Proteases involved in long-term potentiation

Much attention has been paid to proteases involved in long-term potentiation (LTP). Calpains, Ca-dependent cysteine proteases, have first been demonstrated to be the mediator of LTP by the proteolytic cleavage of fodrin, which allows glutamate receptors located deep in the postsynaptic membrane to move to the surface. It is now generally considered that calpain activation is necessary for LTP formation in the cleavage of substrates such as protein kinase Czeta, NMDA receptors, and the glutamate receptor-interacting protein. Recent studies have shown that serine proteases such as tissue-type plasminogen activator (tPA), thrombin, and neuropsin are involved in LTP. tPA contributes to LTP by both receptor-mediated activation of cAMP-dependent protein kinase and the cleavage of NMDA receptors. Thrombin induces a proteolytic activation of PAR-1, resulting in activation of protein kinase C, which reduces the voltage-dependent Mg2+ blockade of NMDA receptor-channels. On the other hand, neuropsin may act as a regulatory molecule in LTP via its proteolytic degradation of extracellular matrix protein such as fibronectin. In addition to such neuronal proteases, proteases secreted from microglia such as tPA may also contribute to LTP. The enzymatic activity of each protease is strictly regulated by endogenous inhibitors and other factors in the brain. Once activated, proteases can irreversibly cleave peptide bonds. After cleavage, some substrates are inactivated and others are activated to gain new functions. Therefore, the issue to identify substrates for each protease is very important to understand the molecular basis of LTP.     

Targeted disruption of LIG-1 gene results in psoriasiform epidermal hyperplasia

We have designed a chimeric promoter that can be stimulated by various pro-inflammatory mediators and so drive the expression of therapeutic genes under inflammatory conditions. The promoter has two parts, the [-247/+20] fragment of the human type IIA secreted phospholipase A2 gene promoter, which is stimulated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), and a double peroxisome proliferator-activated receptor response element that is activated by some eicosanoids and by non-steroidal anti-inflammatory drugs (NSAIDs). Transfection experiments using rabbit articular chondrocytes in primary culture showed that this chimeric promoter produced a low basal activity and was induced by NSAIDs, WY-14643, IL-1beta, and 15-deoxy Delta12,14 prostaglandin J2. The latter two compounds stimulated the promoter synergistically.     

Identification of a soluble form phospholipase A2 receptor as a circulating endogenous inhibitor for secretory phospholipase A2

Venomous snakes have various types of phospholipase A(2) inhibitory proteins (PLIs) in their circulatory system to protect them from attack by their own phospholipase A(2)s (PLA(2)s). Here we show the first evidence for the existence of circulating PLI against secretory PLA(2)s (sPLA(2)s) in mammals. In mouse serum, we detected specific binding activities of group IB and X sPLA(2)s, which was in contrast with the absence of binding activities in serum prepared from mice deficient in PLA(2) receptor (PLA(2)R), a type I transmembrane glycoprotein related to the C-type animal lectin family. Western blot analysis after partial purification with group IB sPLA(2) affinity column confirmed the identity of serum sPLA(2)-binding protein as a soluble form of PLA(2)R (sPLA(2)R) that retained all of the extracellular domains of the membrane-bound receptor. Both purified sPLA(2)R and the recombinant soluble receptor having all of the extracellular portions blocked the biological functions of group X sPLA(2), including its potent enzymatic activity and its binding to the membrane-bound receptor. Protease inhibitor tests with PLA(2)R-overexpressing Chinese hamster ovary cells suggested that sPLA(2)R is produced by cleavage of the membrane-bound receptor by metalloproteinases. Thus, sPLA(2)R is the first example of circulating PLI that acts as an endogenous inhibitor for enzymatic activities and receptor-mediated functions of sPLA(2)s in mice.     

Characterization and expression of embryonic and adult globins of the teleost Oryzias latipes (medaka)

Using the teleost Oryzias latipes (medaka), we isolated three embryonic globin cDNAs (em.alpha-0, em.alpha-1, and em.beta-1) from the embryos 5 days after fertilization (at 30 degrees C) and two adult globin cDNAs (ad.alpha-1 and ad.beta-1) from the kidney of the fully-grown adult fish, and predicted their amino acid sequences. Molecular phylogenetic analysis showed that the embryonic globins were highly homologous in amino acid sequence to the embryonic globins previously identified in rainbow trout and zebrafish, and that they formed a monophyletic group among the teleostean globin molecules. They were clearly discriminated from the adult globin of the medaka. RT-PCR analysis showed that the embryonic globin mRNAs were intensely expressed in stage 30 and 38 embryos and in young fish 30 days after hatching. The level of expression decreased drastically after the young fish stage, and was low in fully-grown adult fish. The adult alpha globin mRNA ad.alpha-1 was scarcely expressed in the embryos, and the level of expression gradually increased in young to fully-grown adult fish. Unexpectedly, the adult beta globin mRNA ad.beta-1 was expressed throughout life, from the early embryonic stage to the fully-grown adult stage. This expression profile was quite different from that of the rainbow trout previously investigated. Some globins of the medaka were expressed both in primitive hematopoiesis and in definitive hematopoiesis.     

Cytology of basaloid squamous carcinoma of the bronchus. A case report

BACKGROUND: The cytologic appearance of basaloid squamous carcinoma (BSC) arising in the lower respiratory tract has not been described very well because of its rarity. This article describes a surgical case of bronchial BSC and provides the first documentation of the sputum and imprint cytologic features of the tumor. CASE: A 74-year-old man presented with hemoptysis. An abnormal intrabronchial mass was revealed by computed tomography and bronchoscopy. Preoperative cytology and biopsy showed that the mass was composed of small, round, atypical cells, but correct diagnosis was difficult. Under a tentative diagnosis of small round cell carcinoma, a right lobectomy was performed. The resected tumor was composed of small cells showing peripheral palisading and partial epidermoid differentiation. There was no glandular differentiation. Focal necrosis was also noted. Immunohistochemical markers for smooth muscle and neuroendocrine cells were negative. The tumor was eventually diagnosed as BSC or basaloid carcinoma (BC) with squamous differentiation. CONCLUSION: It is important to recognize this disease, especially when undetermined small round cell carcinoma is noted in cytologic specimens, in order to properly assess prognosis. Cytologic detection of nuclear palisading of the neoplastic cells, one of the hallmarks of the disease, may be difficult, however, careful examination to reveal neoplastic cells showing squamous differentiation appears helpful for diagnosis.     

Intracellular signal-responsive artificial gene regulation for novel gene delivery

We describe two types of artificial gene-regulation systems responding to cyclic AMP-dependent protein kinase (PKA) or caspase-3. These molecular systems use newly synthesized cationic polymers, PAK and PAC. The PAK polymer includes substrate oligopeptide for PKA, ARRASLG, as receptor of PKA signal, while the PAC polymer possesses oligopeptide that is comprised of a substrate sequence of caspase-3, DEVD, and a cationic oligolysine, KKKKKK. These polymers formed stable complexes with DNA to totally suppress the gene expression. However, PKA or caspase-3 signal disintegrates the PAK-DNA or the PAC-DNA complex, respectively. This liberates the DNA and activated the gene expression. These systems are the first concept of an intracellular signal-responsive gene-regulation system using artificial polymer. We expect that these systems can be applied to the novel highly cell specific gene delivery strategy that is involved in our previously proposed new drug delivery concept, the drug delivery system based on responses to cellular signals.     

IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-kappaB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-kappaB activation within 45 min after stimulation. A blockade of NF-kappaB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1beta or IL-17 + tumor necrosis factor (TNF)-alpha enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-alpha on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.     

Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life

MDC9 (ADAM9/meltrin gamma) is a widely expressed and catalytically active metalloprotease-disintegrin protein that has been implicated in the ectodomain cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and as an alpha secretase for the amyloid precursor protein. In this study, we evaluated the expression of MDC9 during development and generated mice lacking MDC9 (mdc9(-/-) mice) to learn more about the function of this protein during development and in adults. During mouse development, MDC9 mRNA is ubiquitously expressed, with particularly high expression levels in the developing mesenchyme, heart and brain. Despite the ubiquitous expression of MDC9, mdc9(-/-) mice appear to develop normally, are viable and fertile, and do not have any major pathological phenotypes compared to wild-type mice. Constitutive and stimulated ectodomain shedding of HB-EGF is comparable in embryonic fibroblasts isolated from mdc9(-/-) and wild-type mice, arguing against an essential role of MDC9 in HB-EGF shedding in these cells. Furthermore, there were no differences in the production of the APP alpha and gamma secretase cleavage product (p3) and of beta- and gamma-secretase cleavage product (A beta) in cultured hippocampal neurons from mdc9(-/-) or wild-type mice, arguing against an essential major role of MDC9 as an alpha-secretase in mice. Further studies, including functional challenges and an evaluation of potential compensation by, or redundancy with, other members of the ADAM family or perhaps even with other molecules will be necessary to uncover physiologically relevant functions for MDC9 in mice.