Stress symposium in Johannesburg: plants versus humans

On 18 February 1998, a ‘Stress symposium’ was held at the Rand Afrikaans University (RAU) in Johannesburg, South Africa. The meeting brought together people from both the plant and the human oxidative stress field, which was exemplified by a talk entitled ‘Heat shock proteins in host-pathogen interactions: plants versus humans’. There were moments when it appeared as if the main difference between plants and humans was, as sung by Julos Beaucarne, that ‘the human plant is the only one to be able to water itself…’     

Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest

Centrosomes organize the microtubule cytoskeleton for both interphase and mitotic functions. They are implicated in cell-cycle progression but the mechanism is unknown. Here, we show that depletion of 14 out of 15 centrosome proteins arrests human diploid cells in G1 with reduced Cdk2-cyclin A activity and that expression of a centrosome-disrupting dominant-negative construct gives similar results. Cell-cycle arrest is always accompanied by defects in centrosome structure and function (for example, duplication and primary cilia assembly). The arrest occurs from within G1, excluding contributions from mitosis and cytokinesis. The arrest requires p38, p53 and p21, and is preceded by p38-dependent activation and centrosomal recruitment of p53. p53-deficient cells fail to arrest, leading to centrosome and spindle dysfunction and aneuploidy. We propose that loss of centrosome integrity activates a checkpoint that inhibits G1-S progression. This model satisfies the definition of a checkpoint in having three elements: a perturbation that is sensed, a transducer (p53) and a receiver (p21).     

The spindle assembly checkpoint is satisfied in the absence of interkinetochore tension during mitosis with unreplicated genomes

The accuracy of chromosome segregation is enhanced by the spindle assembly checkpoint (SAC). The SAC is thought to monitor two distinct events: attachment of kinetochores to microtubules and the stretch of the centromere between the sister kinetochores that arises only when the chromosome becomes properly bioriented. We examined human cells undergoing mitosis with unreplicated genomes (MUG). Kinetochores in these cells are not paired, which implies that the centromere cannot be stretched; however, cells progress through mitosis. A SAC is present during MUG as cells arrest in response to nocodazole, taxol, or monastrol treatments. Mad2 is recruited to unattached MUG kinetochores and released upon their attachment. In contrast, BubR1 remains on attached kinetochores and exhibits a level of phosphorylation consistent with the inability of MUG spindles to establish normal levels of centromere tension. Thus, kinetochore attachment to microtubules is sufficient to satisfy the SAC even in the absence of interkinetochore tension.     

Hyperthermia assists survival of astrocytes from oxidative-mediated necrotic cell death.

In response to many stresses and pathologic states, including different models of nervous system injury, cells synthesize a variety of proteins, most notably the inducible 72 kDa heat shock protein 70 (Hsp70), which plays important roles in maintaining cellular integrity and viability. We report here that cultured astrocytes from rat diencephalon express high levels of Hsp70 upon exposure to elevated temperatures, and are less vulnerable to a subsequent oxidative stress. Complex oxidative stress was induced by exposure of astrocytes to an aqueous extract of tobacco smoke. This resulted in both glutathione and ATP depletion, along with cell death that proceeded through a necrotic pathway. Pretreatment of cultures with the glutathione replenishing agent, N-acetyl-L-cysteine, prevented glutathione and ATP loss as well as necrotic cell death. Thermal stress also protected astrocytes from necrotic cell death but without affecting glutathione or ATP levels. We propose that heat shock protects astrocytes from necrosis induced by oxidative stress, probably as a result of Hsp70 synthesis, through an antioxidant-ATP independent mechanism. As Hsp70 may transfer from glial to neuronal cells, its synthesis by astrocytes may represent an important survival mechanism by which astrocytes protect neurons against oxidative-mediated cell death.     

Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

Effects of LTB4 and Ca++ ionophore A23187 on the release by human alveolar macrophages of factors controlling fibroblast functions

Cultured human alveolar macrophages from smokers with lung cancer produced spontaneously variable amounts of factors stimulating fibroblast proliferation and production of prostaglandin E2 and collagenase by fibroblasts. These biological activities belong to molecules similar or identical to interleukin 1. Exogenous leukotriene B4 added to alveolar macrophage cultures increased the production of these factors. The Ca++ ionophore A23187 was found to have similar effects. By the control of monokine production, leukotriene B4 locally released by inflammatory cells may modulate lung fibroblast functions.     

In vitro activity of N-benzenesulfonylbenzotriazole on Trypanosoma cruzi epimastigote and trypomastigote forms

Chagas disease is still an important health problem in Central and South America. However, the only drugs currently available for specific treatment of this disease may induce toxic side effects in the host. The aim of this work was to determine the activity of N-benzenesulfonylbenzotriazole (BSBZT) against the protozoan parasite Trypanosoma cruzi. The effects of BSBZT and benzotriazole (BZT) were compared to those of benznidazole (BZL) on epimastigote and trypomastigote forms. BSBZT was found to have an in vitro growth inhibitory dose-dependent activity against epimastigotes, with flow cytometry analysis confirming that the treated parasites presented size reduction. BSBZT showed an IC(50) of 21.56 μg/mL (81.07 μM) against epimastigotes at 72 h of incubation, whereas BZT did not affect the growth of this parasite form. Furthermore, the toxic effect of BSBZT, was stronger and appeared earlier (at 24h) in trypomastigotes than in epimastigotes, with the LC(50) of this compound being 28.40 μg/mL (106.79 μM) against trypomastigotes. The concentrations of BSBZT used in this study presented low hemolytic activity and cytotoxicity. Consequently, at concentrations near IC(50) and LC(50) (25μg/mL), BSBZT caused only 2.4% hemolysis and 15% of RAW 264.7 cell cytotoxicity. These results reveal the potential of BSBZT as a prototype in drug design for developing new anti-T. cruzi compounds.     

Modified natural porcine surfactant modulates tobacco smoke-induced stress response in human monocytes

Tobacco smoke (TS) is a potent source of oxidants and oxidative stress is an important mechanism by which TS exerts its toxicity in the lung. We have shown that TS induces heat shock (HS)/stress protein (HSP) synthesis in human monocytes. Pulmonary surfactant (PS) whose major physiological function is to confer mechanical stability to alveoli, also modulates oxidative metabolism and other pro-inflammatory functions of monocytes-macrophages. In order to determine whether PS alters the stress response induced by TS, we incubated human peripheral blood monocytes overnight with modified natural porcine surfactant (Curosurf) (1 mg/ml) before exposure to TS. Curosurf decreased TS-induced, but not HS-induced, expression of the major cytosolic, inducible 72 kD HSP (Hsp70). Furthermore, TS-generated superoxide anions production was significantly decreased by Curosurf in an acellular system, suggesting a direct scavenging effect of PS. We also examined the effects of TS and PS on monocytes ultrastructure. Monocytes incubated with Curosurf presented smoother cell membranes than control monocytes, while TS-induced monocyte vacuolization was, at least in part, prevented by Curosurf. Taken together, our data suggest that PS plays a protective role against oxygen radical-mediated, TS-induced cellular stress responses.