A sensitive chemiluminescence assay for pertussis toxin and for evaluation of cell-free pertussis toxoids

Abstract Pertussis toxin (PT) inhibited luminol-enhanced chemiluminescence induced in rabbit peritoneal neutrophils by N'-formyl- l -methionyl- l -leucyl- l -phenylalanine (fMLP) at doses as low as 0.8 ng·ml⁻¹, even in the presence of a 10-fold higher concentration of filamentous haemagglutinin (FHA). A cell-free extract of Bordetella pertusis , containing predominantly PT and FHA, suppressed the neutrophil response to fMLP. After toxoiding with carbodiimide, the inhibitory activity of the extract was abolished and an enhancement of neutrophil chemiluminescence was observed due to FHA activity. Abrogation of the chemiluminescent response of neutrophils to fMLP is proposed as a sensitive, in vitro assay for PT, and may be useful for monitoring the residual toxin activity in pertussis toxoids and for determining the anti-toxic effects of anti-PT antibodies.     

Study of the binding of jatrophone to Escherichia coli s-ribonucleic acid

The interaction of jatrophone with sRNA from Escherichia coli has been investigated through UV, CD, and 1H NMR measurements. The results obtained show that the interaction with jatrophone increases the stability of the polynucleotide. It appears that the optical properties of jatrophone depend upon the jatrophone/nucleotide ratio. The observed behaviour can only be explained by the existence of different types of interaction between jatrophone and sRNA. Even for a jatrophone/nucleotide ratio as low as 0.17 the 1H NMR spectra show a multiplicity of resonances that can only be explained by the simultaneous existence of two different binding modes involving the jatrophone molecules.     

The Catecholaminergic Polymorphic Ventricular Tachycardia Mutation R33Q Disrupts the N-terminal Structural Motif That Regulates Reversible Calsequestrin Polymerization

Calsequestrin undergoes dynamic polymerization with increasing calcium concentration by front-to-front dimerization and back-to-back packing, forming wire-shaped structures. A recent finding that point mutation R33Q leads to lethal catecholaminergic polymorphic ventricular tachycardia (CPVT) implies a crucial role for the N terminus. In this study, we demonstrate that this mutation resides in a highly conserved alternately charged residue cluster (DGKDR; cluster 1) in the N-terminal end of calsequestrin. We further show that this cluster configures itself as a ring system and that the dipolar arrangement within the cluster brings about a critical conformational flip of Lys³¹-Asp³² essential for dimer stabilization by formation of a H-bond network. We additionally show that Ca²⁺-induced calsequestrin aggregation is nonlinear and reversible and can regain the native conformation by Ca²⁺ chelation with EGTA. This study suggests that cluster 1 works as a molecular switch and governs the bidirectional transition between the CASQ2 monomer and dimer. We further demonstrate that mutations disrupting the alternating charge pattern of the cluster, including R33Q, impair Ca²⁺-CASQ2 interaction, leading to altered polymerization-depolymerization dynamics. This study provides new mechanistic insight into the functional effects of the R33Q mutation and its potential role in CPVT.     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

Expression of CD40, CD44, bcl-2 antigens and rate of cell proliferation on fine needle aspirates from metastatic melanoma

The clinical behaviour of melanoma is often unpredictable using clinical and histological criteria. Tumour cell markers related to cell cycle regulation, apoptosis, cell-cell interactions and cell proliferation might improve the possibility of predicting the clinical course of melanoma. The aim of the present study was to refine prognostic criteria by an immunocytochemical investigation of CD44, CD40, bcl-2 antigens and cell proliferation in tumour cells aspirated from metastases of malignant melanoma. CD40 is a cell surface receptor shown to be expressed by lymphomas as well as carcinomas, and is thought to play a central role in the process of tumour progression. CD44 is a transmembrane glycoprotein, which is involved in growth signal transmission of importance in the binding of tumour cells to endothelium, cell migration and enhancement of cell motility, which makes it of interest to study in relation to the metastasizing capacity of tumours. The bcl-2 protein is active in the process of programmed cell death (apoptosis) as an antiapoptotic agent and its expression may reflect tumour progression. Mean/median percentages of tumour cell positivity were 8.5/3.0 for CD40, 76.1/86.3 for CD44 and 7.4/3.3 for bcl-2. A significant correlation was observed between expression of apoptosis-associated bcl-2 antigen and overall survival (r = 0.33). The CD44 positive cell fraction was higher in patients with short overall survival than those with long survival but this difference was not statistically significant. The expression of CD40 did not correlate with overall survival. The mean/median proliferation fraction assessed by MIB-1 monoclonal antibody was 25.8/23.9 and showed a significant correlation with survival after diagnosis of melanoma metastasis (r = 0.32). Lack of bcl-2 expression and a high proportion of tumour cells expressing Ki-67 antigen are predictors of poor prognosis that are independent of the traditionally accepted Breslow's thickness of the primary melanomas.     

Systemic lupus erythematosus onset in lupus-prone B6.MRL/lpr mice Is influenced by weight gain and Is preceded by an increase in neutrophil oxidative burst activity

In this study, we assessed whether weight gain influenced the systemic lupus erythematosus (SLE) onset and/or outcome, and examined the role that reactive oxygen species (ROS) production by neutrophils played in the SLE onset and/or outcome. Female control (C57BL/6) and lupus-prone B6.MRL/lpr mice (CM and LPM, respectively) at 4 weeks old were fed standard diet or standard diet plus cafeteria diet during 12 weeks. SLE diagnosis relied on the presence of both antinuclear antibodies (ANA) and renal abnormalities. We found that the percentage of weight gain in CM and LPM increased as a function of the length of cafeteria diet feeding period, but it was not associated with energy intake. Cafeteria diet-fed CM and LPM at 8 and 12 weeks old were overweight, while CM and LPM at 16 weeks old were obese. Compared with standard diet-fed CM and LPM, cafeteria diet-fed CM and LPM exhibited elevated glucose and total cholesterol levels, and diminished triglycerides levels. Standard diet-fed 16-week-old LPM and cafeteria diet-fed 12-week-old LPM had nephritis, characterized by the increased interstitial infiltration of leukocytes. Cafeteria diet-induced weight gain rose the frequency of homogeneous and speckled ANA staining patterns in the 12- and 16-week-old LPM groups. Together, these results indicated that weight gain anticipated the SLE onset. In addition, neutrophils from cafeteria diet-fed 8-week-old LPM exhibited augmented ROS production capacity; in standard diet-fed LPM, such rise occurred only in the 16-week-old group. Thus, the neutrophil ROS production capacity was increased before the SLE onset and during its outcome. Overweight and obese CM and LPM displayed elevated levels of kidney, liver, heart, and spleen lipid peroxidation. In conclusion, cafeteria diet-induced weight gain is associated with the increased production of ANA and neutrophil-derived ROS, which may contribute to accelerate the SLE onset.     

Ligand Independence of the T618I Mutation in the Colony-stimulating Factor 3 Receptor (CSF3R) Protein Results from Loss of O-Linked Glycosylation and Increased Receptor Dimerization

Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.     

Adherence of human neutrophils changes Ca signaling during activation with opsonized particles

Changes in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) upon activation of human neutrophils by opsonized particles (serum-treated zymosan; STZ) were evaluated by three different methods: (i) measurement of total fluorescence changes in indo-1 loaded neutrophils activated in suspension; (ii) measurement of fluorescence changes in individual indo-1 loaded neutrophils in a flow cytometer and (iii) measurement of fluorescence changes in individual fura-2 loaded neutrophils adherent to serum-coated coverslips. Our study shows that the opsonized particle-induced change in [Ca²⁺]_i in neutrophils is altered during adherence of the cells to a serum-coated surface. These observations might be of importance for neutrophil function in vivo, since adherence is a prerequisite for diapedesis and chemotaxis.     

Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism

Previous work from us and others has suggested that cholesterol is an important lipid in the context of the organization of the actin cytoskeleton. However, reorganization of the actin cytoskeleton upon modulation of membrane cholesterol is rarely addressed in the literature. In this work, we explored the signaling crosstalk between cholesterol and the actin cytoskeleton by using a high-resolution confocal microscopic approach to quantitatively measure changes in F-actin content upon cholesterol depletion. Our results show that F-actin content significantly increases upon chronic cholesterol depletion, but not during acute cholesterol depletion. In addition, utilizing inhibitors targeting the cholesterol biosynthetic pathway at different steps, we show that reorganization of the actin cytoskeleton could occur due to the synergistic effect of multiple pathways, including prenylated Rho GTPases and availability of membrane phosphatidylinositol 4,5-bisphosphate. These results constitute one of the first comprehensive dissections of the mechanistic basis underlying the interplay between cellular actin levels and cholesterol biosynthesis. We envision these results will be relevant for future understating of the remodeling of the actin cytoskeleton in pathological conditions with altered cholesterol.