Mosaicism in amniotic fluid cell cultures: Classification and significance

The last decade has witnessed increasing application of human cytogenetic technology to prenatal chromosome analysis. However, unlike the rather uniform peripheral blood T-lymphocyte system which has provided most of our experience in human cytogenetics, long-term amniotic-fluid cell cultures display extreme cellular heterogeneity and disproportionate growth of certain cell types as a consequence of clonal amplification. When they enter cell culture, many of these cells are approching the terminal stages of their respective life spans and may have accumulated chromosomal aberrations. Concern about the possibility of true fetal mosaicism seems warranted chiefly in situations were multiple colonies display potentially viable aberrations. Clonal analysis, preferable of multiple clonal types, and attention to details of clonal morphology are likely to minimize diagnostic errors and undue apprehension resulting from mosaicism in amniotic-fluid cell cultures.     

Muscle satellite (stem) cell activation during local tissue injury and repair

In post-mitotic tissues, damaged cells are not replaced by new cells and hence effective local tissue repair mechanisms are required. In skeletal muscle, which is a syncytium, additional nuclei are obtained from muscle satellite (stem) cells that multiply and then fuse with the damaged fibres. Although insulin-like growth factor-I (IGF-l) had been previously implicated, it is now clear that muscle expresses at least two splice variants of the IGF-I gene: a mechanosensitive, autocrine, growth factor (MGF) and one that is similar to the liver type (IGF-IEa). To investigate this activation mechanism, local damage was induced by stretch combined with electrical stimulation or injection of bupivacaine in the rat anterior tibialis muscle and the time course of regeneration followed morphologically. Satellite cell activation was studied by the distribution and levels of expression of M-cadherin (M-cad) and related to the expression of the two forms of IGF-I. It was found that the following local damage MGF expression preceded that of M-cad whereas IGF-IEa peaked later than M-cad. The evidence suggests therefore that an initial pulse of MGF expression following damage is what activates the satellite cells and that this is followed by the later expression of IGF-IEa to maintain protein synthesis to complete the repair.     

Age and sex influence on oxidative damage and functional status in human skeletal muscle

A reduction in muscle mass, with consequent decrease in strength and resistance, is commonly observed with advancing age. In this study we measured markers of oxidative damage to DNA, lipids and proteins, some antioxidant enzyme activities as well Ca²⁺ transport in sarcoplasmic reticulum membranes in muscle biopsies from vastus lateralis of young and elderly healthy subjects of both sexes in order to evaluate the presence of age- and sex- related differences. We found a significant increase in oxidation of DNA and lipids in the elderly group, more evident in males, and a reduction in catalase and glutathione transferase activities. The experiments on Ca²⁺ transport showed an abnormal functional response of aged muscle after exposure to caffeine, which increases the opening of Ca²⁺ channels, as well a reduced activity of the Ca²⁺ pump in elderly males. From these results we conclude that oxidative stress play an important role in muscle aging and that oxidative damage is much more evident in elderly males, suggesting a gender difference maybe related to hormonal factors.This revised version was published online in September 2005 with corrections to the Cover Date.     

Heterogeneous Subcellular Morphology of Lung Adenocarcinoma Cells: Identification of Different Cytotypes on Cytological Material

We performed an electron microscopic study of cytologic material from 20 cases of primary lung adenocarcinoma (pleural effusion, 13 cases; fine needle aspiration biopsy, 7 cases). Ultrastructural characteristics related to secretory and storing activity of adenocarcinoma cells were evaluated semi-quantitatively. Data analysis identified three basic cell types (secretory, well or poorly differentiated; storing; and indifferent). We could classify our cases in five groups of pure or mixed cytotypes. Our results demonstrated a subcellular morphological heterogeneity manifested by the presence of different basic cell types: secretory, storing or indifferent features (or both); and different secretory and storage products in the same cell. This heterogeneity of lung adenocarcinoma cells suggests that the neoplasm could arise from a single cell type capable of differentiating along different lines. Cases lacking secretory differentiation seemed to be characterized by more aggressive biologic behavior. A clear correlation between the ultrastructural cytotypes identified and the clinical and prognostic data on the patients was not observed. This may be due to the fact that 75% of the patients were in clinical stage III at the time of diagnosis; also, in this series only five cases did not have characteristics that indicated secretory activity.     

Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets

Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P(2) membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 microM SNP), and a peroxynitrite group (exposed to 100 microM peroxynitrite). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 microM bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198+/-25 nmol/mg protein/h in the normoxic group, 177+/-30 nmol/mg protein/h in the SNP group (p=NS versus normoxic) and 77+/-20 nmol/mg protein/h in the peroxynitrite group (p<0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 microm), a product of peroxynitrite, decreased the enzyme activity from 926+/-102 to 200+/-77 (p<0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain.     

Phosphorylated KDR is expressed in the neoplastic and stromal elements of human renal tumours and shuttles from cell membrane to nucleus

Vascular endothelial growth factor (VEGF)-A is an important angiogenic factor in establishing the vasculature in renal cell carcinomas (RCCs). Since little is known about VEGF signalling in RCCs, the profile of phosphorylated KDR (pKDR) has been investigated and the intracellular location of the receptor has been examined in the present study. Using two monoclonal antibodies raised against the phosphorylated KDR epitopes (Y1059 and Y1214) known to mediate different VEGF functions, together with a commercial anti-KDR antibody and immunohistochemistry, the expression of pKDR was investigated in a series of normal (n = 25) and neoplastic kidneys (n = 54; clear cell n = 35; papillary n = 10; oncocytomas n = 8). pKDR was present in many tissue elements of both normal and neoplastic renal tissues, with strong expression in the cell membrane, cytoplasm, and nuclei of normal kidney and tumour cells, as well as endothelial cells in tumours of all histological types. Patterns and intensity were similar using both anti-pKDR antibodies. There was no significant correlation in clear cell carcinomas between pKDR expression and age (p = 0.57), tumour size (p = 0.2), gender (p = 0.59), grade (p = 0.2) or histological type (p = 0.36). To delineate further the intracellular processing that might account for the cellular distribution, confocal microscopy was also performed. Antibodies to the different phosphorylated epitopes demonstrated different intracellular staining patterns. This study shows that pKDR is present in a wide variety of renal tumours, suggesting that anti-VEGF therapy might have direct effects on tumour cells. It further suggests that cells traffic pKDR depending on the precise KDR tyrosines that are autophosphorylated in a manner that enables receptor activation to result in different functions.     

Circulating stem cell factor in patients with chronic idiopathic urticaria.

BACKGROUND: The nature of histamine-releasing factors involved in the pathogenesis of chronic idiopathic urticaria (CIU) is still controversial, since functional IgG autoantibodies specific for the high-affinity IgE receptor, Fc(epsilon)RI, can be detected in only 20% of patients showing a strong skin reactivity on the autologous serum skin test. The absence of systemic eosinophilia in CIU patients, along with the increase in mast cells in skin biopsy specimens, suggests a possible role for stem cell factor (SCF), the only cytokine/growth factor known to induce mediator release from human mast cells. OBJECTIVE: To investigate the possible role of SCF as a histamine-releasing factor in patients with CIU. METHODS: The SCF levels were measured in serum samples from 65 patients with CIU who scored strongly positive on the autologous serum skin test; of these patients, 32 had negative results and 33 had positive results on in vitro histamine release assay by a quantitative commercial sandwich immunoassay technique. Serum samples from 40 healthy subjects were used as controls. RESULTS: Serum SCF levels in all 65 CIU patients did not differ from those found in healthy controls. No difference in SCF levels was found between patients with positive and negative results on histamine release assay. CONCLUSIONS: An increase in serum SCF levels does not play a pathogenic role in CIU.