Molecular events associated with acquisition of heat tolerance by the yeast Saccharomyces cerevisiae

Abstract: The heat shock response is an inducible protective system of all living cells. It simultaneously induces both heat shock proteins and an increased capacity for the cell to wisthstand potentially lethal temperatures (an increased thermotolerance). This has lead to the suspicion that these two phenomena must be inexorably linked. However, analysis of heat shock protein function in Saccharomyces cerevisiae by molecular genetic techniques has revealed only a minority of the heat shock proteins of this organism having appreciable influences on thermotolerance. Instead, physiological perturbations and the accumulation of trehalose with heat stress may be more important in the development of thermotolerance during a preconditioning heat shock. Vegetative S. cerevisiae also acquires thermotolerance through osmotic dehydration, through treatment with certain chemical agents and when, due to nutrient limitation, it arrests growth in the GI phase of the cell cycle. There is evidence for the activities of the cAMP-dependent protein kinase and plasma membrane ATPase being very important in thermotolerance determination. Also, intracellular water activity and trehalose probably exert a strong influence over thermotolerance through their effects on stabilisation of membranes and intracellular assemblies. Future investigations should address the unresolved issue of whether the different routes to thermotolerance induction cause a common change to the physical state of the intracellular environment, a change that may result in an increased stabilisation of cellular structures through more stable hydrogen bonding and hydrophobic interactions.     

Effects of recombinant human gamma interferon on intracellular survival of Bordetella pertussis in human phagocytic cells

Abstract Several studies have demonstrated that Bordetella pertussis has the ability to enter and survive intracellularly within human polymorphonuclear leukocytes (PMNL) and human monocytes/macrophages. The effects of human recombinant gamma interferon (IFN-γ) on the survival of B. pertussis in PMNL and human monocytes, and on the oxidative burst activity of PMNL and human monocytes in response to B. pertussis were assessed in this study. IFN-γ partially increased intracellular killing of phagocytosed B. pertussis in human monocytes, as determined by an orange acridine-crystal violet assay. In contrast, IFN-γ did not enhance intracellular killing of B. pertussis in PMNL. No significant increase of superoxide production was noted in human monocytes in response to B. pertussis when stimulated with various concentrations of IFN-γ. The partial increase of B. pertussis killing by IFN-γ within monocytes, together with poor production of superoxide may explain how B. pertussis can survive within human phagocytic cells, and thus cause a more prolonged course of the disease.     

Activation of the Na-K(NH4)-2Cl-cotransporter from rat submandibular glands in response to VIP

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pH_i) was estimated with 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH₄Cl, the pH_i transiently increased (diffusion of NH₃) and then dropped (influx of NH₄⁺). Isoproterenol increased 2.5-fold the rate of NH₄⁺ influx; bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 μmol/L) mimicked the response to isoproterenol. VIP (1 nmol/L-1 μmol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca²⁺]_i fivefold increased the uptake of NH₄⁺ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca²⁺]_i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na⁺-K⁺-2Cl⁻-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.¹     

Ca2+ Signals Generated by CatSper and Ca2+ Stores Regulate Different Behaviors in Human Sperm

[Ca²⁺]_i signaling regulates sperm motility, enabling switching between functionally different behaviors that the sperm must employ as it ascends the female tract and fertilizes the oocyte. We report that different behaviors in human sperm are recruited according to the Ca²⁺ signaling pathway used. Activation of CatSper (by raising pH_i or stimulating with progesterone) caused sustained [Ca²⁺]_i elevation but did not induce hyperactivation, the whiplash-like behavior required for progression along the oviduct and penetration of the zona pellucida. In contrast, penetration into methylcellulose (mimicking penetration into cervical mucus or cumulus matrix) was enhanced by activation of CatSper. NNC55-0396, which abolishes CatSper currents in human sperm, inhibited this effect. Treatment with 5 μm thimerosal to mobilize stored Ca²⁺ caused sustained [Ca²⁺]_i elevation and induced strong, sustained hyperactivation that was completely insensitive to NNC55-0396. Thimerosal had no effect on penetration into methylcellulose. 4-Aminopyridine, a powerful modulator of sperm motility, both raised pH_i and mobilized Ca²⁺ stored in sperm (and from microsomal membrane preparations). 4-Aminopyridine-induced hyperactivation even in cells suspended in Ca²⁺-depleted medium and also potentiated penetration into methylcellulose. The latter effect was sensitive to NNC55-039, but induction of hyperactivation was not. We conclude that these two components of the [Ca²⁺]_i signaling apparatus have strikingly different effects on sperm motility. Furthermore, since stored Ca²⁺ at the sperm neck can be mobilized by Ca²⁺-induced Ca²⁺ release, we propose that CatSper activation can elicit functionally different behaviors according to the sensitivity of the Ca²⁺ store, which may be regulated by capacitation and NO from the cumulus.     

Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB

Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.     

Ricin A chain reaches the endoplasmic reticulum after endocytosis

Ricin is a potent ribosome inactivating protein and now has been widely used for synthesis of immunotoxins. To target ribosome in the mammalian cytosol, ricin must firstly retrograde transport from the endomembrane system to reach the endoplasmic reticulum (ER) where the ricin A chain (RTA) is recognized by ER components that facilitate its membrane translocation to the cytosol. In the study, the fusion gene of enhanced green fluorescent protein (EGFP)-RTA was expressed with the pET-28a (+) system in Escherichia coli under the control of a T7 promoter. The fusion protein showed a green fluorescence. The recombinant protein can be purified by metal chelated affinity chromatography on a column of NTA. The rabbit anti-GFP antibody can recognize the fusion protein of EGFP-RTA just like the EGFP protein. The cytotoxicity of EGFP-RTA and RTA was evaluated by the MTT assay in HeLa and HEP-G2 cells following fluid-phase endocytosis. The fusion protein had a similar cytotoxicity of RTA. After endocytosis, the subcellular location of the fusion protein can be observed with the laser scanning confocal microscopy and the immuno-gold labeling Electro Microscopy. This study provided important evidence by a visualized way to prove that RTA does reach the endoplasmic reticulum.     

Involvement of Inositol 1,4,5-Trisphosphate-Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV-1 Protein Tat

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.     

Peptide loading in the endoplasmic reticulum accelerates trafficking of peptide: MHC class II complexes in B cells

In a combination of biochemical and immunoelectron-microscopical approaches we studied intracellular trafficking and localization of the endoplasmic-reticulum (ER)-formed complexes of murine MHC class II molecule I-A^b and an antigenic peptide E52–68 covalently linked to its -chain. The association with the peptide in the ER leads to sharp acceleration of the intracellular trafficking of the complexes to the plasma membrane. Within the cells, E52–68:I-A^b complexes accumulate in the multivesicular MHC class II compartment (MIIC), but not in denser multilaminar or intermediate type MIICs. The changes in the trafficking of ER-formed complexes result solely from the presence of the tethered peptide, since wild-type class II molecules traffic similarly in bare lymphocyte syndrome cells and in wild-type antigen-presenting cells.     

Effect of vesicle traps on traffic jam formation in fast axonal transport

The purpose of this paper is to develop a model for simulation of the formation of organelle traps in fast axonal transport. Such traps may form in the regions of microtubule polar mismatching. Depending on the orientation of microtubules pointing toward the trap region, these traps can accumulate either plus-end or minus-end oriented vesicles. The model predicts that the maximum concentrations of organelles occur at the boundaries of the trap regions; the overall concentration of organelles in the axon with traps is greatly increased compared to that in a healthy axon, which is expected to contribute to mechanical damages of the axon. The organelle traps induce hindrance to organelle transport down the axon; the total organelle flux down the axon with traps is found to be significantly reduced compared to that in a healthy axon.     

Redox-state of intactNitella cells: dependency on intracellular pH and photosynthesis

Summary The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (E_RED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, E_RED values of+237±93mVand+419±51 mV with respect to a normal H⁺-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state E_RED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of E_RED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic E_RED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic E_RED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10M 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), E_RED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.Abbreviations E_M transmembrane voltage - E_RED redox-state voltage - E₀ midpoint-redox-voltage - APW artificial pond water - CEF cytoplasm enriched fragment