Regulation of production and secretion of adrenomedullin in the cardiovascular system

Adrenomedullin (AM) has multi-functional properties, of which the vasodilatory hypotensive effect is the most characteristic. AM and its gene are ubiquitous in a variety of tissues and organs, in the cardiovascular system, as well as the adrenal medulla. AM secretion, especially in cardiovascular tissues, is regulated mainly by mechanical stressors such as shear stress, inflammatory cytokines such as interleukin (IL)-1, tumor necrosis factor (TNF), and lipopolysaccharide (LPS), hormones such as angiotensin (Ang) II and endothelin (ET)-1, and metabolic factors such as hypoxia, ischemia, or hyperglycemia. Elevation of plasma AM due to overproduction in response to one or more of these stimuli in pathological conditions may explain the raised plasma AM levels present in cardiovascular and renal diseases such as congestive heart failure, myocardial infarction, hypertension, chronic renal failure, stroke, diabetes mellitus, and septic shock. In addition to shear stress, stretching of cardiomyocytes may be another mechanical stimulus for AM synthesis and secretion. Our recent studies have shown the importance of aldosterone and additional hormonal factor on AM secretion in vascular wall.     

IGSF4: a new intercellular adhesion molecule that is called by three names,TSLC1, SgIGSF and SynCAM,by virtue of its diverse function

Members of the immunoglobulin superfamily often play key roles in intercellular adhesion. IGSF4 is a novel immunoglobulin (Ig)-like intercellular adhesion molecule. Three Ig-like domains are included in the extracellular domain of IGSF4 and mediate homophilic or heterophilic interactions independently of Ca2+. The cytoplasmic domain of IGSF4 contains the binding motifs that connect to actin fibers. Since IGSF4 has been characterized by several independent research groups, this molecule is called by three names, TSLC1, SgIGSF and SynCAM. IGSF4 was first characterized as a tumor suppressor of non-small cell lung cancer and termed TSLC1, although how IGSF4 suppresses tumor growth remains unknown. Silencing of the IGSF4 gene was primarily achieved by allelic loss and promoter methylation in this type of cancers. Soon after this discovery, IGSF4 was found to have roles in adhesion of spermatogenic cells to Sertoli cells and mast cells to fibroblasts and termed SgIGSF. Other researchers revealed that IGSF4 drives synaptic formation of neural cells and termed it SynCAM.     

Wnt2b controls retinal cell differentiation at the ciliary marginal zone

The ciliary marginal zone of the vertebrate retina contains undifferentiated progenitor cells that continue to proliferate and add new neurons and glia peripherally during the embryonic stages - even after the formation of a functional retina. To understand the molecular mechanism that controls the prolonged progenitor cell proliferation in the ciliary marginal zone, we employed a candidate molecule approach, focusing on Wnt2b (formerly know as Wnt13), which is expressed in the marginal most tip of the retina. Frizzled 4 and 5, seven-pass transmembrane Wnt receptors, were expressed in the peripheral and central part of the retina, respectively. LEF1, a downstream Wnt signaling component, was expressed at high levels in the ciliary marginal zone with expression gradually decreasing towards the central retina. The LEF1-expressing region, which is where Wnt signaling is supposedly activated, expressed a set of molecular markers that are characteristic of the progenitor cells in the ciliary marginal zone. Overexpression of Wnt2b by use of in ovo electroporation in the central retina inhibited neuronal differentiation and induced the progenitor cell markers. Blocking of the Wnt downstream signaling pathway by a dominant-negative LEF1 inhibited proliferation of the cells in the marginal area, which resulted in their premature neuronal differentiation. The progenitor cells in the ciliary marginal zone differentiated into all the neuronal and glial cell types when cultured in vitro, and they proliferated for a longer period than did centrally located progenitor cells that underwent a limited number of cell divisions. In addition, the proliferation of these progenitor cells was promoted in the presence of Wnt2b. These results suggest that Wnt2b functions to maintain undifferentiated progenitor cells in the ciliary marginal zone, and thus serves as a putative stem cell factor in the retina.     

Identification and quantification of N(epsilon)-(Hexanoyl)lysine in human urine by liquid chromatography/tandem mass spectrometry

The identification and quantification of N(epsilon)-(hexanoyl)lysine (N(epsilon)-HEL), which was found from the reactions between lipid hydroperoxide and lysine, from human urine was examined using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The N(epsilon)-HEL in the partially purified urine fraction was identified using LC/MS/MS by several approaches including precursor/product ion scans. The peak found by the multiple-reaction monitoring (MRM) of the collision-induced fragmentation of N(epsilon)-HEL was clearly observed in urine, and the elution position coincided with the synthetic standard N(epsilon)-HEL. The product, estimated N(epsilon)-HEL, was absorbed by a specific antibody to N(epsilon)-HEL. Moreover, N(alpha)-HEL, one of the plausible hexanoyl adducts from the reaction between the N(alpha) moiety of L-lysine and the peroxidized lipid, was hardly detected in urine samples, suggesting that the origin of the N(epsilon)-HEL is the peroxidized lipid-modified proteins but not artificial hexanoylated L-lysine. Using the MRM technique, the amount of urinary N(epsilon)-HEL from the control subjects (observed healthy) was estimated to be 1.58 +/- 0.23 mumol/mol of creatinine. A comparative study of the urinary N(epsilon)-HEL with an oxidative stress marker, 8-oxo-7,8-dihydro-2'-deoxyguanosine, showed a high correlation (r = 0.844) between the two biomarkers. Furthermore, the quantification of N(epsilon)-HEL in the control and diabetic urines revealed that the urinary N(epsilon)-HEL from diabetic subjects (3.21 +/- 0.65 mumol/mol of creatinine) was significantly higher than that from the control subjects.     

Chondrosarcoma of the urinary bladder and establishment of a human chondrosarcoma cell line (OCUU-6)

Chondrosarcoma is a very rare tumor of the urinary bladder, with only 4 cases reported to date. In this study, we report on a case of a 73-year-old male who presented bladder mass and right hydroureteronephrosis. Radical cystectomy, right nephrectomy and left ureterocutaneoustomy were performed, and histological study disclosed chondrosarcoma of the urinary bladder. As reported in other cases, the tumor was highly aggressive with a short clinical course, and the patient died of carcinomatous pleuritis at one month after surgery. Subsequently, we successfully established a human chondrosarcoma cell line (OCUU-6) from the pleural effusion of the patient.     

Early bone marrow hematopoietic defect in simian/human immunodeficiency virus C2/1-infected macaques and relevance to advance of disease

To clarify hematological abnormalities following infection with human immunodeficiency virus (HIV), we examined the hematopoietic capability of bone marrow by using cynomolgus monkeys infected with pathogenic simian/human immunodeficiency virus (SHIV) strain C2/1, an animal model of HIV infection. The relationship between the progress of the infection and the CD4/CD8 ratio of T lymphocytes or the amount of SHIV C2/1 viral load in the peripheral blood was also investigated. A colony assay was performed to assess the hematopoietic capability of bone marrow stem cells during the early and advanced phases of the infection. Colonies of granulocytes-macrophages (GM) were examined by PCR for the presence of the SIVmac239 gag region to reveal direct viral infection. There was a remarkable decrease in the CFU-GM growth on days 1 and 3 postinoculation, followed by recovery on day 56. During the more advanced stage, the CFU-GM growth decreased again. There was minimal evidence of direct viral infection of pooled cultured CFU-GM despite the continuously low CD4/CD8 ratios. These results indicate that the decrease in colony formation by bone marrow stem cells is reversible and fluctuates with the advance of the disease. This decrease was not due to direct viral infection of CFU-GM. Our data may support the concept that, in the early phase, production of inhibitory factors or deficiency of a stimulatory cytokine is responsible for some of the bone marrow defects described in the SHIV C2/1 model.     

Determination of sugar phosphates and nucleotides related to photosynthetic metabolism by high-performance anion-exchange liquid chromatography with fluorometric and ultraviolet detection

A high-performance anion-exchange liquid chromatography system was constructed to identify sugar phosphates and nucleotides involved in photosynthetic metabolism. First sugar phosphates and nucleotides were separated by a gradient elution with boric acid and sodium phosphate, then they were detected by a fluorescence detector (as fluorescent derivatives with arginine) and UV detector, respectively. Eight authentic sugar phosphates and 11 authentic nucleotides could be analyzed using the system. The applicability of the system to the determination of the corresponding sugar phosphates and nucleotides in extracts from only five soybean leaf discs (8.95 cm2) was shown.     

Inhibitory and enhancing effects of insertion of central polypurine tract sequence on gene expression with vectors derived from human immunodeficiency virus type 1

Reportedly, in human immunodeficiency virus type 1 (HIV) vectors, insertion of central polypurine tract (cPPT) increased expression of transgenes for a short period. To test this for a stable condition, we constructed a series of vectors carrying a Neo(r) gene as a stable marker driven by a synthetic thymidine kinase (hTK) promoter. Transduction efficiency was increased in about 2-fold and decreased in about 8-fold by insertion of the reported 178bp and our 282bp cPPTs, respectively. PCR analyses revealed that insertion of 282bp cPPT, but not 178bp cPPT, impaired integration, although it did not deteriorate nuclear transport much. Furthermore, we found that insertion of 282bp cPPT between hTK promoter and an upstream LTR sequence reduced reporter gene activity in about 5-fold. This inhibitory effect of 282bp cPPT may partly account for the observed decrease in transduction efficiency. We suggest that actual effect of cPPT insertion should be examined in each HIV vector.     

Disruption of a single copy of the p38alpha MAP kinase gene leads to cardioprotection against ischemia-reperfusion

The p38 mitogen-activated protein kinase (p38) is activated in the heart during ischemia-reperfusion. However, it is not clear whether the activation of p38 is the protective response or the kinase mediates the cellular damage by ischemia-reperfusion. We examined the role of p38alpha in ischemia-reperfusion injury by studying p38alpha(+/-) mice. The p38alpha protein level in the p38alpha(+/-) heart was 50+/-8.7% compared with that in the p38alpha(+/+) heart. Upon reperfusion following ischemia for 25min, p38alpha activity was transiently increased. The maximum level of p38 activity in p38alpha(+/-) was 60+/-10.5% compared with that in p38alpha(+/+). In the p38alpha(+/+) heart, 25min ischemia and 2h reperfusion resulted in necrotic injury (37.1+/-2.7% of the area at risk), whereas infarct size was drastically reduced to 7.2+/-0.7% in the p38alpha(+/-) heart. These suggested that p38alpha plays a pivotal role in the signal transduction pathway mediating myocardial cell death caused by ischemia-reperfusion.