Lipopolysaccharides derived from Porphyromonas gingivalis and Escherichia coli: Differential and interactive effects on novelty-induced hyperlocomotion, blood cytokine levels and TLR4-related processes.

Lipopolysaccharide (LPS), a component of the Gram-negative bacterial cell wall, activates Toll-like receptors (TLRs). Porphyromonas gingivalis (Pg) may be involved in the progression of periodontal disease. Mice exposed to a novel environment show hyperlocomotion that is inhibited by systemic administration of LPS derived from Escherichia coli (Ec-LPS). However, whether Pg-LPS influences novelty-induced locomotion is unknown. Accordingly, we carried out an open field test to analyse the effects of Pg-LPS. For comparison, effects of Ec-LPS were also studied. We additionally investigated the influence of systemic administration of Pg-LPS or Ec-LPS on IL-6, TNF-alpha, and IL-10 levels in blood, as they could be involved in the changes in locomotion. The TLR4 receptor antagonist TAK-242 was used to study the involvement of TLR4. Since Pg-LPS may block TLR4 in vitro, we analysed the effects of Pg-LPS on Ec-LPS-induced changes in behavioural and biochemical parameters. Male ddY mice were used. Pg- or Ec-LPS and TAK-242 were administered intraperitoneally. Ec-LPS (840 µg/kg), but not Pg-LPS (100, 500 and 840 µg/kg), inhibited novelty-induced locomotion, which was antagonized by TAK-242 (3.0 mg/kg). Ec-LPS (840 µg/kg) increased blood levels of IL-6 and IL-10, which were antagonized by TAK-242 (3.0 mg/kg). However, TAK-242 did not inhibit Ec-LPS-induced increases in TNF-alpha levels in blood. Pg-LPS (100, 500, and 840 µg/kg) did not alter blood IL-6, TNF-alpha, or IL-10 levels. The Ec-LPS-induced increase in blood IL-10, but not IL-6 and TNF-alpha, levels was inhibited by Pg-LPS (500 µg/kg). These results suggest that TLR4 stimulation mediates the inhibition of novel environment-induced locomotion in mice following systemic administration of Ec-LPS, while also increasing blood IL-6 and IL-10 levels. In contrast, Pg-LPS did not exhibit these effects. The present study also provides in vivo evidence that Pg-LPS can inhibit TLR4-mediated increases in blood levels of IL-10, a cytokine thought to prevent the development of periodontal disease.

Secondary structure of the amino-terminal region of HCV NS3 and virological response to pegylated interferon plus ribavirin therapy for chronic hepatitis C.

The aim of the study was to identify a predictive marker for the virological response in hepatitis C virus 1b (HCV-1b)-infected patients treated with pegylated interferon plus ribavirin therapy. A total of 139 patients with chronic hepatitis C who received therapy for 48 weeks were enrolled. The secondary structure of the 120 residues of the amino-terminal HCV-1b non-structural region 3 (NS3) deduced from the amino acid sequence was classified into two major groups: A and B. The association between HCV NS3 protein polymorphism and virological response was analyzed in patients infected with group A (n = 28) and B (n = 40) isolates who had good adherence to both pegylated interferon and ribavirin administration (>95% of the scheduled dosage) for 48 weeks. A sustained virological response (SVR) representing successful HCV eradication occurred in 33 (49%) in the 68 patients. Of the 28 patients infected with the group A isolate, 18 (64%) were SVR, whereas of the 40 patients infected with the group B isolate only 15 (38%) were SVR. The proportion of virological responses differed significantly between the two groups (P < 0.05). These results suggest that polymorphism in the secondary structure of the HCV-1b NS3 amino-terminal region influences the virological response to pegylated interferon plus ribavirin therapy, and that virus grouping based on this polymorphism can contribute to prediction of the outcome of this therapy.

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate.

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.

Differentiation of rat bone marrow cells cultured on artificial basement membrane containing extracellular matrix into a liver cell lineage.

BACKGROUND/AIMS: Bone marrow (BM) cells have been shown to be capable of differentiating into a liver cell lineage in vitro. However, their differentiation and proliferation is poor, and the cell characteristics are poorly understood. METHODS: We cultured rat BM cells on an artificial basement membrane containing extracellular matrix (ECM) with hepatocyte growth factor (HGF). The expression of mRNA for liver-specific genes was analyzed by reverse transcription PCR. The expression of albumin and Musashi-1 by cultured cells was analyzed using a fluorescence-activated cell sorter (FACS). The proportions of albumin-positive cells when culture was performed with different concentrations of HGF were analyzed by FACS. RESULTS: On culture day 21, polygonal cells proliferated and formed cell colonies. These cells expressed mRNA for all the liver-specific genes analyzed, and showed heterogeneous differentiation, some cells expressing albumin, others expressing Musashi-1. Albumin-positive differentiated cells were large and rich in intracellular structures, while Musashi-1-positive undifferentiated cells had the opposite characteristics. Culturing cells with higher concentrations of HGF induced an increased proportion of albumin-positive cells. CONCLUSIONS: The results suggest that cell culture on an ECM with a high concentration of HGF increases the extent to which BM cells differentiate into a liver cell lineage and proliferate in vitro.