Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.     

A PAF receptor antagonist inhibits acute airway inflammation and late-phase responses but not chronic airway inflammation and hyperresponsiveness in a primate model of asthma

We have examined the effects of a PAF receptor antagonist, WEB 2170, on several indices of acute and chronic airway inflammation and associated changes in lung function in a primate model of allergic asthma. A single oral administration WEB 2170 provided dose related inhibition of the release of leukotriene C(4) (LTC(4)) and prostaglandin D(2) (PGD(2)) recovered and quantified in bronchoalveolar lavage (BAL) fluid obtained during the acute phase response to inhaled antigen. In addition, oral WEB 2170 treatment in dual responder primates blocked the acute influx of neutrophils into the airways as well as the associated late-phase airway obstruction occurring 6 h after antigen inhalation. In contrast, a multiple dosing regime with WEB 2170 (once a day for 7 consecutive days) failed to reduce the chronic airway inflammation (eosinophilic) and associated airway hyperresponsiveness to inhaled methacholine that is characteristic of dual responder monkeys. Thus, we conclude that the generation of PAF following antigen inhalation contributes to the development of lipid mediators, acute airway inflammation and associated late-phase airway obstruction in dual responder primates; however, PAF does not play a significant role in the maintenance of chronic airway inflammation and associated airway hyperresponsiveness in this primate model.     

ADP-ribosylation of gelsolin-actin complexes by clostridial toxins.

ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied. Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin. The Km for NAD (4 microM) was identical for both substrates. C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol. In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin. The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated. Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex. ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex. When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min. According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex. This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex.     

Binding of sugar phosphates, inositol phosphates and phosphorylated amino acids to actin.

Binding of biological phosphate compounds to actin was investigated by the effect of these compounds on the critical concentration of the pointed ends of gelsolin-capped actin filaments. According to this assay millimolar concentrations of glucose 6-phosphate and the bisphosphorylated sugars fructose 1,6-bisphosphate, fructose 2,6-bisphosphate, glucose 1,6-bisphosphate, sedoheptulose 1,7-bisphosphate and 2,3-bisphosphoglycerate were found to associate with actin. Glycerophosphoinositol phosphates bound to actin if they were present in millimolar concentrations, and if carbon atom 4 of the inositol ring was phosphorylated and carbon atom 5 was free of phosphate. Also phosphoserine and phosphotyrosine were found to interact with actin. Most of the actin-binding compounds stabilized actin filaments by decreasing the critical concentration suggesting that these compounds had a higher affinity for the subunits along actin filaments than for actin monomers. However, 2,3-bisphosphoglycerate and fructose 2,6-bisphosphate increased the critical concentration probably because these sugar phosphates bound to actin monomers thereby inhibiting actin polymerization.     

Rate constants and equilibrium constants for binding of actin to the 1:1 gelsolin-actin complex.

The rate constant and equilibrium constant of association of an actin monomer with 1:1 gelsolin-actin complex isolated from chicken were measured by using fluorescently labeled actin. According to fluorescence stopped-flow experiments, the rate constant of formation of the 1:2 gelsolin-actin complex from 1:1 gelsolin-actin complex and actin was found to be about 2 x 10(7) M-1 s-1 under conditions where gelsolin binds Ca2+. The rate of dissociation of one actin molecule from the 1:2 gelsolin-actin complex was determined by exchange of actin for fluorescently labeled actin. The rate constant of dissociation was about 0.02 s-1. Thus, the equilibrium constant for association of actin with 1:1 gelsolin-actin complex can be calculated to be in the range of 10(9) M-1. The rate of dissociation of actin from 1:2 gelsolin-actin complex was independent of the Ca2+ concentration. Ca2+ affects only the rate of association of actin with 1:1 gelsolin-actin complex.     

Expression of Krox Proteins During Differentiation of the O-2A Progenitor Cell Line CG-4

Abstract: Krox proteins are important regulators of development and terminal differentiation. Using the rat glial progenitor cell line CG-4 as a model system for oligodendrocyte differentiation, we show that on the RNA level Krox-24 is the predominant member of the Krox family in these cells. Similar results were also obtained on the protein level as the major Krox protein from CG-4 cell extracts reacted specifically with an antibody against Krox-24. Whereas Krox-24 RNA and protein were abundant in undifferentiated CG-4 cells, a dramatic decrease in expression was detected after a 3–5-day period of differentiation during which we observed a reciprocal increase in the levels of myelin basic protein expression. Importantly, regulation of Krox-24 expression was very similar in CG-4 cells and primary oligodendrocyte cultures. When expression of Krox-24 in differentiating CG-4 cells was followed on a closer time scale, we observed a sharp and transient increase in Krox-24 RNA, protein, and DNA binding activity immediately after the onset of differentiation followed by an equally rapid decrease. This expression pattern implicates Krox-24 both in maintenance of the undifferentiated state and in the immediate early phase of differentiation of CG-4 cells and possibly oligodendrocytes.     

The gene for the ataxia-telangiectasia variant,Nijmegen breakage syndrome,maps to a 1-cM interval on chromosome 8q21.

Nijmegen breakage syndrome (NBS; Seemanová II syndrome) and Berlin breakage syndrome (BBS), also known as ataxia-telangiectasia variants, are two clinically indistinguishable autosomal recessive familial cancer syndromes that share with ataxia-telangiectasia similar cellular, immunological, and chromosomal but not clinical findings. Classification in NBS and BBS was based on complementation of their hypersensitivity to ionizing radiation in cell-fusion experiments. Recent investigations have questioned the former classification into two different disease entities, suggesting that NBS/BBS is caused by mutations in a single radiosensitivity gene. We now have performed a whole-genome screen in 14 NBS/BBS families and have localized the gene for NBS/BBS to a 1-cM interval on chromosome 8q21, between markers D8S271 and D8S270, with a peak LOD score of 6.86 at D8S1811. This marker also shows strong allelic association to both Slavic NBS and German BBS patients, suggesting the existence of one major mutation of Slavic origin. Since the same allele is seen in both former complementation groups, genetic homogeneity of NBS/BBS can be considered as proved.     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997