Monoclonal antibodies in the analysis of fibronectin isoforms generated by alternative splicing of mRNA precursors in normal and transformed human cells

Recent results showing that a single fibronectin gene can give rise to several different mRNAs by alternative splicing have offered an explanation for fibronectin polymorphism. Here we report on monoclonal antibodies that show specificity for a fibronectin segment (ED) that can be included or omitted from the molecule depending on the pattern of splicing of the mRNA precursors. Using these monoclonals, we have quantitatively analyzed the expression of the ED sequence in human fibronectin from different sources. The results demonstrated that, at the protein level, the ED segment is not expressed in plasma fibronectin and that, in fibronectin from the tissue culture medium of tumor-derived or simian virus-40-transformed human cells, the percentage of fibronectin molecules containing the ED segment is about 10 times higher than in fibronectin from normal human fibroblasts. These results suggest that in malignant cells the mechanisms that regulate the splicing of mRNA precursors are altered.     

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.     

Structural differences in the cell binding region of human fibronectin molecules isolated from cultured normal and tumor-derived human cells

Fibronectins isolated from human plasma (pFN) and from the conditioned media of normal (N-cFN) and tumor (T-cFN) human cells were compared by cathepsin D digestion followed by immunostaining of released fragments with the monoclonal antibody 3E3, specific for the cell binding site. Two different staining patterns were obtained, one specific for pFN and N-cFN, the second common to fibronectins from the 3 different kinds of tumors studied. This indicates structural differences between N-cFN and T-cFN in the cell binding region of the fibronectin molecule.     

Proteoglycan modifications in cultured osteogenesis imperfecta skin fibroblasts

The PGs produced in the growth medium by skin fibroblast cultures from two O.I. affected patients were investigated. After density gradient centrifugation, in the most dense fraction two main families of molecules appeared. The patient with the more severe clinical picture showed a lower content of the PGs with the highest molecular weight. The GAG composition of PGs was different in the two patients. The more severely affected one showed an increase of HS and a decrease of ChS content, in agreement with the lower value of galactosamine to glucosamine ratio in urinary GAGs.     

Intravenous vs. oral rehydration: effects on subsequent exercise-heat stress

Castellani, John W., Carl M. Maresh, Lawrence E. Armstrong,Robert W. Kenefick, Deborah Riebe, Marcos Echegaray, Douglas Casa, andV. Daniel Castracane. Intravenous vs. oral rehydration: effects onsubsequent exercise-heat stress. J. Appl.Physiol. 82(3): 799-806, 1997.This studycompared the influence of intravenous vs. oral rehydration afterexercise-induced dehydration during a subsequent 90-min exercisebout. It was hypothesized that cardiovascular, thermoregulatory, and hormonal variables would be the same between intravenous and oral rehydration because of similar restoration ofplasma volume (PV) and osmolality (Osmo). Eight non-heat-acclimated menreceived three experimental treatments (counterbalanced design) immediately after exercise-induced dehydration (33°C) to 4%body weight loss. Treatments were intravenous 0.45% NaCl (iv; 25 ml/kg), no fluid (NF), and oral saline (Oral; 25 ml/kg).After rehydration and rest (2 h total), subjects walked at 50% maximalO₂ consumption for up to 90 min at36°C. The following observations were made: 1) heart rate was higher(P < 0.05) in Oral vs. ivat minutes 45, 60, and75 of exercise;2) rectal temperature, sweat rate, percent change in PV, and change in plasma Osmo were similar between ivand Oral; 3) change in plasmanorepinephrine decreased less (P < 0.05) in Oral compared with iv at minute45; 4) changes in plasma adrenocorticotropic hormone and cortisol were similar between ivand Oral after exercise was initiated; and5) exercise time was similar betweeniv (77.4 ± 5.4 min) and Oral (84.2 ± 2.3 min). These datasuggest that after exercise-induced dehydration, iv and Oral wereequally effective as rehydration treatments. Thermoregulation, changein adrenocorticotropic hormone, and change in cortisol were notdifferent between iv and Oral after exercise began; this is likely dueto similar percent change in PV and change in Osmo.

     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain

The product of rat gene 33 was identified as an ErbB-2-interacting protein in a two-hybrid screen employing the ErbB-2 juxtamembrane and kinase domains as bait. This interaction was reproduced in vitro with a glutathione S-transferase fusion protein spanning positions 282 to 395 of the 459-residue gene 33 protein. Activation of ErbB-2 catalytic function was required for ErbB-2-gene 33 physical interaction in living cells, whereas ErbB-2 autophosphorylation was dispensable. Expression of gene 33 protein was absent in growth-arrested NIH 3T3 fibroblasts but was induced within 60 to 90 min of serum stimulation or activation of the ErbB-2 kinase and decreased sharply upon entry into S phase. New differentiation factor stimulation of mitogen-deprived mammary epithelial cells also caused accumulation of gene 33 protein, which could be found in a complex with ErbB-2. Overexpression of gene 33 protein in mouse fibroblasts inhibited (i) cell proliferation driven by ErbB-2 but not by serum, (ii) cell transformation induced by ErbB-2 but not by Ras or Src, and (iii) sustained activation of ERK 1 and 2 by ErbB-2 but not by serum. The gene 33 protein may convey inhibitory signals downstream to ErbB-2 by virtue of its association with SH3-containing proteins, including GRB-2, which was found to associate with gene 33 protein in living cells. These data indicate that the gene 33 protein is a feedback inhibitor of ErbB-2 mitogenic function and a suppressor of ErbB-2 oncogenic activity. We propose that the gene 33 protein be renamed with the acronym RALT (receptor-associated late transducer).     

Integrating management techniques to restore subtropical forests invaded by Hedychium coronarium J. Köenig (Zingiberaceae) in a biodiversity hotspot

The restoration of areas invaded by non‐native plants is challenging as invasive plants may affect both biotic and abiotic components of ecosystems, leading to impacts that constrain recolonization by native species after invaders are eliminated. In such a scenario, restoration techniques as topsoil transposition might accelerate colonization by native species in forests. Hedychium coronarium J. Koenig (Zingiberaceae) is a Himalayan herbaceous rhizomatous plant recognized as invasive in several countries. This study aimed to experimentally evaluate the response of plant assemblages to topsoil transposition on a site invaded by H. coronarium after chemical control. Four treatments were applied: chemical control integrated with topsoil transposition, chemical control of H. coronarium alone, topsoil transposition alone, and no intervention (control). Plots were evaluated prior to the application of treatments and then monthly for 11 months after treatments. Parameters were measured for H. coronarium (number of ramets, ramet height, and cover) and other species (species richness, abundance, and cover). Plots treated with chemical control (regardless of topsoil transposition) were similar in terms of all parameters measured and species composition, with dominance of herbs and shrubs. Plots managed solely with topsoil transposition had lower species richness, abundance, and cover, but more diverse life‐forms, being equally rich in climbers, trees, and herbs. Chemical control was effective to control invasion by H. coronarium and increase species richness and abundance on the managed site. Topsoil transposition promoted colonization by species that might accelerate restoration.     

Environmental impacts of household goods in Europe: a process-based life cycle assessment model to assess consumption footprint

The International Journal of Life Cycle Assessment - Current patterns of household goods consumption generate relevant environmental pressures and impacts. Environmental impacts are not only...