Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.     

DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Chloroplast ontogenesis inHelianthus annuus L.

Summary Research carried out into successively older sunflower leaves has allowed us to follow the different ontogenetic stages leading to the building up of a well-differentiated chloroplast. In all the intrathylakoid compartments of the young plastids there is very electron-dense material whose accumulation causes the dilatation of some thylakoid profiles and, consequently, the forming of roundish bodies. The thylakoid membranes appear as Lithtly Stained Membranes which, according to the most recent interpretation, are considered as incompletely organized ones. At a subsequent stage there is an increase of the thylakoid membrane staining along with a decrease of the electron-density of the intrathylakoid compartments that, at the end of the ontogenetic process, appear electrontransparent and flattened. Therefore, the intrathylakoid material appears to have been used up in the forming of well-organized membranes. This particular situation, present in the early ontogenetic stages of the plastids, may be attributed to an initial disequilibrium between the synthesis speed of membranal material and the capability to build up well-organized membranes.     

Biological insights in the pathogenesis of hypermobile Ehlers-Danlos syndrome from proteome profiling of patients' dermal myofibroblasts

Hypermobile Ehlers-Danlos syndrome (hEDS), mainly characterized by generalized joint hypermobility and its complications, minor skin changes, and apparently segregating with an autosomal dominant pattern, is still without a known molecular basis. Hence, its diagnosis is only clinical based on a strict set of criteria defined in the revised EDS nosology. Moreover, the hEDS phenotypic spectrum is wide-ranging and comprises multiple associated signs and symptoms shared with other heritable or acquired connective tissue disorders and chronic inflammatory diseases. In this complex scenario, we previously demonstrated that hEDS patients' skin fibroblasts show phenotypic features of myofibroblasts, widespread extracellular matrix (ECM) disarray, perturbation of ECM-cell contacts, and dysregulated expression of genes involved in connective tissue architecture and related to inflammatory and pain responses. Herein, the cellular proteome of 6 hEDS dermal myofibroblasts was compared to that of 12 control fibroblasts to deepen the knowledge on mechanisms involved in the disease pathogenesis. Qualitative and quantitative differences were assessed based on top-down and bottom-up approaches and some differentially expressed proteins were proofed by biochemical analyses. Proteomics disclosed the differential expression of proteins principally implicated in cytoskeleton organization, energy metabolism and redox balance, proteostasis, and intracellular trafficking. Our findings offer a comprehensive view of dysregulated protein networks and related pathways likely associated with the hEDS pathophysiology. The present results can be regarded as a starting point for future in-depth investigations aimed to decipher the functional impact of potential bioactive molecules for the development of targeted management and therapies.