The Localization of Bullous Pemphigoid Antigen 180 (BP180) in Hemidesmosomes Is Mediated by Its Cytoplasmic Domain and Seems to be Regulated by the β4 Integrin Subunit

Bullous pemphigoid antigen 180 (BP180) is a component of hemidesmosomes, i.e., cell-substrate adhesion complexes. To determine the function of specific sequences of BP180 to its incorporation in hemidesmosomes, we have transfected 804G cells with cDNA-constructs encoding wild-type and deletion mutant forms of human BP180. The results show that the cytoplasmic domain of BP180 contains sufficient information for the recruitment of the protein into hemidesmosomes because removal of the extracellular and transmembrane domains does not abolish targeting. Expression of chimeric proteins, which consist of the membrane targeting sequence of K-Ras fused to the cytoplasmic domain of BP180 with increasing internal deletions or lacking the NH₂ terminus, indicates that the localization of BP180 in hemidesmosomes is mediated by a segment that spans 265 amino acids. This segment comprises two important regions located within the central part and at the NH₂ terminus of the cytoplasmic domain of BP180.

To investigate the effect of the α6β4 integrin on the subcellular distribution of BP180, we have transfected COS-7 cells, which lack α6β4 and BP180, with cDNAs for BP180 as well as for human α6A and β4. We provide evidence that a mutant form of BP180 lacking the collagenous extracellular domain as well as a chimeric protein, which contains the entire cytoplasmic domain of BP180, are colocalized with α6β4. In contrast, when cells were transfected with cDNAs for α6A and mutant forms of β4, either lacking the cytoplasmic COOH-terminal half or carrying phenylalanine substitutions in the tyrosine activation motif of the cytoplasmic domain, the recombinant BP180 molecules were mostly not colocalized with α6β4, but remained diffusely distributed at the cell surface. Moreover, in cells transfected with cDNAs for α6A and a β4/β1 chimera, in which the cytoplasmic domain of β4 was replaced by that of the β1 integrin subunit, BP180 was not colocalized with the α6β4/β1 chimera in focal adhesions, but remained again diffusely distributed. These results indicate that sequences within the cytoplasmic domain of β4 determine the subcellular distribution of BP180.

     

Subnuclear localization of S/MAR-binding proteins is differently affected by in vitro stabilization with heat or Cu2+

The nuclear matrix, a proteinaceous entity thought to be a scaffolding structure that determines the higher order organization of eukaryotic chromatin, is usually prepared from intact nuclei by a series of extraction steps. In most cell types investigated, the nuclear matrix does not spontaneously resist these extractions, but must rather be stabilized before the application of extracting agents such as high salt solutions or lithium diiodosalicylate. We have examined the effect of two widely used stabilization procedures on the localization of nuclear matrix proteins. Four individual polypeptides were studied, all of which are scaffold or matrix-associated region (S/MAR)-binding proteins: SATB1, SAF-A/hnRNP-U, NuMA , and topoisomerase II α. Nuclei were isolated from K562 human erythroleukemia cells in a buffer containing spermine, spermidine, KCl and EDTA, and the nuclear matrix or scaffold was obtained by extraction with lithium diiodosalicylate after stabilization by heat treatment (37° or 42°C) or incubation with Cu²⁺ ions. When the localization of individual proteins was determined by immunofluorescent staining and confocal scanning laser microscopy, markedly different consequences of the two stabilization strategies became evident, ranging from a total maintenance of the localization (NuMA and topoisomerase II α) to a marked redistribution (SATB1 and SAF-A/hnRNP-U). Our results seem to indicate that a reevaluation of stabilization protocols employed for the preparation of the nuclear matrix is desirable, especially by performing morphological controls. Received: 22 January 1997; in revised form: 17 February 1997 / Accepted: 21 February 1997     

Modifications of proteoglycans extracted from monolayer cultures of young and senescent human skin fibroblasts

Proteoglycans (PGs) were extracted from culture monolayers of human skin fibroblasts (HFs) at early and late passages. Total PGs from senescent cells had markedly reduced abilities to bind type I collagen and hyaluronic acid, but retained normal binding properties with fibronectin and laminin. The constituent polysaccharides of PGs were comparatively characterised. PGs recovered from young and senescent HF cultures had equivalent total polyanionic charges and similar size distributions of the glycosaminoglycan chains. This applied to both types of polysaccharide chains found in PGs, namely the galactosaminoglycuronans (GalN-GAGs) and the glucosaminoglycuronans (GlcN-GAGs). However, senescent HFs produced a greater proportion of PGs containing GlcN-GAG chains and increased the sulphation of the remainding PG fraction with GalN-GAG moieties, yielding a major gain of C6-sulphate groups in the galactosamine residues.     

Increased Secretion of the Amino-Terminal Fragment of Amyloid Precursor Protein in Brains of Rats with a Constitutive Up-Regulation of Protein Kinase C

Abstract: Protein kinase C (PKC) activation stimulates release of secreted amyloid precursor protein (APP_s) in several cell lines. To ascertain the role of PKC in regulating APP metabolism in vivo, we used an animal model (methylazoxymethanol-treated rats; MAM rats) in which PKC is permanently hyperactivated in selected brain areas, i.e., cortex and hippocampus. A significant decrease in membrane-bound APP concentration was found in synaptosomes derived from cortex and hippocampus of MAM rats, where PKC is up-regulated, with a concomitant increase in APP_s production in soluble fractions of the same brain areas. In contrast, in a brain area not affected by MAM treatment (i.e., cerebellum), APP secretion is similar in control and MAM rats, indicating that altered metabolism of APP is restricted to only those areas in which the PKC system is up-regulated. In addition, phorbol esters or H-7 modulate APP_s release in hippocampal slices from both control and MAM rats, further supporting an in vivo role for this enzyme in regulating metabolism of mature APP.     

Preliminary results in HIV-1-infected patients treated with transfer factor (TF) and Zidovudine (ZDV)

The efficiency of HIV-1 specific transfer factor (TF) administration, combined with Zidovudine (ZDV), in asymptomatic persistent generalised lymphadenopaty, or AIDS related complex (ARC) patients was evaluated. Twenty patients were randomly assigned to receive only ZDV (1st group) or ZDV together with HIV-1-specific TF (2nd group). HIV-1-specific TF was administered orally at 2 × 10⁷ cell equivalent daily for 15 days, and thereafter once a week for up to 6 months. There were no significant differences between the two groups in clinical evolution, red blood cells, haemoglobin, lymphocytes, CD20 subset, transaminases,β-2-microglobulin, p24 antigen. White blood cells, CD8 lymphocytes as well as IL-2 levels increased in the second group, while the CD4 subset increased in the first group. The combination treatment with ZDV and TF appeared to be safe and well tolerated. Furthermore, levels of serum cytokines were investigated in 10 patients (8 asymptomatic and 2 ARC) treated with ZDV, and compared with 5 patients of the 2nd group (3 asymptomatic and 2 ARC) treated with ZDV plus HIV-1-specific TF. Peripheral lymphocytes, CD4, CD8 subsets, IL-2, TNFα, IL-6, p24 antigen, IL-2 soluble lymphocyte receptors (sR), CD4sR, CD8sR and ß-2-microglobulin were evaluated at the baseline and at the 3rd month. The CD4 subset was not significantly different in the two groups, whilst IL-2 increased in the 2nd group receiving ZDV plus TF, suggesting an activation of the Th1 secretion pattern.     

Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy

 DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations. Accepted: 5 September 1996     

Water- and nutrient-use efficiency of a deciduous species, Vaccinium myrtillus, and an evergreen species, V. vitis-idaea, in a subalpine dwarf shrub heath in the southern Alps, Italy

Periodic measurements of gas‐exchange rates and determinations of foliar N and P concentrations were used for evaluating instantaneous water‐use efficiency and photosynthetic nutrient‐use efficiency in two co‐existing dwarf shrubs of different growth form (V. myrtillus, deciduous, and V. vitis‐idaea, evergreen) in a subalpine heath in the southern Alps of Italy. Those data were compared with cumulative assessments of water‐use efficiency and photosynthetic nutrient‐use efficiency obtained by measuring leaf carbon isotope discrimination in leaf tissues and by estimating nutrient resorption from senescing leaves. V. myrtillus presented higher dry‐weight based rates of net photosynthesis (A_weight) compared to V. vitis‐idaea. A_weight was positively correlated with foliar‐nutrient status and intercellular‐to‐ambient gradient in CO₂ concentrations. A_weight was, furthermore, negatively correlated with leaf specific mass. Instantaneous photosynthetic nutrient‐use efficiency did not differ between the two species but the percentages of N and P pools resorbed from senescing leaves were somewhat higher in the deciduous species. The evergreen species showed lower P concentrations in senescing leaves which indicated a higher proficiency in resorbing phosphorus compared to the deciduous species. In addition, the evergreen species achieved a higher carbon gain per unit foliar N and P, due to a longer mean residence time of both nutrients. The two species did not differ from each other with respect to both instantaneous and long‐term water‐use efficiency. This was consistent with the climatic pattern, showing no sign of water deficiency through the growing season. Current‐year V. vitis‐idaea leaves had a significantly higher Δ¹³C compared to previous‐year leaves, possibly mirroring a long term acclimation of evergreen leaves, as far as they age, to the habitat conditions in the understory where evergreen species are usually confined within mixed dwarf‐shrub communities.     

Photosynthetic performance of cold-sensitive mutants of maize at low temperature

The virescent character is a genetic variant in pigmentation characterized by a delay in greening. Seedlings of the virescent mutants v1, v2, v3, v4, v13, v16, v18, v19 and v26 of maize exhibit chlorosis when grown at low temperature. Chlorotic leaves contain plastids that appear to have been arrested at an early stage of development. The results indicated that V16, V2, V3 and V4 loci control early stages of chloroplast development while V1, V13 and V19 may play a role at the end of development. The mutations in the V18 and V26 loci may control an intermediate step. At the pigment level, the virescent mutants of maize differ widely from analogous mutations existing in other plants. The mutations were characterized by a reduced amount of chlorophyll a and b (up to 100 times in v16) and chlorophyll a/b ratio above normal (up to 13.7 in v16). Lutein content was reduced in all mutants (less than 3% in v16 compared to wild type) but v13, while pigments of the xanthophyll cycle were found at higher levels in v1 and v13 (more than 10 and 90%, respectively). The v2, v3, v4, v16 and v18 mutants that are most depleted in β-carotene (36 times less in average than wild type) are also deprived in D1 and D2 polypeptides. Moreover, the v2, v3, v4, v16 and v18 mutants characterized by a lower accumulation in lutein are most depleted of light-harvesting complex II. All mutants possess a functioning, fully reversible, non-photochemical quenching mechanism. This is most developed in the v13 and v19 mutants (φ_n = 0.48 and 0.44, respectively). These two mutants also have a relatively high primary photochemical yield for photosystem II and a functioning photosystem I (φ_p = 0.23 and 0.39, respectively). The most interesting mutant is v13 that shows severe chlorosis and possesses the most effective non-photochemical quenching mechanism(s), which is thought to provide protection against excess photon absorption by photosystem II.     

Nitrogen concentration and δ15N signature of ombrotrophic Sphagnum mosses at different N deposition levels in Europe

Alteration of the global nitrogen (N) cycle because of human‐enhanced N fixation is a major concern particularly for those ecosystems that are nutrient poor by nature. Because Sphagnum‐dominated mires are exclusively fed by wet and dry atmospheric deposition, they are assumed to be very sensitive to increased atmospheric N input. We assessed the consequences of increased atmospheric N deposition on total N concentration, N retention ability, and δ¹⁵N isotopic signature of Sphagnum plants collected in 16 ombrotrophic mires across 11 European countries. The mires spanned a gradient of atmospheric N deposition from about 0.1 up to about 2 g m^?2 yr^?1. Mean N concentration in Sphagnum capitula was about 6 mg g^?1 in less polluted mires and about 13 mg g^?1 in highly N‐polluted mires. The relative difference in N concentration between capitulum and stem decreased with increasing atmospheric N deposition, suggesting a possible metabolic mechanism that reduces excessive N accumulation in the capitulum. Sphagnum plants showed lower rates of N absorption under increasing atmospheric N deposition, indicating N saturation in Sphagnum tissues. The latter probably is related to a shift from N‐limited conditions to limitation by other nutrients. The capacity of the Sphagnum layer to filter atmospheric N deposition decreased exponentially along the depositional gradient resulting in enrichment of the mire pore water with inorganic N forms (i.e., NO₃^?+NH₄⁺). Sphagnum plants had δ¹⁵N signatures ranging from about ?8‰ to about ?3‰. The isotopic signatures were rather related to the ratio of reduced to oxidized N forms in atmospheric deposition than to total amount of atmospheric N deposition, indicating that δ¹⁵N signature of Sphagnum plants can be used as an integrated measure of δ¹⁵N signature of atmospheric precipitation. Indeed, mires located in areas characterized by greater emissions of NH₃ (i.e., mainly affected by agricultural activities) had Sphagnum plants with a lower δ¹⁵N signature compared with mires located in areas dominated by NO_x emissions (i.e., mainly affected by industrial activities).