Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.     

Light-harvesting chlorophyll a-b complex requirement for regulation of Photosystem II photochemistry by non-photochemical quenching

Recently, it has been suggested (Horton et al. 1992) that aggregation of the light-harvesting a-b complex (LHC II) in vitro reflects the processes which occur in vivo during fluorescence induction and related to the major non-photochemical quenching (qE). Therefore the requirement of this chlorophyll a-b containing protein complex to produce qN was investigated by comparison of two barley mutants either lacking (chlorina f2) or depressed (chlorina¹⁰⁴) in LHC II to the wild-type and pea leaves submitted to intermittent light (IL) and during their greening in continuous light. It was observed that qN was photoinduced in the absence of LHC II, i.e. in IL grown pea leaves and the barley mutants. Nevertheless, in these leaves qN had no (IL, peas) or little (barley mutants) inhibitory effect on the photochemical efficiency of Q_A reduction measured by flash dosage response curves of the chlorophyll fluorescence yield increase induced by a single turn-over flash During greening in continuous light of IL pea leaves, an inhibitory effect on Q_A photoreduction associated to qN developed as Photosystem II antenna size increased with LHC II synthesis. Utilizing data from the literature on connectivity between PS II units versus antenna size, the following hypothesis is put forward to explain the results summarized above. qN can occur in the core antenna or Reaction Center of a fraction of PS II units and these units will not exhibit variable fluorescence. Other PS II units are quenched indirectly through PS II-PS II exciton transfer which develops as the proportion of connected PS II units increases through LHC II synthesis.     

Herbivory on calcicolous lichens: different food preferences and growth rates in two co-existing land snails

A total of 32 calcicolous lichen species, one alga and one bryophyte were recorded on a limestone wall in the grassland Great Alvar on the Baltic island of Öland, Sweden. Fourteen (41%) of these 34 species and free-living cyanobacteria showed herbivore damage, most probably due to grazing by the land snails Chondrina clienta and Balea perversa which inhabited the limestone wall. Three laboratory experiments were conducted to examine the food preferences of individuals of C. clienta and B. perversa collected at this site and to evaluate any association between their preference and the net food quality of the lichens to the snails. Chondrina clienta and B. perversa exhibited food preferences, which differed significantly between species. Within species, variation in food choice was similar among individuals. This indicates that snail populations may be composed of polyphagous individuals with similar food preferences. Different lichen species were of different net food quality to the snails as indicated by growth rate differences. In both snail species the most preferred lichen species of the choice experiment caused the largest weight increase in juveniles, viz. Caloplaca flavovirescens for C. clienta and Aspicilia calcarea for B. perversa. This suggest that the snail species studied differ in their abilities to deal with secondary compounds and physical characteristics of certain lichens or that they can utilize the energy and nutrients of these lichens to a different extent. It is suggested that differential food preferences might reduce the intensity of interspecific competition for resources (lichens) between C. clienta and B. perversa.     

Skewed inactivation of an X chromosome deleted at the dystrophin gene in an asymptomatic mother and her affected daughter

A girl with severe Becker muscular dystrophy and apparently normal chromosomes had a heterozygous deletion for exons 51, 52, and 53 of the dystrophin gene. This deletion was transmitted by her mother, who was unaffected. To differentiate the normal and the deleted X chromosomes, fluorescence in situ hybridization (FISH) was applied to metaphase chromosomes, using probes for both exons 51 and 52, which are only 388 and 113 base pairs long, respectively. FISH signals were observed in one or both chromatids of one chromosome, but never on both chromosomes, suggesting the lack of hybridization on the deleted X chromosome. Using 5-bromodeoxyuridine incorporation to differentiate the late (inactive) and the early replicating (active) X chromosomes, 77% of the signals were observed on the active X chromosomes in the mother. This percentage was only 18% in the daughter, suggesting that skewed inactivation of the X chromosomes was responsible for the phenotypic differences.     

Induction of an immune network cascade in cancer patients treated with monoclonal antibodies (ab1)

The antitumor effector functions of unconjugated monoclonal antibodies (mAb) in cancer therapy are not fully understood. Direct cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent cytolysis and apoptosis have been suggested. Induction of anti-idiotypic (ab₂) and anti-anti-idiotypic (ab₃) antibodies as well as the corresponding T cells (T₂ and T₃) has also been proposed to be of therapeutic significance. In this study induction of an immune network cascade in ten patients with colorectal carcinoma, treated with mAb 17-1A (ab₁) was assessed. After treatment, all ten patients had anti-idiotypic antibodies and anti-anti-idiotypic antibodies with ab₁-like binding specificity while only five of ten patients had T cells corresponding to ab₃ (T₃) as assessed by a proliferation assay (DNA synthesis), and an assay of interferon production (ELISPOT) (Enzyme-linked immuno SPOT) in vitro or by a delayed-type hypersensitivity reaction in vivo. Purified T cells from four of the five patients with a positive T₃ test responded with DNA synthesis after stimulation using human anti-mAb 17-1A anti-idiotypic monoclonal antibodies. These four patients had a clinical response showing a tumor reduction after therapy, while all six patients lacking a proliferative response failed to show tumor regression. Induction of a cell-mediated immune network cascade might accordingly be an important anti-tumor effector function of mAb and should be considered in the future design of mAb-based therapy protocols in cancer patients.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

The significance of partial migration for food web and ecosystem dynamics

Migration is ubiquitous and can strongly shape food webs and ecosystems. Less familiar, however, is that the majority of life cycle, seasonal and diel migrations in nature are partial migrations: only a fraction of the population migrates while the other individuals remain in their resident ecosystem. Here, we demonstrate different impacts of partial migration rendering it fundamental to our understanding of the significance of migration for food web and ecosystem dynamics. First, partial migration affects the spatiotemporal distribution of individuals and the food web and ecosystem-level processes they drive differently than expected under full migration. Second, whether an individual migrates or not is regularly correlated with morphological, physiological, and/or behavioural traits that shape its food-web and ecosystem-level impacts. Third, food web and ecosystem dynamics can drive the fraction of the population migrating, enabling the potential for feedbacks between the causes and consequences of migration within and across ecosystems. These impacts, individually and in combination, can yield unintuitive effects of migration and drive the dynamics, diversity and functions of ecosystems. By presenting the first full integration of partial migration and trophic (meta-)community and (meta-)ecosystem ecology, we provide a roadmap for studying how migration affects and is affected by ecosystem dynamics in a changing world.     

The genome of the non-cultured,bacterial-like organism associated with citrus greening disease contains thenusG-rplKAJL-rpoBC gene cluster and the gene for a bacteriophage type DNA polymerase

We have recently cloned three DNA fragments (In-2.6, In-1.0, and In-0.6) of the noncultured, bacterial-like organism (BLO) associated with citrus greening disease. Nucleotide sequence determination has shown that fragment In-2.6 is part of therplKAJL-rpoBC gene cluster, a well-known operon in eubacteria. The DNA fragment upstream of and partially overlapping with In-2.6 could be isolated and was shown to be thenusG gene. InEscherichia coli, nusG is also immediately upstream ofrplKAJL-rpoBC. Fragment In-1.0 carries the gene for a bacteriophage type DNA polymerase. Fragment In-0.6 could not be identified.When In-2.6 was used, at high stringency, as a probe to detect greening BLO strains in infected plants, hybridization was obtained with all Asian strains tested, but not with the African strain examined. At lower stringencies, In-2.6 was able to detect also the African strain. The implications of these reults in the taxonomical position of the greening BLO are discussed.