Effects of nitric oxide on matrix metalloproteinase-2 production by rheumatoid synovial cells

Nitric oxide (NO) is a multifunctional messenger molecule generated from L-arginine by a family of enzymes, including nitric oxide synthase (NOS). This study was performed to examine whether NO modulates the production of matrix metalloproteinases (MMPs), which degrade all components of extracellular matrix (ECM), in rheumatoid synovial cells. We investigated the effects of exogenously generated NO by a NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP), on the MMPs production by rheumatoid synovial cells. Culture media conditioned by SNAP-treated synovial cells were examined by gelatin zymography and immunoblot analysis. Incubation of synovial cells with SNAP resulted in gelatinase A production in a dose-dependent fashion. Furthermore, RT-PCR analysis demonstrated that MMP-2 mRNA expression was induced in SNAP-treated synovial cells. In contrast, SNAP did not influence the production of TIMP-1 and TIMP-2, which preferentially inhibit MMP-2, by rheumatoid synovial cells. Our data indicate that NO could modulate MMP production by rheumatoid synovial cells and therefore contribute to ECM degradation of articular components in RA.     

Three-dimensional image analysis of the hepatic restructuring process in chronic active hepatitis

OBJECTIVE: To elucidate the process of hepatic restructuring in the course of chronic hepatitis from a morphologic viewpoint. STUDY DESIGN: The three-dimensional (3-D) liver structure was investigated by computer-aided reconstruction in five cases (one autopsy and four surgical cases) of chronic active hepatitis (type C), including early to late stages of restructuring. RESULTS: Our 3-D reconstruction revealed the following. At an early stage, portal and periportal inflammation and fibrosis widened the portal tracts, giving rise to the formation of portal-to-portal and portal-to-hepatic venous connections, although most central veins were still located at an almost normal site in the hepatic lobules. In a middle stage, bridging fibrosis developed to create a network of interstitium where the central veins were rather decreased in number, with regenerative nodules multiplying in the parenchyma. At the late stage, the lobular structure was destroyed, and the parenchyma consisted uniformly of regenerative nodules, with remaining but rearranged lobules among them. CONCLUSION: The above changes of liver structure suggest that in cirrhogenesis from chronic hepatitis, a combination of nodular regeneration and formation of an interstitial network come to replace the normal lobular structure, hastening the development of liver cirrhosis.     

Bruton's tyrosine kinase (Btk) associates with protein kinase C mu

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B-cells. Mutational defects are associated with a severe immunodeficiency syndrome, X-chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCmu, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B-cells with phorbol ester or H(2)O(2) affected Btk/PKCmu interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCmu. Transient overexpression of PKCmu deletion mutants as well as expression of selected PKCmu domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH-TH domain. These data show the existence of a PKCmu/Btk complex in vivo and identify two PKCmu domains that participate in Btk interaction.     

Determination of the entrapped volume of liposomes: dilution method

A novel method was developed for the determination of the entrapped volume of liposomes. The obtained values of the entrapped volume by our "dilution method" agreed very well with those of the conventional "quenching method." The dilution method also offered the great advantages of simple procedure and high reproducibility. The principle and validity of our method are discussed.     

Activation of human T lymphocytes by ganglioside-containing liposomes

We investigated the in vitro stimulatory effect of ganglioside (G_M3, G_D1a, G_D1b, G_T1b, or G_Q1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca²⁺]₁) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The G_D1a- and G_T1b-containing liposomes significantly increased [Ca²⁺]₁ of human T lymphocytes compared with the G_M3-, G_D1b- and G_Q1b-containing ones. The response of CD8⁺ and CD4⁺ cells was significantly higher than that of CD20⁺ cells. Our results show that the increase in [Ca²⁺]_i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8⁺ and CD4⁺ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.     

Structure and chromosome mapping of the human SIX4 and murine Six4 genes

Six4, a member of the homeobox gene subfamily (Six), is expressed in a developmentally regulated fashion, and supposed to be involved in embryogenesis. We cloned the human SIX4 and murine Six4 genomic DNAs and determined their structures. The structure, including the 5' upstream region of both genes, was well conserved suggesting the conserved function and regulation of these genes. Human SIX4 was mapped to chromosome 14q23.     

Tumor marker measurements of cells in a fine needle used for aspiration cytology

OBJECTIVE: To investigate whether the needle washing could yield sufficient cells for tumor marker (TM) measurements as an ancillary technique to ensure the accuracy of fine needle aspiration cytology (FNAC) of tumors. STUDY DESIGN: After obtaining preliminary data that aspirated tumor cells within a 22-gauge needle could be collected by washing it with distilled water for TM measurements, we studied tumor cell numbers and TM values obtained by washing a 22-gauge needle directly after tumor aspiration and another needle after FNAC. RESULTS: Using 8 resected hepatobiliary and pancreatic carcinomas, the used needles yielded 16.8+/-10.5 x 10(4) cells per milliliter. Used needles from 6 adenocarcinomas expelled 479.2+/-406.5 ng/mL of carcinoembryonic antigen, and 6,561.3+/-5,713.1 ng/mL of CA 19-9, while the needles from 2 hepatomas showed normal values of those markers. CONCLUSION: A needle used for FNAC contains sufficient cells for TM measurements, which can be ancillary to the differential diagnosis.