Lipid composition and synthesis of HaCaT cells, an immortalized human keratinocyte line, in comparison with normal human adult keratinocytes

Abstract Cultured keratinocytes are frequently employed for studies of epidermal lipid metabolism. Interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime and variations between passages, problems thai are not encountered to the same extent with immortalized cell lines. The present study was undertaken to compare the lipid composition and synthesis of normal human adult keratinocytes (NHAK) with HaCaT cells, a long-lived. spontaneously immortalized human keratinocyte line, in relation to proliferation and differentiation. No differences between the two cell types were observed: a) in total lipid content; b) in the distribution of major lipid classes during growth at 50%, 75% and 100% confluence: c) in cultures grown at 0.6 mM calcium, at which differentiation is retarded, or at 1.6 mM calcium, at which some differentiation takes place; d) in the incorporation of [¹⁴C] acetate into cellular lipids at confluence, or e) in the fatty acid composition of major cellular lipid classes. At 100% confluence NHAK and HaCaT cells differ in their cholesterol metabolism. At all stages of growth, cholesterol synthesis in HaCaT cells is more LDL-dependent than in NHAK. Furthermore, NHAK become less LDL-dependent at confluence whereas HaCaT cells do not. HaCaT cells also revealed a significantly larger fraction of phosphatidyl-ethanolamine, -serine and -inositol at 0.6 mM calcium concentration than NHAK. These findings suggest that HaCaT cells do not differentiate as well as NHAK in vitro and may therefore serve as a model for the study of lipid metabolism in cells defective in terminal differentiation.     

Bovine seminal ribonuclease is cytotoxic for both malignant and normal telomerase-positive cells

Bovine seminal-ribonuclease (BS-RNase) is a member of the 'ribonucleases with special biological actions' family since it possesses specific anti-tumour, anti-spermatogenic and embryotoxic activities and exerts an immunosuppressive effect on T lymphocytes. In previous studies it was demonstrated that BS-RNase induced apoptosis in proliferating, malignant and normal cells and that telomerase activity loss also caused apoptotic death in neoplastic cells. Since an obvious relationship between cell proliferation and telomerase activity exists, the aim of this work was to study if the pro-apoptotic cytotoxic action exerted by BS-RNase on proliferating malignant cells (HT29) and proliferating normal cells (PHA-stimulated lymphocytes) could be linked to a possible BS-RNase effect on telomerase activity. In BS-RNase-treated HT29 cells (Na-butyrate-differentiated or not) and human lymphocytes (proliferating or not), we investigated cell vitality (MTT method) and morphology (SEM), BS-RNase localization (immunofluorescence), telomerase activity (TRAP-ELISA method), hTR mRNA expression (RT-PCR), and hTERT levels (western blot). While no BS-RNase effect was detectable on not proliferating cells, a clear relationship was noticed between the diminished number of vital elements of both proliferating cell populations after treatment (48 h and 72 h for HT29 and PHA-stimulated lymphocytes, respectively) with 50 microg/ml BS-RNase and the decrease of their telomerase activity. At the same time, we found that hTR levels, the RNA subunit of telomerase, in proliferation-inhibited BS-RNase-treated cells were diminished. Moreover, by immunofluorescence technique, we detected BS-RNase in the HT29 cell nucleolus after 3-h treatment. Therefore, as hTR has been recently proven to co-fractionate with nucleoli, we hypothesize that a BS-RNase direct action on the telomerase hTR subunit could be a possible mechanism of action by which BS-RNase exerts its pro-apoptotic effects only on proliferating cells.     

When should we discontinue antiarrhythmic therapy for atrial fibrillation after coronary artery bypass grafting? A prospective randomized study

BACKGROUND: New-onset atrial fibrillation after coronary artery bypass grafting is common. Medical therapy includes various antiarrhythmic drugs to control heart rate and restore sinus rhythm. The purpose of this study was to determine the duration of antiarrhythmic therapy after discharge from the hospital. METHODS: One hundred twenty-nine patients in whom new atrial fibrillation after coronary artery bypass grafting developed and successfully reverted to sinus rhythm were prospectively randomized at dismissal to receive antiarrhythmic therapy for 1 week (group A; n = 44), 3 weeks (group B; n = 42), or 6 weeks (group C; n = 43). Patients were followed up for an additional 4 weeks after discontinuation of antiarrhythmic therapy for detection of recurrent atrial fibrillation. RESULTS: The incidence of new atrial fibrillation during the study period was 21.2% (256/1206). Among the 129 patients who consented to the study, conversion to sinus rhythm was accomplished with the following medications: amiodarone (group A, 82%; group B, 93%; group C, 88%; P = .29), digoxin (group A, 16%; group B, 7%; group C, 7%; P = .29), beta-blockers (group A, 27%; group B, 19%; group C, 14%; P = .30), calcium channel blockers (group A, 2%; group B, 2%; group C, 0%; P = .60), quinidine (group A, 2%; group B, 2%; group C, 7%; P = .44), and procainamide (group A, 4.5%; group B, 2%; group C, 0%; P = .37). Follow-up was completed in 128 patients (99.2%). There was no significant difference in the recurrence of atrial fibrillation among groups (0%, 2%, and 0% for groups A, B, and C, respectively). CONCLUSIONS: Patients with new atrial fibrillation after coronary artery bypass grafting, converted to normal sinus rhythm before hospital discharge, have a benign course. Antiarrhythmic therapy as short as 1 week may be appropriate in these patients.     

Patients with antinuclear antibody-positive juvenile idiopathic arthritis constitute a homogeneous subgroup irrespective of the course of joint disease

OBJECTIVE: We recently hypothesized that in the International League of Associations for Rheumatology (ILAR) classification of juvenile idiopathic arthritis (JIA), the presumably homogeneous patient group characterized by early onset of disease, a female predilection, the presence of antinuclear antibodies (ANA), asymmetric arthritis, and the risk for iridocyclitis is classified into different categories. We sought to investigate whether ANA-positive patients belonging to the ILAR categories of oligoarthritis and rheumatoid factor (RF)-negative polyarthritis share homogeneous features and to compare these features with those of ANA-negative patients with JIA in the same categories. METHODS: We identified patients who were followed up during a 15-year period. All patients had JIA according to the ILAR criteria, with oligoarticular or polyarticular onset. ANA positivity was defined as 2 or more positive results at a titer of >or=1:160. Demographic and clinical features, including the number of joints involved over time and measures of JIA severity at the last followup visit, were recorded retrospectively. RESULTS: A total of 256 patients were included: 190 were ANA positive (109 had persistent oligoarthritis, 48 had extended oligoarthritis, and 33 had RF-negative polyarthritis), and 66 were ANA negative (35 had RF-negative polyarthritis, and 31 had oligoarthritis). All patients who were positive for ANA were similar in terms of age at disease presentation, female-to-male ratio, and frequency of symmetric arthritis and iridocyclitis. Compared with ANA-positive patients with polyarticular disease, ANA-negative patients with polyarticular arthritis were older at disease presentation and had a lower frequency of iridocyclitis, a higher frequency of symmetric arthritis, a greater cumulative number of joints affected over time, and a different pattern of joint disease, with a greater frequency of shoulder and hip involvement. The strong relationship between the presence of ANA and younger age at disease presentation, asymmetric arthritis, and development of iridocyclitis was confirmed by multivariate regression analysis. CONCLUSION: Our results support the hypothesis that patients with similar characteristics are currently classified into different JIA categories. The value of ANA positivity as a possible modifier of the current classification system deserves consideration.     

Distribution of inhaled mercury (203Hg) in various organs

Mercury and its derivatives have long been known to be toxic for the brain and other organs in both animals and man. The distribution of inhaled radioactive mercury (203Hg) in body tissues of rats and mice was investigated by means of a micro-autoradiographic technique. Animals were exposed to 203Hg vapours 6 h daily for 10 days, and then sacrificed at different times after the last exposure. Whole-body autoradiograms showed significant uptake of labelled mercury by the kidney, brain, myocardium, intestine and liver, in decreasing order. Micro-autoradiography demonstrated selective localization of 203Hg in the cytoplasm and processes of neurons, whereas little radioactivity was found in the glial cells of the gray and white matter. High levels of 203Hg were detected in nuclei of the cerebellum, midbrain, pons and medulla, in the Purkinje cells of the cerebellar cortex, and in the epithelium of the ependyma and choroid plexus. In the lung, radioactivity appeared to be confined to the erythrocytes of small blood vessels, which may be the carriers of this metal to the brain. In the liver, ingested but not inhaled radioactivity was concentrated in the reticulo-endothelium. In the kidney, proximal and distal convoluted tubules, but not the medulla or the glomeruli, took up large amounts of inhaled 203Hg. Mercury is distributed to many organs in addition to the brain. It may be transported by circulating red blood cells and it concentrates in the cytoplasm of parenchymal cells.