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…’     

Residue cluster additivity of thermodynamic stability in the hydrophobic core of mesophile vs. hyperthermophile rubredoxins

The branched sidechain residues 24 and 33 in the hydrophobic core of rubredoxin differ between the Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) sequences. Their X-ray structures indicate that these two sidechains are in van der Waals contact with each other, while neither appears to significantly interact with the other nonconserved residues. The simultaneous interchange of residues 24 and 33 between the Cp and Pf rubredoxin sequences yield a complementary pair of hybrid proteins for which the sum of their thermodynamic stabilities equals that of the parental rubredoxins. The 1.2 kcal/mol change arising from this two residues interchange accounts for 21% of the differential thermodynamic stability between the mesophile and hyperthermophile proteins. The additional interchange of the sole nonconserved aromatic residue in the hydrophobic core yields a 0.78 kcal/mol deviation from thermodynamic additivity.     

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).     

Effect of 2,4-d, hydric stress and light on indica rice (Oryza sativa) somatic embryogenesis

With the purpose of increasing the embryogenesis regeneration process in vitroplants obtained from somatic embryos of the indica rice variety CR-5272 (Oryza sativa L.), two independent experiments were performed. The first experiment consisted in the effect of combination of three concentrations of the gelling agent Phytagel (1.8. 2.4. and 3 gL(-1)) and four 2,4-D concentrations (2.26, 4.52. 6.78, and 9.05 microM) on the induction and subsequent regeneration of embryogenic calli. On the second experiment, the pre-regeneration phase was modified: calli were subjected to darkness or diffuse light conditions for one, two, and three weeks. In embryogenesis induction, 35% calligenesis was obtained using the MS culture medium supplemented with 6.78 microM of 2.4-D and 2.4 gL(-1) Phytagel, whereas on the control treatment (MS medium supplemented with 9.05 microM of 2.4-D and 3 gL(-1) Phytagel ) 24% calligenesis was obtained. In addition, regeneration percentages were improved (22% and 16% for calli induced with the above treatments, respectively). Furthermore, in light exposure experiments, the best result was obtained by exposing the embryogenic calli to darkness for one week in pre-regeneration, followed by direct light exposure during the regeneration phase.     

Enhanced thermal stability achieved without increased conformational rigidity at physiological temperatures: spatial propagation of differential flexibility in rubredoxin hybrids

The extreme thermal stability of proteins from hyperthermophilic organisms is widely believed to arise from an increased conformational rigidity in the native state. In apparent contrast to this paradigm, both Pyrococcus furiosus (Pf) rubredoxin, the most thermostable protein characterized to date, and its Clostridium pasteurianum (Cp) mesophile homolog undergo a transient conformational opening of their multi-turn segments, which is more favorable in hyperthermophile proteins below room temperature. Substitution of the hyperthermophile multi-turn sequence into the mesophile protein sequence yields a hybrid, (14-33(Pf)) Cp, that exhibits a 12 degrees increase in its reversible thermal unfolding transition midpoint. Nuclear magnetic resonance (NMR) magnetization transfer-based hydrogen exchange was used to monitor backbone conformational dynamics in the subsecond time regime. Despite the substantially increased thermostability, flexibility throughout the entire main chain of the more thermostable hybrid is equal to or greater than that of the wild type mesophile rubredoxin near its normal growth temperature. In comparison to the identical core residues of the (14-33(Pf)) Cp rubredoxin hybrid, six spatially clustered residues in the parental mesophile protein exhibit a substantially larger temperature dependence of exchange. The exchange behavior of these six residues closely matches that observed in the multi-turn segment, consistent with a more extensive conformational process. These six core residues exhibit a much weaker temperature dependence of exchange in the (14-33(Pf)) Cp hybrid, similar to that observed for the multi-turn segment in its parental Pf rubredoxin. These results suggest that differential temperature dependence of flexibility can underlie variations in thermostability observed for mesophile versus hyperthermophile homologs.     

Tetrathiolate coordination of germanium(IV) in a protein active site

The tetracysteine metal coordination site of the rubredoxins from Clostridium pasteurianum (Cp) and Pyrococcus furiosus (Pf) are shown to stably bind the inorganic Ge(IV) ion. This is the first characterized coordination complex of tetravalent germanium with a biological macromolecule. Zn(II), Ga(III) and Ge(IV) substitution yields differential NMR chemical shifts for the 1H and 15N amide resonances throughout much of the protein structure. The differential shifts for the six backbone amides that hydrogen bond to the metal-coordinated sulfurs indicate that the pseudo 2-fold symmetry of the active site is more closely maintained in the hyperthermophile Pf rubredoxin than in its mesophile Cp homolog. These three metal substitutions form an isoelectronic series of small diamagnetic proteins for which reference structures are known to 1A resolution. These series provide a promising system to analyze theoretical predictions of the effects of differential charge distribution on chemical shifts from both proximal and long range interactions.     

Lung squamous cell carcinoma and adenocarcinoma cell lines use different mediators to induce comparable phenotypic and functional changes in human monocyte-derived dendritic cells

Tumor-derived immunosuppressive factors contribute to the evasion of malignant cells from the immune response, partially by hampering dendritic cell (DC) differentiation. Here, we analyze whether soluble mediators released by the most frequent histological types of non-small cell lung carcinoma, squamous cell carcinoma (SCC), and adenocarcinoma (AD) cells, affect the development and functionality of DC. Monocytes from healthy donors were differentiated in vitro into DC with granulocyte–macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, in the absence or presence of soluble factors (SF) from SCC or AD cell lines. Monocytes were differentiated in parallel into macrophages (MΦ s) with macrophage colony-stimulating factor (M-CSF). SF-treated DC were phenotypically and functionally more similar to MΦ s than to untreated DC [control DC (Ctrl-DC)]. Both tumors increased myelomonocytic markers (CD14, CD16, CD32, and CD163) and impaired CD1a expression on DC. SF-treated DC increased their endocytic capacity, and released higher levels of IL-6, IL-10, and lower levels of IL-12, compared to Ctrl-DC. SF-treated DC were poor stimulators in mixed lymphocyte reactions, and naïve CD4⁺ T lymphocytes stimulated by SF-treated DC secreted lower levels of interferon (IFN)-γ and higher amounts of IL-10 than controls. In contrast to AD, the effects caused by SCC were mostly abolished by IL-6 neutralization during monocyte differentiation. However, tumor-derived prostanoid blockade recovered the IFN-γ levels secreted by lymphocytes stimulated with SF-treated DC, whereas prostanoid/IL-6 or prostanoid/IL-10 blockade decreased IL-10 production only by SCC-DC-stimulated lymphocytes. Thus, we provide evidence that lung SCC and AD cause comparable deficiencies on DC in vitro, skewing monocyte differentiation from DC to MΦ -like cells, but most of these changes occurred via different mediators.     

The acute asymmetric effects of hemiovariectomy on testosterone secretion vary along the estrous cycle. The participation of the cholinergic system

The presence of asymmetry in the capacity of the left and right ovaries to secrete testosterone was analyzed by studying the effects of hemiovariectomy along the estrus cycle one hour after surgery. The effects of ether anesthesia on hormone serum levels were also analyzed.     

Homocysteine, grey matter and cognitive function in adults with cardiovascular disease

Background

Elevated total plasma homocysteine (tHcy) has been associated with cognitive impairment, vascular disease and brain atrophy.

Methods

We investigated 150 volunteers to determine if the association between high tHcy and cerebral grey matter volume and cognitive function is independent of cardiovascular disease.

Results

Participants with high tHcy (≥15 µmol/L) showed a widespread relative loss of grey matter compared with people with normal tHcy, although differences between the groups were minimal once the analyses were adjusted for age, gender, diabetes, hypertension, smoking and prevalent cardiovascular disease. Individuals with high tHcy had worse cognitive scores across a range of domains and less total grey matter volume, although these differences were not significant in the adjusted models.

Conclusions

Our results suggest that the association between high tHcy and loss of cerebral grey matter volume and decline in cognitive function is largely explained by increasing age and cardiovascular diseases and indicate that the relationship is not causal.