Endogenous Blocker Research Articles

Non-invasive diagnosis of acute rejection in kidney transplants with delayed graft function

Delayed graft function (DGF) often occurs in kidney transplants from deceased donors. We wanted to provide studies giving more accurate non-invasive tests for acute rejection (AR). Using real-time PCR, we examined the expression of cytolytic molecules such as perforin, granzyme B, and fas-ligand along with serpin proteinase inhibitor-9. We also measured the expression of FOXP3, a characteristic gene of T-regulatory cells known to be involved in AR. These studies were conducted on peripheral blood monocytes, urinary cells, and 48 surveillance kidney biopsies taken from a total of 35 patients with DGF. Of these patients, 20 had a histopathological diagnosis of AR, whereas other 28 had characteristics of acute tubular necrosis (ATN). Expression of cytolytic and apoptotic-associated genes in the biopsy tissue, peripheral blood leukocytes, and urinary cells was significantly higher in patients with AR than that in patients with ATN. Diagnostic parameters associated with FOXP3 gene expression were most accurate in peripheral blood leukocytes and urine cells with sensitivity, specificity, positive and negative predictive values, and accuracy between 94 and 100%. Our study shows that quantification of selected genes in peripheral blood leukocytes and urinary cells from renal transplant patients with DGF may provide a useful and accurate non-invasive diagnosis of AR.

The molecular basis of chloroquine block of the inward rectifier Kir2.1 channel

Although chloroquine remains an important therapeutic agent for treatment of malaria in many parts of the world, its safety margin is very narrow. Chloroquine inhibits the cardiac inward rectifier K(+) current I(K1) and can induce lethal ventricular arrhythmias. In this study, we characterized the biophysical and molecular basis of chloroquine block of Kir2.1 channels that underlie cardiac I(K1). The voltage- and K(+)-dependence of chloroquine block implied that the binding site was located within the ion-conduction pathway. Site-directed mutagenesis revealed the location of the chloroquine-binding site within the cytoplasmic pore domain rather than within the transmembrane pore. Molecular modeling suggested that chloroquine blocks Kir2.1 channels by plugging the cytoplasmic conduction pathway, stabilized by negatively charged and aromatic amino acids within a central pocket. Unlike most ion-channel blockers, chloroquine does not bind within the transmembrane pore and thus can reach its binding site, even while polyamines remain deeper within the channel vestibule. These findings explain how a relatively low-affinity blocker like chloroquine can effectively block I(K1) even in the presence of high-affinity endogenous blockers. Moreover, our findings provide the structural framework for the design of safer, alternative compounds that are devoid of Kir2.1-blocking properties.

About the possible role of endogenous modulators of β-adrenoreactivity and M-cholinoreactivity in the pathogenesis of asthma

Ability of blood sera from 19 asthmatic children and 56 asthmatic adults to modify reactivity of β-adrenoreceptors and M-cholinoreceptors in isolated smooth muscles of the uterus horns from nonpregnant rats (n = 234) and cow trachea (n = 36) have shown that asthma presence did not change serum myocyte-stimulating activity but reduced the sensitizing activity of β-adrenoreceptors and the blocking activity of M-cholinoreceptors and enhanced the blocking activity of β-adrenoreceptors. The similar changes were also seen in child urine and indicated the decreased level of the endogenous sensitizer of β-adrenoreceptors and the growing level of the endogenous blocker of β-adrenoreceptors in asthma patients and the decreased level of endogenous blocker of M-cholinoreceptors in certain asthmatics. These events could provide asthma occurrence.

Transplant: immunology and treatment of rejection

Transplant: immunology and treatment of rejection

Production of resurgent current in NaV1.6-null Purkinje neurons by slowing sodium channel inactivation with beta-pompilidotoxin.

Voltage-gated tetrodotoxin-sensitive sodium channels of Purkinje neurons produce "resurgent" current with repolarization, which results from relief of an open-channel block that terminates current flow at positive potentials. The associated recovery of sodium channels from inactivation is thought to facilitate the rapid firing patterns characteristic of Purkinje neurons. Resurgent current appears to depend primarily on NaV1.6 alpha subunits, because it is greatly reduced in "med" mutant mice that lack NaV1.6. To identify factors that regulate the susceptibility of alpha subunits to open-channel block, we voltage clamped wild-type and med Purkinje neurons before and after slowing conventional inactivation with beta-pompilidotoxin (beta-PMTX). beta-PMTX increased resurgent current in wild-type neurons and induced resurgent current in med neurons. In med cells, the resurgent component of beta-PMTX-modified sodium currents could be selectively abolished by application of intracellular alkaline phosphatase, suggesting that, like in NaV1.6-expressing cells, the open-channel block of NaV1.1 and NaV1.2 subunits is regulated by constitutive phosphorylation. These results indicate that the endogenous blocker exists independently of NaV1.6 expression, and conventional inactivation regulates resurgent current by controlling the extent of open-channel block. In Purkinje cells, therefore, the relatively slow conventional inactivation kinetics of NaV1.6 appear well adapted to carry resurgent current. Nevertheless, NaV1.6 is not unique in its susceptibility to open-channel block, because under appropriate conditions, the non-NaV1.6 subunits can produce robust resurgent currents.

Serine proteinase inhibitor-9, an endogenous blocker of granzyme B/perforin lytic pathway, is hyperexpressed during acute rejection of renal allografts.

Serine proteinase inhibitor (PI)-9 with a reactive center P1 (Glu)-P1' is a natural antagonist of granzyme B and is expressed in high levels in cytotoxic T lymphocytes (CTL). In view of the role of CTL in acute rejection, we explored the hypothesis that PI-9 would be hyperexpressed during acute rejection. Because PI-9 can protect CTL from its own fatal arsenal and potentially enhance the vitality of CTL, we examined whether PI-9 levels correlate with the severity of rejection as well as predict subsequent graft function. We obtained 95 urine specimens from 87 renal allograft recipients. RNA was isolated from the urinary cells and mRNA encoding PI-9, granzyme B, or perforin and a constitutively expressed 18S rRNA was measured with the use of real-time quantitative polymerase chain reaction assay, and the level of expression was correlated with allograft status. The levels of PI-9 (P=0.001), granzyme B (P<0.0001), and perforin mRNAs (P<0.0001), but not the levels of 18S rRNA (P=0.54), were higher in the urinary cells from the 29 patients with a biopsy-confirmed acute rejection than in the 58 recipients without acute rejection. PI-9 levels were significantly higher in patients with type II or higher acute rejection changes compared with those with less than type II changes (P=0.01). Furthermore, PI-9 levels predicted subsequent graft function (r=0.43, P=0.01). PI-9 mRNA levels in urinary cells are diagnostic of acute rejection, predict renal allograft histology grade, and predict functional outcome following an acute rejection episode.

食品成分の化学的解析とその機能に関する栄養生化学的研究

Chemical and biochemical aspects of various food constituents were investigated in detail to elucidate their physiological functions and nutritional significance. 1) A nitrogen-protein conversion factor of 4.0 was employed to calculate the crude protein contents of cultivated mushrooms. 4-Aminobenzoate hydroxylase newly isolated from Agaricus bisporus was shown to be useful for selective determination of 4-aminobenzoate in pancreatic function diagnosis. 2) NG-Methylarginine derived from proteins methylated post-translationally was demonstrated to be actively metabolized by a newly identified enzyme, dimethylargininase, suggesting that this metabolic system might take part in the regulation of nitric oxide synthesis by removing methylated arginine as an endogenous blocker. The major origin of endogenous dimethylamine as a possible precursor of potent carcinogens in animals was demonstrated to be a degradation product of NG-monomethylarginine by dimethylargininase. 3) The major food allergenic protein occurring in soybean was identified as a 34-kDa oil-body-associated protein, which is classified as a member of the papain-like thiol proteinase family highly homologous to house dust mite allergen, Der p I, the most frequent causative allergen for young patients with atopic dermatitis.

Specific inhibition of long-lasting, L-type calcium channels by synthetic parathyroid hormone.

The effect of an active synthetic N-terminal fragment of bovine parathyroid hormone (bPTH), bPTH-(1-34), on Ca2+ channels was studied in mouse neuroblastoma cells (N1E-115). With the whole-cell variation of the patch-clamp technique, T (transient) and L (long-lasting) types of Ca2+ currents were identified. Pharmacological characterization showed that the L current was amplified by the Ca2+ channel stimulator BAY K-8644, but the T current was unaffected. The administration of bPTH-(1-34) produced dose-related inhibition of the L current, which could be reversed by BAY K-8644. The peptide had no effect on the T current. In addition, use of the fluorescent indicator fura-2 showed that bPTH-(1-34) inhibited the KCl-stimulated increase in intracellular free Ca2+ in neuroblastoma cells with L channels but not in cells with T channels. An inactivated (oxidized) preparation of bPTH-(1-34) failed to affect the L current. High-affinity binding of labeled PTH analog to these neuroblastoma cells was also demonstrated. In addition, bPTH-(1-34) inhibited the L current in cultured vascular smooth muscle cells from rat tail artery. These data indicate that, in some tissues, PTH can act as an endogenous blocker of Ca2+ entry.

Fluorimetric micro-determination of kynurenic acid, an endogenous blocker of neurotoxicity, by high-performance liquid chromatography

Fluorimetric micro-determination of kynurenic acid, an endogenous blocker of neurotoxicity, by high-performance liquid chromatography