Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling

Toll-like receptors (TLRs) are a family of proteins playing important roles in host defense. Mice defective of functional TLR4 are hyporesponsive to LPS, suggesting that TLR4 is essential for LPS signaling. Here we report the cloning of an alternatively spliced mouse TLR4 (mTLR4) mRNA. The additional exon exists between the second and third exon of the reported mTLR4 gene and contains an in-frame stop codon. The alternatively spliced mRNA encodes 86 aa of the reported mTLR4 and an additional 36 aa. This alternatively spliced mTLR4 mRNA expressed a partially secretary 20-kDa protein, which we named soluble mTLR4 (smTLR4). In a mouse macrophage cell line, the exogenously expressed smTLR4 significantly inhibited LPS-mediated TNF-alpha production and NF-kappaB activation. Additionally, in mouse macrophages, LPS increased the mRNA for smTLR4. Taken together, our results indicate that smTLR4 may function as a feedback mechanism to inhibit the excessive LPS responses in mouse macrophages.     

Isolation of a CDC28 homologue from Cryptococcus neoformans that is able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae

A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.     

Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs, there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis, bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably, purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs, and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.     

Interleukin-1α: Its possible roles in cancer therapy

Our studies on recombinant human IL-1 polypeptide were summarized with respect to molecular cloning, production, quantitative assay systems, antitumor activity, myelorestorative activity and augmentation of host resistance to infections.Recombinant human IL-1 (18 kDa) was produced through the expression of the cloned human IL-1 cDNA inEscherichia coli and purified to an endotoxin-free homogeneous polypeptide. The human IL-1 inhibited dose-dependently the growth of syngeneic murine tumors transplanted in mice and completely regressed the tumors in some cases, and its antitumor activity was significantly enhanced in combination with indomethacin. The human IL-1 accelerated the recovery of the numbers of peripheral leukocytes and neutrophils in a dose-dependent manner at a dose as low as 10 ng/mouse/day in myelo suppressed mouse model produced by administering anticancer chemotherapeutic drugs. The myelorestorative effect of IL-1 was observed not only on leukocytes/neutrophils, but also on platelets in myelosuppressed mice. In addition, the human IL-1 markedly augmented dose-dependently resistance of normal and leukopenic mice to various microbial infections.These results suggested that recombinant human IL-1 might be useful for cancer therapy from the viewpoints of improving adverse effects such as myelosuppression caused by chemotherapy and/or radiation therapy and preventing infections. In addition, use of IL-1 may permit more intensive chemo- and radiation therapies using higher doses. Finally, the antitumor activity of the IL-1 itself may play an important role.     

EMG-angle relationship of the hamstring muscles during maximum knee flexion.

The aim of the present study was to investigate the EMG-joint angle relationship during voluntary contraction with maximum effort and the differences in activity among three hamstring muscles during knee flexion. Ten healthy subjects performed maximum voluntary isometric and isokinetic knee flexion. The isometric tests were performed for 5 s at knee angles of 60 and 90 degrees. The isokinetic test, which consisted of knee flexion from 0 to 120 degrees in the prone position, was performed at an angular velocity of 30 degrees /s (0.523 rad/s). The knee flexion torque was measured using a KIN-COM isokinetic dynamometer. The individual EMG activity of the hamstrings, i.e. the semitendinosus, semimembranosus, long head of the biceps femoris and short head of the biceps femoris muscles, was detected using a bipolar fine wire electrode. With isometric testing, the knee flexion torque at 60 degrees knee flexion was greater than that at 90 degrees. The mean peak isokinetic torque occurred from 15 to 30 degrees knee flexion angle and then the torque decreased as the knee angle increased (p<0.01). The EMG activity of the hamstring muscles varied with the change in knee flexion angle except for the short head of the biceps femoris muscle under isometric condition. With isometric contraction, the integrated EMGs of the semitendinosus and semimembranosus muscles at a knee flexion angle of 60 degrees were significantly lower than that at 90 degrees. During maximum isokinetic contraction, the integrated EMGs of the semitendinosus, semimembranosus and short head of the biceps femoris muscles increased significantly as the knee angle increased from 0 to 105 degrees of knee flexion (p<0.05). On the other hand, the integrated EMG of the long head of the biceps femoris muscle at a knee angle of 60 degrees was significantly greater than that at 90 degrees knee flexion with isometric testing (p<0.01). During maximum isokinetic contraction, the integrated EMG was the greatest at a knee angle between 15 and 30 degrees, and then significantly decreased as the knee angle increased from 30 to 120 degrees (p<0.01). These results demonstrate that the EMG activity of hamstring muscles during maximum isometric and isokinetic knee flexion varies with change in muscle length or joint angle, and that the activity of the long head of the biceps femoris muscle differs considerably from the other three heads of hamstrings.     

Suppression of Cytokine Production in T Helper Type 2 Cells by Nitric Oxide in Comparison with T Helper Type 1 Cells

We examined the effect of nitric oxide (NO) on cytokine production in T helper (Th) cell subsets, using murine splenic CD4⁺ T cells and two types of Th clones. Interferon-gamma-treated murine peritoneal exudate cells (IFN-PEC) suppressed DNA synthesis to 60% of the control level in CD4⁺ T cells stimulated with the anti-CD3 monoclonal antibody. The production of IL-2 and IL-4 in the CD4⁺ T cells decreased to 63.2% and 9.2%, respectively, of the control value by co-culture with IFN-PEC. The addition of N^G-monomethyl-L -arginine (L-NMMA) partially recovered the suppression of DNA synthesis. In the presence of indomethacin, the suppression of DNA synthesis was partially inhibited and the reduction in the cytokine production caused by IFN-PEC was partially recovered. The simultaneous addition of N^G-monomethyl-L -arginine (L-NMMA) and indomethacin completely inhibited not only the suppression of DNA synthesis but also the reduction in the cytokine production caused by IFN-PEC. Moreover, DNA synthesis in the Th2 clone was suppressed to a greater extent than that in the Th1 clone by co-culture with IFN-PEC. This suppression in the Th1 clone was inhibited by the addition of L-NMMA, whereas the DNA synthesis in the Th2 clone was not recovered by L-NMMA. In addition, sodium nitroprusside (SNP) suppressed IL-4 production in the Th2 clone but had no effect on IL-2 production in the Th1 clone. In the experiment of the co-culture with IFN-PEC, the inhibitory-effect of NO on T cell activation was not clarified by the influence of prostaglandins. However, in conclusion, cytokine production in Th2 cells may be more susceptible to NO than that in Th1 cells.     

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4 nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by ∼0.1% upon activation relative to the relaxing state and increased by ∼0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca²⁺-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca²⁺-binding and the second induced by actomyosin interaction.     

Neurodegeneration of mouse nigrostriatal dopaminergic system induced by repeated oral administration of rotenone is prevented by 4-phenylbutyrate, a chemical chaperone

Parkinson's disease (PD) is a progressive neurodegenerative disorder that is primarily characterized by the degeneration of dopaminergic neurons in the nigrostriatal pathway. Previous studies have demonstrated that chronic systemic exposure of Lewis rats to rotenone produced many features of PD, and cerebral tauopathy was also detected in the case of severe weight loss. The present study was designed to assess the neurotoxicity of rotenone after daily oral administration for 28 days at several doses in C57BL/6 mice. In addition, we examined the protective effects of 4-phenylbutyrate (4-PBA) on nigral dopamine (DA) neurons in rotenone-treated mice. 4-PBA was injected intraperitoneally daily 30 min before each oral administration of rotenone. Chronic oral administration of rotenone at high doses induced specific nigrostriatal DA neurodegeneration, motor deficits and the up-regulation of alpha-synuclein in the surviving DA neurons. In contrast to the Lewis rat model, cerebral tauopathy was not detected in this mouse model. 4-PBA inhibited rotenone-induced neuronal death and decreased the protein level of alpha-synuclein. These results suggest that this rotenone mouse model may be useful for understanding the mechanism of DA neurodegeneration in PD, and that 4-PBA has a neuroprotective effect in the treatment of PD.     

Rare sugar d-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats

A rare sugar, d-psicose has progressively been evaluated as a unique metabolic regulator of glucose and lipid metabolism, and thus represents a promising compound for the treatment of type 2 diabetes mellitus (T2DM). The present study was undertaken to examine the underlying effector organs of d-psicose in lowering blood glucose and abdominal fat by exploiting a T2DM rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were fed 5% d-psicose or 5% d-glucose supplemented in drinking water, and only water in the control for 13 weeks and the protective effects were compared. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. After 13 weeks feeding, d-psicose treatment significantly reduced the increase in body weight and abdominal fat mass. Oral glucose tolerance test (OGTT) showed the reduced blood glucose and insulin levels suggesting the improvement of insulin resistance in OLETF rats. Oil-red-O staining elucidated that d-psicose significantly reduced lipid accumulation in the liver. Immunohistochemical analysis showed d-psicose induced glucokinase translocation from nucleus to cytoplasm of the liver which enhances glucokinase activity and subsequent synthesis of glycogen in the liver. d-psicose also protected the pathological change of the β-cells of pancreatic islets. These data demonstrate that d-psicose controls blood glucose levels by reducing lipotoxicity in liver and by preserving pancreatic β-cell function.