Downregulation of mitogen-activated protein kinases by the Bordetella bronchiseptica Type III secretion system leads to attenuated nonclassical macrophage activation.

Bordetella bronchiseptica utilizes a type III secretion system (TTSS) to establish a persistent infection of the murine respiratory tract. Previous studies have shown that the Bordetella TTSS mediated cytotoxicity in different cell types, inhibition of NF-kappaB in epithelial cells, and differentiation of dendritic cells into a semimature state. Here we demonstrate modulation of mitogen-activated protein kinase (MAPK) signaling pathways and altered cytokine production in macrophages and dendritic cells by the Bordetella TTSS. In macrophages, the MAPKs ERK and p38 were downregulated. This resulted in attenuated production of interleukin- (IL-)6 and IL-10. In contrast, the Th-1-polarizing cytokine IL-12 was produced at very low levels and remained unmodulated by the Bordetella TTSS. In dendritic cells, ERK was transiently activated, but this failed to alter cytokine profiles. These results suggest that the Bordetella TTSS modulates antigen-presenting cells in a cell type-specific manner and the secretion of high levels of IL-6 and IL-10 by macrophages might be important for pathogen clearance.

Pseudohypoaldosteronism type 1 and the genes encoding prostasin, alpha-spectrin, and Nedd4.

Pseudohypoaldosteronism type 1 (PHA1), a rare disorder of infancy, presents with potential life-threatening salt wasting and failure to thrive. Thus far, PHA1 has been attributed to mutations affecting the mineralocorticoid receptor or any of the three subunits assembling the amiloride-sensitive epithelial sodium channel (ENaC). However, a lot of patients with a phenotype resembling PHA1, show no defects in these proteins, making it likely that further genes are involved in the aetiology of this disease. Recent studies have elucidated additional participants (alpha-spectrin and members of the families of transmembrane serine proteases, ubiquitin-protein ligases, and serum- and glucocorticoid-regulated kinases, respectively) regulating and/or interacting in the complex pathway of sodium retention in the amiloride-sensitive distal nephron. This led us to investigate whether PHA1 can also be associated with mutations in some of these genes. Our data suggest that at least the prostasin gene might be excluded as a causative locus.

Bordetella type III secretion and adenylate cyclase toxin synergize to drive dendritic cells into a semimature state.

Bordetella bronchiseptica establishes persistent infection of the murine respiratory tract. We hypothesize that long-term colonization is mediated in part by bacteria-driven modulation of dendritic cells (DCs) leading to altered adaptive immune responses. Bone marrow-derived DCs (BMDCs) from C57BL/6 mice infected with live B. bronchiseptica exhibited high surface expression of MHCII, CD86, and CD80. However, B. bronchiseptica-infected BMDCs did not exhibit significant increases in CD40 surface expression and IL-12 secretion compared with BMDCs treated with heat-killed B. bronchiseptica. The B. bronchiseptica type III secretion system (TTSS) mediated the increase in MHCII, CD86, and CD80 surface expression, while the inhibition of CD40 and IL-12 expression was mediated by adenylate cyclase toxin (ACT). IL-6 secretion was independent of the TTSS and ACT. These phenotypic changes may result from differential regulation of MAPK signaling in DCs. Wild-type B. bronchiseptica activated the ERK 1/2 signaling pathway in a TTSS-dependent manner. Additionally, ACT was found to inhibit p38 signaling. These data suggest that B. bronchiseptica drive DC into a semimature phenotype by altering MAPK signaling. These semimature DCs may induce tolerogenic immune responses that allow the persistent colonization of B. bronchiseptica in the host respiratory tract.

Steroid sulfatase (STS) expression in the human temporal lobe: enzyme activity, mRNA expression and immunohistochemistry study.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) are suggested to be important neurosteroids. We investigated steroid sulfatase (STS) in human temporal lobe biopsies in the context of possible cerebral DHEA(S) de novo biosynthesis. Formation of DHEA(S) in mature human brain tissue has not yet been studied. 17 alpha-Hydroxylase/C17-20-lyase and hydroxysteroid sulfotransferase catalyze the formation of DHEA from pregnenolone and the subsequent sulfoconjugation, respectively. Neither their mRNA nor activity were detected, indicating that DHEA(S) are not produced within the human temporal lobe. Conversely, strong activity and mRNA expression of DHEAS desulfating STS was found, twice as high in cerebral neocortex than in subcortical white matter. Cerebral STS resembled the characteristics of the known placental enzyme. Immunohistochemistry revealed STS in adult cortical neurons as well as in fetal and adult Cajal-Retzius cells. Organic anion transporting proteins OATP-A, -B, -D, and -E showed high mRNA expression levels with distinct patterns in cerebral neocortex and subcortical white matter. Although it is not clear whether they are expressed at the blood-brain barrier and facilitate an influx rather than an efflux, they might well be involved in the transport of steroid sulfates from the blood. Therefore, we hypothesize that DHEAS and/or other sulfated 3beta-hydroxysteroids might enter the human temporal lobe from the circulation where they would be readily converted via neuronal STS activity.

Infection of barley roots by Chaetomium globosum: evidence for a protective role of the exodermis.

Plant pathogenesis by fungi is known to be dependent on the host genotype, the virulence of the pathogen and certain environmental conditions influencing fungal establishment. Previously, it has been shown that Chaetomium globosum, a fungus well-characterized for its biocontrol potential, causes necrosis on barley roots grown in Murashige and Skoog (MS)-agar. Using MS-agar and aeroponic culture as axenic plant growth systems, C. globosum pathogenesis was analyzed with serological and histological methods. Irrespective of the growth system, C. globosum infected the root epidermis. Roots grown in MS-agar were extensively colonized intercellularly and intracellularly up to the inner cortex and the tissue underwent necrosis. In contrast, roots grown in aeroponic culture were not colonized beyond the epidermis and the roots appeared healthy. Histochemical analyses revealed that hypodermal suberization stopped fungal invasion. In root tips known to lack suberization, epidermal papilla formation reduced overall infection frequency. The results indicate that specific environmental conditions are important for infection and disease expression in barley roots. Infection is restricted by two spatial and temporal distinct defence mechanisms: (1) papillae in root tips retarding fungal invasion; and (2) suberization of hypodermal cells blocking fungal radial growth.

Characterisation of estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in the human brain.

Estrogens play a crucial role in multiple functions of the brain and the proper balance of inactive estrone and active estradiol-17beta might be very important for their cerebral effects. The interconversion of estrone and estradiol-17beta in target tissues is known to be catalysed by a number of human 17beta-hydroxysteroid dehydrogenase (17beta-HSD) isoforms. The present study shows that enzyme catalysed interconversion of estrone and estradiol-17beta occurs in the human temporal lobe. The oxidative cerebral pathway preferred estradiol-17beta to Delta(5)-androstenediol and testosterone, whereas the reductive pathway preferred dehydroepiandrosterone (DHEA) to Delta(4)-androstenedione and estrone. An allosteric Hill kinetic for NAD-dependent oxidation of estradiol-17beta was observed, whereas a typical Michaelis-Menten kinetic was shown for NADPH-dependent reduction of estrone. Investigations of the interconversion of estrogens in cerebral neocortex (CX) and subcortical white matter (SC) preparations of brain tissue from 12 women and 10 men revealed no sex-differences, but provide striking evidence for the presence of at least one oxidative membrane-associated 17beta-HSD and one cytosolic enzyme that catalyses both the reductive and the oxidative pathway. Membrane-associated oxidation of estradiol-17beta was shown to be significantly higher in CX than in SC (P<0.05), whereas the cytosolic enzyme activities were significantly higher in SC than in CX (P<0.0005). Finally, real-time RT-PCR analyses revealed that besides 17beta-HSD types 4 and 5 also the isozymes type 7, 8, 10 and 11 show substantial expression in the human temporal lobe. The characteristics of the isozymes lead us to the conclusion that cytosolic 17beta-HSD type 5 is the best candidate for the observed cytosolic enzyme activities, whereas the data gave no clear answer to the question, which enzyme is responsible for the membrane-associated oxidation of estradiol-17beta. In conclusion, the study strongly suggests that different cell types and different isozymes are involved in the cerebral interconversion of estrogens, which might play a pivotal role in maintaining the functions of the central nervous system.

Four additional CLCN5 exons encode a widely expressed novel long CLC-5 isoform but fail to explain Dent's phenotype in patients without mutations in the short variant.

BACKGROUND: Dent's disease is caused by mutations in the CLCN5 gene coding for the chloride channel CLC-5. However, sequencing of CLCN5 exonic regions in some patients presenting with low-molecular-weight proteinuria and hypercalciuria - the hallmarks of Dent's disease - failed to identify causative mutations. AIM: Given the observation that some species harbour a CLCN5 mRNA encoding an extended CLC-5 aminoterminus compared with the so far known human form, we worked on the presumption that an orthologous (longer) CLCN5 transcript is also present in humans and that our patients may have mutations herein. METHODS: Extensive databank mining, reverse transcription polymerase chain reaction (RT-PCR) and automated sequencing were used in the search for novel CLCN5 transcripts. The human CLCN5 gene was investigated in 7 patients out of five families by direct automated sequencing of PCR-amplified DNA products. RESULTS: Two new human CLCN5 transcripts expressed in kidney and various other tissues could be identified. These arise from a novel site of transcription initiation, alternative splicing and the use of four additional CLCN5 exons. If being translated, both these mRNAs would lead to an enlarged CLC-5 protein consisting of 816 amino acids by adding 70 aminoterminal residues to the so far known 746-amino-acid-long isoform. Sequence analysis of the henceforward 17 CLCN5 exons revealed no mutation in the patients with a phenotype resembling Dent's disease. CONCLUSIONS: Despite the identification of further targets to explain Dent's disease, the molecular defect in our patients remains to be elucidated. Hence, their phenotype may be explained by mutations that affect so far unknown regulating elements of the CLCN5 gene or another gene(s), probably encoding CLC-5 accessory protein(s).

Characterization of the dehydroepiandrosterone (DHEA) metabolism via oxysterol 7alpha-hydroxylase and 17-ketosteroid reductase activity in the human brain.

Dehydroepiandrosterone and its sulphate are important factors for vitality, development and functions of the CNS. They were found to be subjects to a series of enzyme-mediated conversions within the rodent CNS. In the present study, we were able to demonstrate for the first time that membrane-associated dehydroepiandrosterone 7alpha-hydroxylase activity occurs within the human brain. The cytochrome P450 enzyme demonstrated a sharp pH optimum between 7.5 and 8.0 and a mean KM value of 5.4 micro m, corresponding with the presence of the oxysterol 7alpha-hydroxylase CYP7B1. Real-time RT-PCR analysis verified high levels of CYP7B1 mRNA expression in the human CNS. The additionally observed conversion of dehydroepiandrosterone via cytosolic 17beta-hydroxysteroid dehydrogenase activity could be ascribed to the activity of an enzyme with a broad pH optimum and an undetectably high KM value. Subsequent experiments with cerebral neocortex and subcortical white matter specimens revealed that 7alpha-hydroxylase activity is significantly higher in the cerebral neocortex than in the subcortical white matter (p < 0.0005), whereas in the subcortical white matter, 17beta-hydroxysteroid dehydrogenase activity is significantly higher than in the cerebral neocortex (p < 0.0005). No sex differences were observed. In conclusion, the high levels of CYP7B1 mRNA in brain tissue as well as in a variety of other tissues in combination with the ubiquitous presence of 7alpha-hydroxylase activity in the human temporal lobe led us to assume a neuroprotective function of the enzyme such as regulation of the immune response or counteracting the deleterious effects of neurotoxic glucocorticoids, rather than a distinct brain specific function such as neurostimulation or neuromodulation.

Novel NCCT gene mutations as a cause of Gitelman's syndrome and a systematic review of mutant and polymorphic NCCT alleles.

BACKGROUND: Gitelman's syndrome (GS) is characterized by hypokalemic metabolic alkalosis, hypomagnesemia and hypocalciuria and these phenotypic features have been shown to be attributable to mutations in the gene encoding the thiazide-sensitive Na/Cl cotransporter (NCCT). Until now, 55 different mutations have been reported and most of the families affected with GS exhibit autosomal recessive inheritance. METHODS: All 26 exons of the human NCCT gene were investigated in 2 German NCCT-deficient patients and their families. Mutation detection was performed by either direct automated sequencing of polymerase chain reaction (PCR)-amplified DNA products or by sequence analysis of cloned PCR products. RESULTS: In a 47-year-old German GS female a novel non-conservative missense mutation (S314F) and a complex deletion/insertion in the NCCT gene were found to be associated with the disorder. A further novel non-conservative substitution (S402F) together with a frequently observed R209W exchange were found in a 19-year-old German GS female. CONCLUSIONS: The observation of a compound heterozygote state in both females affected and the absence of a GS phenotype in their relatives carrying a single mutant allele is consistent with an autosomal recessive pattern of inheritance.