Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

Characterization and microarray analysis of genes in human lymphatic endothelial cells from patients with breast cancer

We successfully isolated human lymphatic endothelial cells from afferent lymph vessels (HALEC) of sentinel lymph nodes in patients with breast cancer by using trypsin digestion. The cells were cultured in EGM-2 medium with 10% FBS under the condition of 5% O2, 5% CO2, and 90% N2 at 37 degrees C. The cultured cells exhibited a monolayer with cobblestone appearance and a marked phagocytosis of Dil-Ac-LDL. Immunohistochemical lymphatic vessel markers were also found, such as podoplanin, LYVE-1, VEGF receptor 3, and Prox-1. Quantitative RT-PCR analysis also showed that podoplanin, VEGF R3, and Prox-1 mRNA were expressed more selectively in the cultured cells. The cells had marked immunoreactivity to antisera of ecNOS, iNOS, COX1, and weak reactivity of COX2. Constitutively expressed cell-type specific genes of the cultured cells were also analyzed by oligonucleotide microarray methods. Compared with human umbilical vein endothelial cells (HUVEC), the cells selectively expressed 88 known genes such as angiopoietin-like 4, oxygen radicals-related enzymes, and adhesion molecules and the related proteoglycans. The findings suggest that the cultured cells seem to be human lymphatic endothelial cells. In conclusion, the isolated, cannulated and enzymatic digested method we adopted for culture of human lymphatic endothelial cells may be easy and useful for investigating cellular, molecular biological, and genomic properties of the cells.     

Angiotensin receptor blockers shift the circadian rhythm of blood pressure by suppressing tubular sodium reabsorption

Recently, we found that an angiotensin II receptor blocker (ARB) restored the circadian rhythm of the blood pressure (BP) from a nondipper to a dipper pattern, similar to that achieved with sodium intake restriction and diuretics (Fukuda M, Yamanaka T, Mizuno M, Motokawa M, Shirasawa Y, Miyagi S, Nishio T, Yoshida A, Kimura G. J Hypertens 26: 583-588, 2008). ARB enhanced natriuresis during the day, while BP was markedly lower during the night, resulting in the dipper pattern. In the present study, we examined whether the suppression of tubular sodium reabsorption, similar to the action of diuretics, was the mechanism by which ARB normalized the circadian BP rhythm. BP and glomerulotubular balance were compared in 41 patients with chronic kidney disease before and during ARB treatment with olmesartan once a day in the morning for 8 wk. ARB increased natriuresis (sodium excretion rate; U(Na)V) during the day (4.5 ± 2.2 to 5.5 ± 2.1 mmol/h, P = 0.002), while it had no effect during the night (4.3 ± 2.0 to 3.8 ± 1.6 mmol/h, P = 0.1). The night/day ratios of both BP and U(Na)V were decreased. The decrease in the night/day ratio of BP correlated with the increase in the daytime U(Na)V (r = 0.42, P = 0.006). Throughout the whole day, the glomerular filtration rate (P = 0.0006) and tubular sodium reabsorption (P = 0.0005) were both reduced significantly by ARB, although U(Na)V remained constant (107 ± 45 vs. 118 ± 36 mmol/day, P = 0.07). These findings indicate that the suppression of tubular sodium reabsorption, showing a resemblance to the action of diuretics, is the primary mechanism by which ARB can shift the circadian BP rhythm into a dipper pattern.     

Linkage-disequilibrium mapping narrows the Fukuyama-type congenital muscular dystrophy (FCMD) candidate region to <100 kb.

Fukuyama-type congenital muscular dystrophy (FCMD), the second most common form of muscular dystrophy in Japan, is an autosomal recessive severe muscular dystrophy associated with brain anomalies. After our initial mapping of the FCMD locus to chromosome 9q31-33, we have further defined the locus within a approximately 5-cM region between D9S127 and D9S2111 and have found linkage disequilibrium between FCMD and D9S306 in this candidate region on 9q31. The high prevalence of FCMD among the Japanese, who are a relatively isolated population, provides an opportunity to utilize linkage-disequilibrium mapping. We developed three new microsatellites, near D9S306, from the FCMD YAC contig, determined their positions on YACs, and performed linkage-disequilibrium mapping with these markers and other newly published loci. The maximum value of p(excess), which represents the strength of linkage disequilibrium, was obtained at D9S2107; and this value showed a relatively steady rise and fall across the region that is likely to contain FCMD. Distances between FCMD and each marker were presumed to be approximately 1 Mb, approximately 350 kb, approximately 140 kb, approximately 20 kb, approximately 280 kb, approximately 450 kb, and approximately 740 kb for D9S306, A107XF9, D9S2105, D9S2107, D9S172, D9S299, and D9S2109, respectively. Haplotype analysis using the three closest markers D9S2105, D9S2107, and D9S172 indicated that most FCMD-bearing chromosomes are derived from a single ancestral founder and suggested that these markers can be used for the diagnosis of sporadic FCMD. Thus, the FCMD gene is most likely to lie within a region of <100 kb containing D9S2107.     

Purificaton and characterization of a pepsinogen and its pepsin from proventriculus of the Japanese quail

A crude extract of the proventriculus of the Japanese quail gave at least five bands of peptic activity at pH 2.2 on polyacrylamide gel electrophoresis. The main component, constituting about 40% of the total acid protease activity, was purified to homogeneity by hydroxyapatite and DEAE-Sepharose column chromatographies. At below pH 4.0, the pepsinogen was converted to a pepsin, which had the same electrophoretic mobility as one of the five bands of peptic activity present in the crude extract. The molecular weights of the pepsinogen and the pepsin were 40 000 and 36 000, respectively. Quail pepsin was stable in alkali up to pH 8.5. The optimal pH of the pepsin on hemoglobin was pH 3.0. The pepsin had about half the milk-clotting activity of purified porcine pepsin, but the pepsinogen itself had no activity. The hydrolytic activity of quail pepsin on N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine was about 1% of that of porcine pepsin. Among the various protease inhibitors tested, only pepstatin inhibited the proteolytic activity of the pepsin. The amino acid composition of quail pepsinogen was found to be rather similar to that of chick pepsinogen C, and these two pepsinogens possessed common antigenicity.     

Gene expressions and activities of protein phosphatases PP1 and PP2A in rat liver regeneration after partial hepatectomy.

We have examined the levels of gene expressions and activities of protein phosphatases, PP1 and PP2A, in rat regenerating livers. PP1 alpha mRNA started to increase from 6 h after partial hepatectomy (PH) and showed two peaks at 12 and 48 h. PP2A mRNA level showed two peaks at 6 and 10-12 h. Protein phosphatase activities were determined both in non-nuclear fraction and in nuclei. While spontaneous PP1 activity in non-nuclear fraction was nearly constant, potential PP1 activity revealed by Co(2+)-trypsin treatment showed a small peak between 7 and 12 h. In nuclei, both spontaneous and potential PP1 activity began to increase from 4-7 h after PH, reached a maximum (about 2.5-fold over control levels) at 12 h, the time which corresponds to the G1 to S transition in the cell cycle, and then declined back to control levels by 7 days. PP2A activity in non-nuclear fraction was nearly constant in both spontaneous and potential forms. PP2A activity in both forms in nuclei was very low throughout. These results suggest the possibility that PP1 in nuclei plays some role in the G1 to S transition in the cell cycle of hepatocyte proliferation.     

In Vitro Induction of Antibody-Dependent Cytotoxic Macrophages by the Local Anesthetic Lidocaine

Normal macrophages were activated to antibody-dependent cytotoxic effector cells by in vitro treatment with the local anesthetic lidocaine. Experiments on the dose-response and time course of the effect of lidocaine showed that incubation of normal macrophages with 10 mM lidocaine for 10 min at 28 C was enough for induction of antibody-dependent cellular cytotoxicity. The activation by lidocaine was accompanied by enhanced phagocytosis of sheep red blood cells (SRBC) sensitized with anti-SRBC antiserum, but not enhanced ingestion of polystyrene latex particles (PLP). These findings suggest that lidocaine, which has various effects on cell membranes, induces some perturbation of macrophage membranes, resulting in activation of Fc receptor functions in antibody-dependent cytotoxicity and phagocytosis.