Effects of TNF-alpha/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes.

Tumour necrosis factor alpha (TNF-alpha) kills Daudi cells (Human Burkitt Lymphoma), inducing either necrosis or apoptosis without DNA fragmentation. Therefore, we were interested in studying the molecular and ultrastructural events occurring when the nucleus is more accessible and cells are blocked in mitosis, following colchicine treatment. In fact, as early as after 1 h treatment a typical ladder pattern was shown by means of DNA gel electrophoresis. In parallel the quantitative analysis of the different morphological patterns observed gave evidence of an increased percentage of primary necrosis after 6 h treatment, and a higher incidence of cells in late apoptosis as well as in secondary necrosis after 24 h treatment. Our findings show that Daudi cells respond to the combined treatment with an increased formation of micronuclei and nuclear alterations which follow a number of early mitochondrial changes and result in enhanced cell death. These data imply that TNF-alpha-induced apoptosis of Daudi cells can be triggered by mitochondrial changes and is somehow related to microtubule organization.

Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry.

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.

Immunocytochemical detection of phosphatidylinositol 3 kinase in Burkitt lymphoma cells.

PI 3-kinase, an enzyme responsible for the phosphorylation of the D3 position of the inositol ring of phosphatidylinositol (PI), is recognized to be involved in the regulation of many cellular processes such as mitogenic signalling, inhibition of apoptosis, intracellular vesicle trafficking/secretion, regulation of actin and integrin functions and regulation of protein kinases induced by tumour necrosis factor, oncoproteins and ultraviolet light. Here we report the subcellular distribution and the phosphorylative pattern of p85 alpha subunit of PI 3-kinase in Burkitt lymphoma cells exposed to R interferon alpha treatment. Immunocytochemical analysis of this enzyme, performed by confocal microscopy, revealed an increased expression of this protein at cytoplasmic level after 90 min of interferon alpha treatment. Western blotting analyses performed on nuclear and cytoplasmic fractions confirmed the overexpression found by confocal microscopy at cytoplasmic level in the 90 min interferon alpha treated cells still persisting in the 24 hr treated samples. Such an overexpression was paralleled by an increase of tyrosine phosphorylation both at cytoplasmic and nuclear level suggesting that an enhanced requirement for cytoplasmic expression and phosphorylation of PI 3-kinase might be necessary to the cell for regulating some cytoplasmic-nuclear cross talk involved in the control of Burkitt lymphoma cell metabolism following interferon alpha treatment.

Peptidergic innervation of mesenteric lymphatics in guinea pigs: an immunocytochemical and pharmacological study.

By immunocytochemistry, substance P immunoreactive (SP-IR) and vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibers were examined in guinea pig mesenteric lymph collectors. The immunoreactive nerve fibers, located in the adventitia of lymphatics, were few and were irregularly distributed along the vessel wall. These fibers appeared to be more numerous and more evenly distributed along the corresponding artery and vein walls within the same area. SP immunoreactivity in the vascular nerves was depleted in guinea pigs injected with capsaicin but was unaffected by the injection of 6-hydroxydopamine. By contrast, VIP-IR nerve fibers were unaffected by both treatments. It is concluded that SP-IR nerve fibers in the lymphatics are likely to be of sensory origin and that VIP containing nerves in the lymph collectors are distinct from SP-containing and noradrenergic nerves. It is also suggested that lymph collectors possess a complex although limited innervation pattern not only of autonomic nerve fibers containing classic neurotransmitters but also of peptidergic nerve fibers of a different origin with a vasomotor and/or sensory action.