe to study the effects of nutrient enrichment and fish density on macrophytes and on periphyton chlorophyll a (chl-a) with regard to latitude. Periphyton chl-a densities and plant decline were significantly related to nutrient loading in all countries. Fish effects were significant in a few sites only, mostly because of their contribution to the nutrient pool. A saturation-response type curve in periphyton chl-a with nutrients was found, and northern lakes achieved higher densities than southern lakes. Nutrient concentration and phytoplankton chl-a necessary for a 50% plant reduction followed a latitudinal gradient. Total phosphorus values for 50% plant disappearance were similar from Sweden (0.27 mg L⁻¹) to northern Spain (0.35 mg L⁻¹), but with a sharp increase in southern Spain (0.9 mg L⁻¹). Planktonic chl-a values for 50% plant reduction increased monotonically from Sweden (30 μg L⁻¹) to València (150 μg L⁻¹). Longer plant growing-season, higher light intensities and temperature, and strong water-level fluctuations characteristic of southern latitudes can lead to greater persistence of macrophyte biomass at higher turbidities and nutrient concentration than in northern lakes. Results support the evidence that latitudinal differences in the functioning of shallow lakes should be considered in lake management and conservation policies.     

Gene order and recombination rate in homologous chromosome regions of the chicken and a passerine bird

Genome structure has been found to be highly conserved between distantly related birds and recent data for a limited part of the genome suggest that this is true also for the gene order (synteny) within chromosomes. Here, we confirm that synteny is maintained for large chromosomal regions in chicken and a passerine bird, the great reed warbler Acrocephalus arundinaceus, with few rearrangements, but in contrast show that the recombination-based linkage map distances differ substantially between these species. We assigned a chromosomal location based on sequence similarity to the chicken genome sequence to a set of microsatellite loci mapped in a pedigree of great reed warblers. We detected homologous loci on 14 different chromosomes corresponding to chicken chromosomes Gga1-5, 7-9, 13, 19, 20, 24, 25, and Z. It is known that 2 passerine macrochromosomes correspond to the chicken chromosome Gga1. Homology of 2 different great reed warbler linkage groups (LG13 and LG5) to Gga1 allowed us to locate the split to a position between 20.8 and 84.8 Mb on Gga1. Data from the 5 chromosomal regions (on Gga1, 2, 3, 5, and Z) with 3 or more homologous loci showed that synteny was conserved with the exception of 2 large previously unreported inversions on Gga1/LG5 and Gga2/LG3, respectively. Recombination data from the 9 chromosomal regions in which we identified 2 or more homologous loci (accounting for the inversions) showed that the linkage map distances in great reed warblers were only 6.3% and 13.3% of those in chickens for males and females, respectively. This is likely to reflect the true interspecific difference in recombination rate because our markers were not located in potentially low-recombining regions: several linkage groups covered a substantial part of their corresponding chicken chromosomes and were not restricted to centromeres. We conclude that recombination rates may differ strongly between bird species with highly conserved genome structure and synteny and that the chicken linkage map may not be suitable, in terms of genetic distances, as a model for all bird species.     

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.