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.     

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.     

Metalloproteinase-mediated shedding of heparin-binding EGF-like growth factor and its pathophysiological roles

Heparin-binding EGF-like growth factor (HB-EGF) exists as a membrane-anchored form (proHB-EGF) and as its soluble cleaved product (sHB-EGF). The conversion (ectodomain shedding) of proHB-EGF to sHB-EGF is tightly regulated by specific metalloproteinases. Ectodomain shedding plays a central role in GPCR-mediated EGFR transactivation. Antagonizing metalloproteinases can inhibit EGFR transactivation and might be of therapeutic value, for example in cardiac hypertrophy, skin remodeling and tumor growth.     

Segmental development of reticulospinal and branchiomotor neurons in lamprey: insights into the evolution of the vertebrate hindbrain

During development, the vertebrate hindbrain is subdivided along its anteroposterior axis into a series of segmental bulges called rhombomeres. These segments in turn generate a repeated pattern of rhombomere-specific neurons, including reticular and branchiomotor neurons. In amphioxus (Cephalochordata), the sister group of the vertebrates, a bona fide segmented hindbrain is lacking, although the embryonic brain vesicle shows molecular anteroposterior regionalization. Therefore, evaluation of the segmental patterning of the central nervous system of agnathan embryos is relevant to our understanding of the origin of the developmental plan of the vertebrate hindbrain. To investigate the neuronal organization of the hindbrain of the Japanese lamprey, Lethenteron japonicum, we retrogradely labeled the reticulospinal and branchial motoneurons. By combining this analysis with a study of the expression patterns of genes identifying specific rhombomeric territories such as LjKrox20, LjPax6, LjEphC and LjHox3, we found that the reticular neurons in the lamprey hindbrain, including isthmic, bulbar and Mauthner cells, develop in conserved rhombomere-specific positions, similar to those in the zebrafish. By contrast, lamprey trigeminal and facial motor nuclei are not in register with rhombomere boundaries, unlike those of gnathostomes. The trigeminal-facial boundary corresponds to the rostral border of LjHox3 expression in the middle of rhombomere 4. Exogenous application of retinoic acid (RA) induced a rostral shift of both the LjHox3 expression domain and branchiomotor nuclei with no obvious repatterning of rhombomeric segmentation and reticular neurons. Therefore, whereas subtype variations of motoneuron identity along the anteroposterior axis may rely on Hox-dependent positional values, as in gnathostomes, such variations in the lamprey are not constrained by hindbrain segmentation. We hypothesize that the registering of hindbrain segmentation and neuronal patterning may have been acquired through successive and independent stepwise patterning changes during evolution.     

Expression of equine interleukin-18 by baculovirus expression system and its biologic activity

The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supernatant from AcEIL-18 infected cells showed a synergistic effect with recombinant human interleukin-12 for induction of interferon-gamma gene expression in equine peripheral mononuclear cells, indicating that the recombinant equine IL-18 expressed in this study also has biological activity without any treatment.     

Structure-based virtual screening and biological evaluation of potent and selective ADAM12 inhibitors

We describe a series of potent and selective inhibitors of ADAM12 that were discovered using computational screening of a focused virtual library. The initial structure-based virtual screening selected 64 compounds from a 3D database of 67,062 molecules. Being evaluated by a cell-based ADAM12 activity assay, compounds 5, 11, 14, 16 were further identified as the potent and selective inhibitors of ADAM12 with low nanomolar IC₅₀ values. The mechanism underlying the potency and selectivity of a representative compound, 5, was investigated through molecular docking studies.     

Dual intracellular signaling by proteolytic cleavage of membrane-anchored heparin-binding EGF-like growth factor

Heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a membrane-anchored precursor (proHB-EGF) that is cleaved to release a soluble HB-EGF by specific metalloproteases. Proteolytic cleavage of proHB-EGF yields amino- and carboxy-terminal fragments (HB-EGF and HB-EGF-C). Recent studies indicate that the processing of proHB-EGF is strictly regulated and involved in a variety of biological processes and that not only HB-EGF but also HB-EGF-C functions as a signaling molecule. ProHB-EGF generates dual intracellular signaling molecules by its proteolytic cleavage.