Binding of Nucleic Acids to Intermediate Filaments of the Vimentin Type and Their Effects on Filament Formation and Stability

Abstract

Guanine-rich polynucleotides such as poly(dG), oligo(dG)_12–18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)₂₅ was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG)·poly(dC), and, of those of the RNA type, preferentially poly(rA)·poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.     

Hsp70, a messenger from hyperthermia for the immune system

Heat shock proteins (Hsps) hold a dual role depending on their location. Inside cells, they fulfill essential survival functions as molecular chaperones forming complexes with intracellular polypeptides (self or foreign) to help in protein folding, the resolution of protein aggregates and intracellular protein transport. Released from the cell, they act as messengers communicating the cells’ interior protein composition to the immune system for initiation of immune responses against intracellular proteins. Here we describe the mechanisms by which Hsp70, the heat-inducible Hsp70 family member, crosstalks with the immune system. Further, we discuss that clinical hyperthermia could be a way to initiate the immunologic activity of Hsp70 by upregulating its expression and facilitating release through local necrosis.     

Effects of thermal stress on tumor antigenicity and recognition by immune effector cells

The primary rationale for the application of clinical hyperthermia in the therapy of cancer is based on the direct cytotoxic effect of heat and the radio-chemosensitization of tumor cells. More recently, additional attention is given to the observation that heat and heat-shock proteins can activate the host’s immune system. The expression of heat-shock genes and proteins provides an adaptive mechanism for stress tolerance, allowing cells to survive non-physiologic conditions. However, the same adaptive mechanism can ultimately favor malignant transformation by interfering with pathways that regulate cell growth and apoptosis. Cytoprotection and thermotolerance raised the concern that heat-treated tumor cells might also be resistant to attack by immune effector mechanisms. Many studies, including those from our group, address this concern and document that heat-exposure, although transiently modulating sensitivity to CTL, do not hinder CTL attack. Moreover, there are promising reports of heat-related upregulation of NK-activating ligands, rendering those tumors which have lost MHC class I molecules target for NK cell attack. Heat-induced cytoprotection, therefore, does not necessarily extend protection from cytotoxic immune mechanisms. When interpreting the effects of heat, it is important to keep in mind that thermal effects on cell physiology are strongly dependent on the thermal dose, which is a function of the magnitude of change in temperature and the duration of heat exposure. The thermal dose required to induce cell death in vitro strongly varies from cell type to cell type and depends on microenvironmental factors (Dewey 1994). Therefore, to dissect the immunological behaviour of a given tumor and its micro-environment at different thermal doses, it is essential to characterize the thermosensitivity of every single tumor type and assess the proportion of cells surviving a given heat treatment. In this review, we summarize the pleiotropic effects that heat exposure has on tumor cells. In particular, we focus on the effects of heat on the antigen presentation of tumor cells and their susceptibility to immune effector mechanisms. We emphasize that the response to thermal stress is not a one-time point event, but rather a time period starting with the heat exposure and extending over several days of recovery. In addition, the response of tumor cells and their susceptibility to immune effector cells is strongly dependent on the model system, on the magnitude and duration of the thermal stress and on the time of recovery after heat exposure. Consideration of these aspects might help to explain some of the conflicting results that are reported in the field of thermal stress response. This article forms part of the Symposium in Writing "Thermal stress-related modulation of tumor cell physiology and immune responses", edited by Elfriede Noessner.     

A Synechococcus sp. PCC 7942 mutant with a higher tolerance towards bentazone

In this article we describe the partial characterization of a Synechococcus sp. PCC 7942 mutant Mu1 with an enhanced resistance towards the herbicide bentazone (3-isopropyl-1H-2,1,3-benzothiadiazine-4(3H)-one 2,2-dioxide). The mutant was derived from a random mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (NSG) and exhibited superior growth rates, pigment content and overall photosynthetic activities under regular growth conditions compared to wild type. Whereas Synechococcus PCC 7942 wild type showed significant photoinhibition, especially in the presence of lincomycin, Mu1 was much more robust. A comparative analysis of the content of several photosynthesis-associated proteins revealed that Mu1 had an increased expression of PsbO on mRNA and protein level and that PsbO is tightly bound to Photosystem II, relative to wild type. This result was substantiated by mass spectrometer measurements of photosynthetic water oxidation revealing a higher stability and integrity of the water oxidizing complex in Mu1 cells grown under regular or calcium deficient conditions. Therefore, our results give rise to the possibility that the overexpression of PsbO in mutant Mu1 confers resistance to reactive oxygen species (ROS) formed as a consequence of bentazone binding to the acceptor side of PS II. In addition, we observed a significantly higher tolerance towards bentazone in iron depleted wild type cells, conditions under which the IdiA protein becomes expressed in highly elevated amounts. As we have previously shown, IdiA preferentially protects the acceptor site of PS II against oxidative stress, especially under iron limitation. Thus, it is likely that IdiA due to its topology interferes with bentazone binding or protects PS II against ROS generated in the presence of bentazone. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of mitochondrial respiratory function in small biopsies of liver

Mitochondrial respiratory function was studied in permeabilized pig liver biopsies. The cell membrane was permeabilized mechanically in tissue samples of 2-7 mg, for application of a standardized substrate/inhibitor titration protocol in high-resolution respirometry. Specific respirometric tests demonstrated complete plasma membrane permeabilization and accessibility of substrates to intact mitochondria. High respiratory adenylate control ratios and cytochrome c conservation in the tissue preparation were comparable or even better than in isolated mitochondria. Citrate synthase and cytochrome c oxidase activities remained at 85% of controls after up to 98 h storage of liver tissue at 0 degrees C in histidine-tryptophan-ketoglutarate solution. Multiple mitochondrial defects, however, were indicated after 48 h cold storage by the decline in respiratory capacity, which was lowered to a larger extent with complex I substrates compared to respiration with substrates for complex II or IV, measured in the absence of cytochrome c. After prolonged ischemia, the adenylate control ratio was significantly reduced, and cytochrome c depletion was detected by the stimulatory effect of cytochrome c. High-resolution respirometry allows the assessment of mitochondrial function in a few milligrams of permeabilized liver tissue, without isolation of mitochondria. This provides a basis for the analysis of mitochondrial function in human liver biopsies.     

A fungus-type beta-galactofuranan in the cultivated Trebouxia photobiont of the lichen Ramalina gracilis

A structural characterization of polysaccharides extracted from the aposymbiotically cultured photobiont of the lichen Ramalina gracilis was carried out in order to compare them with those previously found in the symbiotic thallus. The photobiont was isolated from thallus fragments, following the method of Yamamoto, and cultivated in a liquid nutrient medium. Freeze-dried cells were defatted, and the polysaccharides extracted successively with water and aq. 10% KOH, each at 100 degrees C. After purification, the soluble fractions provided a polysaccharide containing a (1-->5)-linked beta-galactofuranosyl backbone, substituted in a small proportion at O-6 by beta-Galf units. Amylose was also found, as insoluble material obtained on freeze-thawing of the alkaline extract. These polysaccharides have not been found in the symbiotic thallus of Ramalina gracilis, which contained only water-soluble (isolichenan) and insoluble glucans (nigeran and laminaran), and galactomannan. Surprisingly, the galactofuranan has similarities with those found in some fungal cell walls.     

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O₂ evolving activity ranging from 290 (for the wild type) to 1100 mol O₂ x mg chlorophyll^-1 x h^-1 (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochrome_b559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochrome_b559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochrome_b559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O₂ evolving photosystem II reaction center core complexes.Abbreviations PS photosystem - chl chlorophyll - LHCP light-harvesting chlorophyll a/b protein complex     

Cyanide formation in preparations from Chlorella vulgaris Beijerinck: Effect of sonication and amygdalin addition

Summary A critical evaluation of a method for recovering HCN from cell extracts is presented. Since crude extracts often bind or metabolize HCN extensively, the HCN recovered by distillation at room temperature represents only the difference between production and consumption. Sonication leads to HCN release from the alga, Chlorella vulgaris Beijerinck. Illumination of extracts at high light intensity in oxygen, with added Mn²⁺ and peroxidase, also stimulates HCN production. In both processes, the HCN is probably formed by oxidation of nitrogenous precursors. Chlorella extracts cause formation of HCN from added amygdalin. No evidence was found, however, for the presence of cyanogenic glycosides in the algae.     

ALGAL CALCIFICATION IN SOME CODIACEAE (CHLOROPHYTA): ULTRASTRUCTURE AND LOCATION OF SKELETAL DEPOSITS1,2

The capitular filaments of Penicillus and Rhipocephalus consist of an inner tube containing the cytoplasm and an outer calcified sheath. The sheath originates at the cell wall and differentiates into several layers which form the outer filament wall. CaCO₃ is deposited between organic layers within the sheath and is not in direct contact with the seawater. Pores within the sheath, usually uncalcified, may facilitate exchange of gases and solutes. The cytoplasm is characterized by vacuolar inclusions of calcium oxalate needles 50–150 μm long. A closed cortical surface is lacking. Udotea cyathiformis Dec. and U. conglutinata (Ellis & Sol.) Lam. are similar to Penicillus and Rhipocephalus, in addition showing some CaCO₃ between filaments (ICS-calcification). Udotea flabellum (Ellis & Sol.) Lam. is different as the filaments are profusely branched giving rise to a fully developed cortical surface. Pores and vacuolar calcium oxalate inclusions are absent. CaCO₃ deposition occurs within cortical filaments in between layers of the filament wall and subcortically in intercellular spares (ICS). Cortex calcification shows primary and secondary deposits bearing some resemblance to sheath calcification and to coralline red algae. In Rhipocephalus phoenix (Ellis & Sol.) Kütz., Penicillus pyriformis A. &E. Gepp, U. cyathiformis and U. conglutinata CaCO₃ is precipitated intracellularly within the sheath, in contrast to Halimeda and Cymopolia where it is deposited extracellularly in between filaments. U. flabellum takes an intermediate position showing both intra- and intercellular calcification. The sheath compartment volume is between 12.5 and 7500 μm³and 5–3 orders of magnitude smaller than the ICS-compartment. Compartment size and location of CaCO₃may bear on calcification mechanisms. One condition for such a mechanism may be restricted exchange of solutes (CO₂, CO₃^2-, HCO₃^-, O₂, Co²⁺). Codiaceae; filament ultrastructure; Penicillus; Rhipocephalus; Udotea