New polymorphisms and markers in the HLA class I region: relevance to hereditary hemochromatosis (HFE)

Hereditary hemochromatosis (HFE) is an inherited disorder whose gene lies in the proximity of the histocompatability antigen (HLA) class I region, on 6p21.3. Despite efforts in refining the HFE region, a number of informative DNA markers, linked to the disease locus and amenable to use in an assay based on the polymerase chain reaction (PCR) is available. The gene content of this region is high, and the HFE gene has not so far been identified. We have used a strategy based on PCR protocols potentially able to detect both polymorphisms and expressed sequences. This approach has been applied to a 700-kb stretch (approximately) of DNA corresponding to the insert of a Centre d'Etude du Polymorphisme Humain yeast artificial chromosome (225 B1) of the possible candidate region. Five new polymorphisms have been detected among 20 specific fragments isolated. Four of them are tightly linked to the HFE locus. Because of the strong linkage disequilibrium with the disease demonstrated by these markers, they could represent starting points for the identification and characterization of the HFE gene. The remaining non-polymorphic fragments, being amplifiable and in most cases linked to NotI sites, may be useful starting points for the generation of a genomic contig of band 6p21.3 and for gene identification.     

Germination and pH of intracellular compartments in seeds of Phacelia tanacetifolia

³¹ P nuclear magnetic resonance spectroscope (NMR) was used to study the response of Phacelia tanacetifolia seeds to dark and light conditions during the first 72 h of incubation. Changes in the chemical shifts (δ) of the pH-dependent ³¹P-NMR signals from the vacuolar and the cytoplasmic orthophosphate pools were correlated with the different incubation conditions. In the dark (favorable to germination), the cytoplasmic pH remained nearly constant over the whole period considered, while the vacuolar pH shitted to more acidic values after the 24th h of incubation. In the light (inhibiting germination), the values of cytoplasmic pH tended to become more acidic than in the dark after the 24th h of incubation, while the vacuolar pH remained practically constant. When seed germination was inhibited in the dark by butyric acid (BA). a permeant weak acid, the values of cytoplasmic and vacuolar pH were similar to those of the ungerminated seeds incubated in the light. When, vice versa, seed germination was promoted in the light by fusicoccin (FC), the values of cytoplasmic and vacuolar pH were similar to those of the dark-germinated seeds. A progressive augmentation of P, metabolism occurred both in the dark and in the light up to the 24th h of incubation. Subsequently, light blocked any further evolution of this parameter. Treatment with butyric acid in the dark again mimicked the effect of light, while FC reversed the negative effect of light. The data show that in Phacelia tanacetifolia seeds germination is linked to a more alkaline cytoplasmic pH. The finding that the light-dependent metabolic inhibition occurs after an early activation of metabolism, i.e. after the first 24 h. suggests that the effects of light on the cytoplasmic and vacuolar pH depend on the early metabolic processes involved in the control of the homeostasis of cell pH and/or on the inhibition of the reactivation of the transport mechanisms.     

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