Effector granules in human T lymphocytes: the luminal proteome of secretory lysosomes from human T cells

Background

The incidence of cancer in patients with neurological diseases, who have been treated with LiCl, is below average. LiCl is a well-established inhibitor of Glycogen synthase kinase-3, a kinase that controls several cellular processes, among which is the degradation of the tumour suppressor protein p53. We therefore wondered whether LiCl induces p53-dependent cell death in cancer cell lines and experimental tumours.

Results

Here we show that LiCl induces apoptosis of tumour cells both in vitro and in vivo. Cell death was accompanied by cleavage of PARP and Caspases-3, -8 and -10. LiCl-induced cell death was not dependent on p53, but was augmented by its presence. Treatment of tumour cells with LiCl strongly increased TNF-α and FasL expression. Inhibition of TNF-α induction using siRNA or inhibition of FasL binding to its receptor by the Nok-1 antibody potently reduced LiCl-dependent cleavage of Caspase-3 and increased cell survival. Treatment of xenografted rats with LiCl strongly reduced tumour growth.

Conclusions

Induction of cell death by LiCl supports the notion that GSK-3 may represent a promising target for cancer therapy. LiCl-induced cell death is largely independent of p53 and mediated by the release of TNF-α and FasL. Key words: LiCl, TNF-α, FasL, apoptosis, GSK-3, FasL     

Concomitant immunity in a rodent model of filariasis: the infection of Meriones unguiculatus with Acanthocheilonema viteae

In an attempt to study the occurrence of concomitant immunity in filarial infections, jirds (Meriones unguiculatus) were experimentally infected with Acanthocheilonema viteae, and patent animals were superinfected with a defined dose of A. viteae stage 3 larvae (L3). Infected animals harbored significantly less worms deriving from the superinfection than the control group (P < 0.05, 56.2%, and 63.4% protection), as shown by analysis of female worms 6 wk after superinfection on the basis of their developmental status and their length. This protection was not due to contact with L3 antigens because a significant reduction of worm burdens deriving of a superinfection was also observed after subcutaneous implantation of a single female worm (P < 0.05, 40.2% and 64.9% protection). The induced protective responses target L3 and restrict their migration because an established infection resulted in a reduction of L3 recovery (95.6% and 94.3%, P < 0.001) from tissues of jirds at day 5 after superinfection. Other data show that L3 from a superinfection are trapped within eosinophil-rich granulomas, which is likely to create unfavorable conditions for the worms and to lead to later death. Taken together, established A. viteae-infections partially protect hosts against homologous superinfection by an immune-mediated mechanism and, thus, regulate the population density of the parasites within the host by concomitant immunity.     

Infection of cultured human endothelial cells by Legionella pneumophila

Legionella pneumophila is a gram-negative pathogen that causes a severe pneumonia known as Legionnaires' disease. Here, we demonstrate for the first time that L. pneumophila infects and grows within cultured human endothelial cells. Endothelial infection may contribute to lung damage observed during Legionnaires' disease and to systemic spread of this organism.     

Specificity inversion of Ochrobactrum anthropi D-aminopeptidase to a D,D-carboxypeptidase with new penicillin binding activity by directed mutagenesis

The serine penicillin-recognizing proteins have been extensively studied. They show a wide range of substrate specificities accompanied by multidomain features. Their adaptation capacity has resulted in the emergence of pathogenic bacteria resistant to beta-lactam antibiotics. The most divergent enzymatic activities in this protein family are those of the Ochrobactrum anthropi D-aminopeptidase and of the Streptomyces R61 D,D-carboxypeptidase/transpeptidase. With the help of structural data, we have attempted to identify the factors responsible for this opposite specificity. A loop deletion mutant of the Ochrobactrum anthropi D-aminopeptidase lost its original activity in favor of a new penicillin-binding activity. D-aminopeptidase activity of the deletion mutant can be restored by complementation with another deletion mutant corresponding to the noncatalytic domain of the wild-type enzyme. By a second step site-directed mutagenesis, the specificity of the Ochrobactrum anthropi D-aminopeptidase was inverted to a D,D-carboxypeptidase specificity. These results imply a core enzyme with high diversity potential surrounded by specificity modulators. It is the first example of drastic specificity change in the serine penicillin-recognizing proteins. These results open new perspectives in the conception of new enzymes with nonnatural specificities. The structure/specificity relationship in the serine penicillin-recognizing proteins are discussed.     

Structure of transfection-active histone H1/DNA complexes

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios r _i (0.1<r _i<30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at r _i=1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.     

Dipetalonema viteae: resistance in Meriones unguiculatus with multiple infections of stage-3 larvae

The jird, Meriones unguiculatus, infected with 80 normal infective larvae of Dipetalonema viteae, revealed a recovery rate of 27.9% 12 weeks after infection. A pretreatment by three injections of 50 normal larvae each and challenge by 80 larvae resulted in a recovery rate of 10.7%. The recovered worms were longer than those from the challenge control animals. When three times 50 irradiated larvae (35 krad) were inoculated, the recovery rate of the challenge decreased to 2.6%, representing a protection of 90.7%. The surviving adult worms were stunted and derived exclusively from the 80 normal larvae given for challenge, since absolutely no adult worms were recovered in eight animals inoculated three times with 50 irradiated larvae only. Sera of all pretreated jirds contained IgG and IgM antibodies which bound in immunoblotting experiments bound predominantly to three proteins of larvae with molecular masses of 68,140, and 165 kDa, respectively. Enzymatic surface iodination revealed that the three antigens were exposed on the larval surface. The coincidence of a partial resistance to a challenge infection and of an antibody response against surface proteins of infective larvae suggests an importance of these antigens for the rejection of D. viteae mediated by an acquired immunological resistance of M. unguiculatus.