Listeria monocytogenes Stimulates Mucus Exocytosis in Cultured Human Polarized Mucosecreting Intestinal Cells through Action of Listeriolysin O

When the intracellular pathogen Listeria monocytogenes infects cultured human mucosecreting polarized HT29-MTX cells apically, it induces the stimulation of mucus exocytosis without cell entry. Using a set of isogenic mutants and purified listeriolysin O (LLO), we identified the L. monocytogenes thiol-activated exotoxin LLO as the agonist of mucus secretion. We demonstrated that the LLO-induced mucus exocytosis did not result from the LLO membrane-damaging activity. We found that LLO-induced mucus exocytosis is an event requiring the binding of LLO to a brush border-associated receptor and membrane oligomerization of the exotoxin. By a pharmacological approach, we demonstrated that no regulatory system or intracellular transducing signal known to be involved in control of mucin exocytosis was activated by LLO. Based on the present data, the stimulatory action of LLO on mucin exocytosis could be accounted for either by an unknown signaling system which remains to be determined or by direct action of LLO with the membrane vesicle components involved in the intracellular vesicular transport of mucins.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

Variation at the ADAM10 gene locus is not associated with Creutzfeldt-Jakob disease

Prion diseases are characterized by the accumulation in the brain of a misfolded and protease-resistant form of the prion protein (PrP(c)). PrP(c) contains an amyloidogenic, neurotoxic sequence that is essential for conversion into PrP(Sc), the pathological isoform. During normal processing, PrP(c) is cleaved at a site within this sequence, and this cleavage is thought to destroy the amyloidogenic potential of the protein. ADAM10, a disintegrin and metalloprotease that plays a key role in the pathogenesis of Alzheimer's disease, was recently shown to use PrP(c) as a substrate. We investigated whether variability in the ADAM10 gene could contribute to the pathogenesis of Creutzfeldt-Jakob disease (CJD), by analyzing a single nucleotide polymorphism (SNP) within ADAM10, as a genetic marker potentially in linkage disequilibrium with a functional polymorphism, in patients with sporadic or variant CJD. We observed no significant differences in ADAM10 genotype or allele frequencies between CJD patients and healthy individuals. Moreover, the distribution of ADAM10 SNP genotypes and alleles did not differ between groups of patients based on genotype at the polymorphic codon 129 of the prion protein gene--the sole major genetic risk factor for CJD identified to date. Our data indicate that ADAM10 is unlikely to confer genetic susceptibility to CJD.     

Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice,a cathepsin L-deficient mutation

We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation.     

Actigraphic assessment of sleep in insomnia: application of the Actigraph Data Analysis Software (ADAS)

The usefulness of the actigraph methodology has been demonstrated in normal individuals. However, the validity of actigraphy has been questioned in insomnia patients because of the considerable measurement error that has been reported between actigraphy (ACT) and polysomnography (PSG). Two independent investigations have reported errors of 48 and 49 min in total sleep time between ACT and PSG. With a new scoring method called the Actigraph Data Analysis Software, a reanalysis of one of these studies was conducted. Based on this reanalysis, we have obtained a measurement error of only 25 min between the two methods. This finding may be an indication of the advantage of this new scoring method. A strong correlation coefficient (r = 0.82, p < 0.0001) was noted between ACT and PSG for total sleep time, thus suggesting a high degree of accuracy of the actigraph methodology in assessing the sleep/wake profile of insomniacs.     

Coxiella burnetii Induces Reorganization of the Actin Cytoskeleton in Human Monocytes

Coxiella burnetii, an obligate intracellular bacterium which survives in myeloid cells, causes Q fever in humans. We previously demonstrated that virulent C. burnetii organisms are poorly internalized by monocytes compared to avirulent variants. We hypothesized that a differential mobilization of the actin cytoskeleton may account for this distinct phagocytic behavior. Scanning electron microscopy demonstrated that virulent C. burnetii stimulated profound and polymorphic changes in the morphology of THP-1 monocytes, consisting of membrane protrusions and polarized projections. These changes were transient, requiring 5 min to reach their maximum extent and vanishing after 60 min of incubation. In contrast, avirulent variants of C. burnetii did not induce any significant changes in cell morphology. The distribution of filamentous actin (F-actin) was then studied with a specific probe, bodipy phallacidin. Virulent C. burnetii induced a profound and transient reorganization of F-actin, accompanied by an increase in the F-actin content of THP-1 cells. F-actin was colocalized with myosin in cell protrusions, suggesting that actin polymerization and the tension of actin-myosin filaments play a role in C. burnetii-induced morphological changes. In addition, contact between the cell and the bacterium seems to be necessary to induce cytoskeleton reorganization. Bacterial supernatants did not stimulate actin remodeling, and virulent C. burnetii organisms were found in close apposition with F-actin protrusions. The manipulation of the actin cytoskeleton by C. burnetii may therefore play a critical role in the internalization strategy of this bacterium.     

Prostate cancer is part of the hereditary non-polyposis colorectal cancer (HNPCC) tumor spectrum

The recognized urologic tumor spectrum in hereditary non-polyposis colon cancer includes ureteral and renal pelvis malignancies. Here, we report a family in which the proband, who had three metachronous adenocarcinomas of the colon and rectum (at ages 54, 57, and 60), presented with an adenocarcinoma of the prostate at age 61. Immunohistochemical (IHC) staining of colonic, rectal, and prostatic tumor tissues demonstrated lack of expression of both MSH2 and MSH6. Accordingly, microsatellite instability (MSI) was found in the rectal, colonic, and prostatic tumors. The kindred complies with the Amsterdam criteria for HNPCC, as five members over three generations had colorectal cancer. Molecular investigations were initiated when the proband's son presented with an adenocarcinoma of the colon at age 35. Southern blotting analysis of genomic DNA led to identification of a novel genomic deletion encompassing exon 5 of the MSH2 gene. Although prostate cancer has occasionally been described in HNPCC families, to the best of our knowledge, this is the first report where the MSI and IHC analysis of the prostatic adenomcarcinoma clearly link its aetiology to the germline mismatch repair mutation. Hence, prostate cancer should be included in the HNPCC tumor spectrum.     

The role of resistance characteristics of viral strains in the prediction of the response to antiretroviral therapy in HIV infection

OBJECTIVE: To study the role of resistance characteristics of viral mutants in the prediction of virologic and immunologic response to antiretroviral therapy in HIV-infection. METHODS: This study is based on a mathematical model that generates viral and immunologic dynamics of HIV infection, taking into account drug-resistant mutants and therapy. We analyzed predictive factors of the increase in CD4 cell count and of the decrease in viral load from baseline after 6 months of HAART on a sample of 300 simulated individuals. The set of potential predictors was constituted by patients' state at initiation of therapy and by resistance characteristics of viral strains at that time. Predictive models, obtained by stepwise regression, were selected and compared using Mallows' Cp criterion. RESULTS: In addition to baseline viral load and CD4 cell count, known to influence response to therapy, baseline CD8 cell count and resistance characteristics of detectable strains are shown to improve the accuracy of the prediction. On the contrary, resistance parameters of low frequency viral mutants have no predictive value. CONCLUSIONS: Characteristics of preexisting detectable resistant mutants as determinants of virologic and immunologic response to antiretroviral therapy increase the capacity to predict the outcome of the treatment. Therefore, the use of phenotypic and genotypic testing could be crucial and should be considered for the choice of therapy.