Multiple downstream signalling pathways from ROCK, a target molecule of Rho small G protein, in reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells

BACKGROUND: In Madin-Darby canine kidney cells, Rho small G protein regulates formation of stress fibres, focal adhesions, and peripheral bundles through reorganization of the actin cytoskeleton. There are two morphologically distinguishable types of Rho-regulated stress fibres: parallel and stellate. Of these, effects of Rho small G protein, mDia1 regulates the formation of parallel stress fibres, whereas ROCK regulates the formation of stellate stress fibres, peripheral bundles and focal adhesions. Both mDia1 and ROCK are direct downstream targets of Rho small G protein. RESULTS: The ROCK-induced formation of stellate stress fibres is regulated mainly through the myosin light chain kinase-dependent phosphorylation of myosin light chain and the LIM-kinase-dependent phosphorylation of cofilin. The ROCK-induced formation of focal adhesions is mainly regulated through a downstream pathway of ROCK other than myosin light chain and cofilin. The ROCK-induced formation of peripheral bundles is regulated at least through ERM proteins, but not through the myosin light chain or cofilin. CONCLUSION: Our present and previous findings suggest the presence of multiple downstream signalling pathways from ROCK to reorganization of the actin cytoskeleton in Madin-Darby canine kidney cells.     

Effect of Pseudomonas aeruginosa exotoxin A on endotoxin-induced tumour necrosis factor production in murine lung

The ability of several Pseudomonas aeruginosa exo-enzymes, including exotoxin A (ETA), to induce inflammation and their influence on endotoxin-induced tumour necrosis factor (TNF) production in murine lung were evaluated. Intratracheal administration of lipopolysaccharide (LPS; 0.1-10 microg/mouse), 2(-1) LD50 of P. aeruginosa alkaline protease (7.5 microg/mouse) and elastase (1.2 microg/mouse) elevated total cell number and the percentage of neutrophils in broncho-alveolar lavage fluid (BALF), whereas ETA (0.1 microg/mouse) did not. LPS induced TNF production in BALF in a dose-dependent manner, whereas the P. aeruginosa exo-enzymes did not. When ETA was inoculated into the respiratory tract before LPS, production of TNF in BALF was significantly suppressed in a dose-dependent manner. ETA also suppressed TNF production by alveolar macrophages (AMs) stimulated with LPS in vitro. Flow cytometric analysis showed that ETA markedly reduced the expression of CD14 and CD11c/CD18 on the surface of AMs. ETA also depressed partially the expression of TNF-alpha mRNA in AMs. These findings suggest that ETA regulates TNF production in murine lung by suppressing LPS receptor expression, mRNA expression and protein synthesis and/or secretion of TNF.     

Unique three-way translocation, t(3;14;18)(q27;q32;q21), in follicular lymphoma

A 43-year-old woman was diagnosed as having stage IV follicular lymphoma. Phenotypically, the lymphoma cells were CD5(-), CD10(+), CD19(+), CD20(+), CD23(-), HLA-DR(+), and IgM-lambda(+). Conventional chromosomal analysis showed a three-way t(3;14;18)(q27;q32;q21) in the lymphoma cells, which was confirmed by spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH). Immunohistochemistry revealed that both BCL2 and BCL6 proteins were expressed in the lymphoma cells, whereas only the BCL6 gene, and not the BCL2 gene, was rearranged by Southern blotting. The patient received combination chemotherapy and has been well for 3 years. This is the first reported case showing a three-way translocation involving 2 major lymphoma-specific abnormalities, 3q27 and t(14;18)(q32;q21).     

Effects of body position on snoring in apneic and nonapneic snorers

STUDY OBJECTIVES: The positional dependency of obstructive sleep apnea (OSA) is well known, but objective evidence for the positional effect on snoring is lacking. The aim of this study is to elucidate the effect of body position on snoring, and that of sleep stage as well. DESIGN: Retrospective analysis of the effects of body position and sleep stage on snoring in nonapneic snorers (snorer group) and OSA patients (apneic group). SETTING: A sleep laboratory in a national hospital in Japan. PATIENTS: Seventy-two patients who complained of habitual snoring and underwent overnight polysomnography. INTERVENTIONS: N/A MEASUREMENTS AND RESULTS: In the lateral position, most subjects in the snorer group showed decreased snoring both in time (p = 0.0004) and intensity (p = 0.0003), but subjects in the apneic group showed variable changes. In the apneic group, the positional dependency of snoring (the ratio of lateral value to supine value) was correlated with supine apnea-hypopnea index (AHI), that is, OSA patients with higher supine AHI tended to show increased snoring in the lateral position. AS to the effect of sleep stage, snoring was increased in deeper non-rapid eye movement sleep and decreased in rapid eye movement sleep in a given position. CONCLUSIONS: This study demonstrated that the positional dependency is different between nonapneic snorers and OSA patients. Most of the nonapneic snorers snore less in the lateral position than in the supine position in contrast to OSA patients who often fail to decrease snoring even in the lateral position.     

The PPARgamma ligand, 15-Deoxy-Delta12,14-PGJ2, regulates apoptosis-related protein expression in cholangio cell carcinoma cells

PPARgamma is known to induce apoptosis in malignant tumor cells, but the mechanism of this induction is not well understood. We investigated induction of apoptosis with 15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), a PPARgamma ligand, in cholangio cell carcinoma (CCC) cells (RBE, ETK-1 or HuCCT-1). Apoptosis was induced in RBE and ETK-1 cells with 15d-PGJ2, but not in HuCCT-1 cells, although PPARgamma was expressed in all CCC cells. Apoptosis-related proteins were also expressed, including FLIP, bclx, Apaf-1 and XIAP, but expression levels differed among the three cell lines. RBE cells treated with 15d-PGJ2 showed caspase activation, and it appeared that PPARgamma-induced apoptosis was dependent on caspase activation. However, neither ETK-1 nor HuCCT-1 cells showed significant activation of caspase-8 or -3 with 15d-PGJ2 treatment, raising the possibility of a caspase-independent apoptosis induction pathway. XIAP was down-regulated by 15d-PGJ2 in all three CCC cell lines. Therefore, 15d-PGJ2 induces apoptosis in CCC cells via caspase-dependent or independent pathways. 15d-PGJ2 may also induce down-regulation of XIAP and may promote caspase cascade activation through TNF-family receptor signaling pathways.     

Bestatin, an inhibitor of aminopeptidase B, suppresses the proliferation and differentiation of human B-cells in vitro

Bestatin, an inhibitor of aminopeptidase B, was examined for its effect on B-cell activation. Small, dense B-cells from human tonsil samples were isolated by Percoll density gradients from non-rosetted (E-) cells and were used as target cells. Although bestatin was not cytotoxic towards B-cells, it inhibited the proliferative response of B-cells induced by SAC- or PMA-stimulation. The inhibition of cell proliferation by bestatin was manifested as cell arrest caused by the selective block of G1b to S phase transition. This inhibitory effect was prevented by the addition of B-cell growth factor (BCGF) or interleukin-2 (IL-2). The presence of BCGF or IL-2 at the initiation of the culture prevented the bestatin-mediated suppressive effect on B-cell proliferation. Bestatin also has a direct inhibitory effect on the differentiation of B-cells independent of its suppressive effect on B-cell proliferation, which was not relieved by T-cell help. Conversely, bestatin suppressed neither proliferation nor Ig secretion of human B lymphoblastoid cell lines, although aminopeptidase activities on the membrane of these cell lines were strongly inhibited by bestatin. These results indicated that bestatin selectively suppressed normal B-cell proliferation and differentiation. Although several studies have demonstrated that bestatin has immunopotentiating effects in tumor-bearing subjects, the above results indicated that the mechanism of immunopotentiation by bestatin is not a direct stimulatory effect on B-cells.     

ELISAs for free light chains of human immunoglobulins using monoclonal antibodies: comparison of their specificity with available polyclonal antibodies

For the measurement of human immunoglobulin free light chains (LCs) in clinical samples, highly specific assays for free LCs are required to discriminate them from LC portions of intact immunoglobulins (bound LCs). To develop specific enzyme-linked immunosorbent assays (ELISAs) for free LCs, two anti-free LC kappa and lambda monoclonal antibodies (MAbs) were raised by a mouse/mouse hybridoma technique. We compared the specificities of these two MAbs with those of six commercially available anti-free LC antisera, which are widely used in free LC immunoassays. Comparative titrations against free LCs and intact IgGs showed the MAbs had less cross-reactivity to intact IgGs, while the four of six antisera had high reactivity to intact IgGs. Using these MAbs, we developed LC kappa and LC lambda ELISAs with ranges from 7.8 to 500 micro g/l of free LCs and less cross-reactivity to intact IgGs (less than 0.12%). On the other hand, ELISAs with anti-free LC antisera showed low specificity and/or sensitivity. Thus, the use of these MAbs may provide reliable methods for specific measurements of free LCs in clinical samples.     

Single sperm analysis of the CAG repeats in the gene for Machado-Joseph disease (MJD1): evidence for non-Mendelian transmission of the MJD1 gene and for the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability

To investigate the mechanism of the meiotic instability of expanded CAG repeats in the gene for Machado-Joseph disease (MJD1), we analyzed the CAG repeat sizes of 1036 single sperm from six individuals with Machado- Joseph disease (MJD). The segregation ratio between single sperm with an expanded allele and those with a normal allele is significantly different (P <0.0001) from the expected 1:1 segregation ratio, which demonstrates segregation distortion of expanded alleles in male meiosis. In single sperm from individuals with the [expanded (CAG)n- CGG]/[normal (CAG)n-GGG] genotype, significantly greater instability of the CAG repeat was observed compared with single sperm from individuals with the [expanded (CAG)n-CGG]/[normal (CAG)n-CGG] genotype (F-test, P <0.001). These findings in single sperm confirm non-Mendelian transmission of the MJD1 gene and the effect of the intragenic CGG/GGG polymorphism on the intergenerational instability of the CAG repeats in the MJD1 gene, which have been observed in clinical and genetic studies. Our results indicate similarities and dissimilarities between MJD and Huntington's disease or myotonic dystrophy in terms of the inter-allelic interaction, segregation distortions and size distribution of trinucleotide repeats in mutant alleles. Further study is required to determine whether there is a common mechanism underlying the instability of the triplet repeats in 'triplet repeat diseases'.