Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1ß, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

Expression of trkB and trkC receptors and their ligands brain-derived neurotrophic factor and neurotrophin-3 in the murine amygdala.

The neurotrophin brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) and their cognate receptors, trkB and trkC, have a variety of physiological brain functions, ranging from cell survival to mechanisms involved in learning and memory and long-term potentiation (LTP). LTP can be induced in the cortex and hippocampus, as well as within the amygdala. However, the role of neurotrophins in amygdalar LTP is largely unknown. Expression patterns of BDNF and NT-3 and their cognate receptors in the adult mouse amygdala have not been analyzed in detail. We have therefore examined the expression of trkB, trkC, BDNF, and NT-3 mRNA and protein in different amygdalar nuclei as well as in the hippocampal areas CA1-CA3 and the dentate gyrus. The distribution pattern of trkB, trkC, BDNF, and NT-3 mRNA in the murine hippocampus is comparable to that seen in rats. Within most amygdalar nuclei, a moderate BDNF mRNA expression was found; however, BDNF mRNA was virtually absent from the central nucleus. No expression of NT-3 mRNA was found within the amygdala, but trkC mRNA-expressing cells were widely distributed within this brain region. trkB mRNA was strongly expressed in the amygdala. Because trkB is expressed in a full-length and a truncated form (the latter form is also expressed by nonneuronal cells), we also investigated the distribution of full-length trkB mRNA-expressing cells and could demonstrate that this version of trkB receptors is also widely expressed in the amygdala. These results can serve as a basis for studies elucidating the physiological roles of these receptors in the amygdala.

Distribution of the iron-regulating protein hepcidin in the murine central nervous system.

Iron serves as an essential trace element for all body tissues, including the central nervous system (CNS). Because iron deficiency as well as iron overload is known to cause damage to the mammalian brain, the maintenance of iron homeostasis is crucial. It has been discovered recently that hepcidin plays an essential role in iron metabolism outside the CNS. A defect in hepcidin expression is responsible for iron accumulation and mice over-expressing hepcidin die postnatally by a severe anemia. We have used RT-PCR, in situ hybridization, and immunohistochemistry to investigate the cellular distribution of hepcidin mRNA and protein in brain, spinal cord, and dorsal root ganglia. Our results show a wide-spread distribution of hepcidin in different brain areas, including the olfactory bulb, cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon, cerebellum, pons, spinal cord, as well as in dorsal root ganglia of the peripheral nervous system. Hepcidin immunoreactivity is not restricted to neurons, but can be detected in both neurons and GFAP-positive glia cells. Because hepcidin action in organs outside the CNS is linked to iron homeostasis, we speculate that it is also involved in such processes in the CNS, putatively together with other iron regulating proteins. Cellular mechanisms and functions of hepcidin in the CNS remain to be elucidated.

FGF-2 deficiency does not alter vulnerability of the dopaminergic nigrostriatal system towards MPTP intoxication in mice.

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2(-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.

Localization of DJ-1 protein in the murine brain.

Mutations in the DJ-1 gene have been identified to cause Parkinson's disease. In humans, nonmutated DJ-1 is expressed in specific brain areas but seems to be expressed by astrocytes rather than by neurons. In contrast, DJ-1 mRNA is mainly found in neurons in the mouse brain. We have investigated the distribution of DJ-1 protein in the mouse brain and found that DJ-1 protein is predominantly expressed by neurons but can also be detected in astrocytes. Consistent with a global role of DJ-1 in the brain, we found immunoreactivity, for example, in cortical areas, hippocampus, basolateral amygdala, the reticular nucleus of the thalamus, zona incerta, and locus coeruleus. Within the substantia nigra, however, DJ-1 is localized in both neuronal and nonneuronal cells, suggesting a distinct role in this area.

Identification and characterization of a novel PrfA-regulated gene in Listeria monocytogenes whose product, IrpA, is highly homologous to internalin proteins, which contain leucine-rich repeats.

The expression of all virulence factors in Listeria monocytogenes characterized to date is controlled by the virulence regulator protein, PrfA. To identify further PrfA-regulated proteins, we examined supernatants of L. monocytogenes EGD harboring additional copies of the PrfA regulator for the presence of novel proteins. This led to the identification and biochemical purification of a hitherto uncharacterized PrfA-dependent 30-kDa protein (A. Lingnau, T. Chakraborty, K. Niebuhr, E. Domann, and J. Wehland, Infect. Immun. 64:1002-1006, 1996). Oligonucleotide primers derived from internal peptide sequences of this protein allowed the cloning and determination of the entire sequence of the respective gene. The protein comprised 297 amino acids with strong overall homology to the internalins, InlA and InlB, particularly in the region harboring the leucine-rich repeats. The gene has been designated irpA for internalin-related protein A gene. Transcriptional studies revealed that the gene was monocistronic and, like the inlA and inlB genes, was transcribed by PrfA-dependent and PrfA-independent mechanisms. Monoclonal antibodies raised against IrpA indicated that it was produced by L. monocytogenes but not by the nonpathogenic species Listeria innocua. To examine the role of IrpA in pathogenesis, we constructed an isogenic in-frame deletion mutant that removed all but 116 amino acids of the IrpA protein. This mutant was neither defective for invasion into many tissue culture cell lines nor did it demonstrate reduced intracellular survival. However, in vivo studies using the mouse infection model revealed that the irpA mutant showed reduced virulence compared to the parental strain. These results suggest a role for IrpA during disseminated infection by L. monocytogenes.