Molecular cloning of Na(+)-ATPase cDNA from a marine alga, Heterosigma akashiwo.

We cloned novel Na(+)-ATPase (HANA) cDNA from marine alga Heterosigma akashiwo. The full-length HANA cDNA was 4467 bp long and coded for a 1330 amino acid protein with a molecular weight of 146,306. The deduced product exhibited around 40% identity in amino acids with Na(+)/K(+)-ATPase alpha-subunits. A hydrophilic sequence of 285 amino acid residues that showed no homology with any sequence listed in databases existed in the M7--M8 junction of HANA. This is the first report on the primary structure of putative Na(+)-transporting ATPase from plant cells.

Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo.

A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.

Preferential subsarcolemmal localization of dystrophin and beta-dystroglycan mRNA in human skeletal muscles.

The intracellular localization of dystrophin and beta-dystroglycan mRNA in skeletal muscles of patients with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and normal subjects was examined by in situ hybridization using biotinylated oligonucleotide probes. These mRNAs were found preferentially in sarcolemma in the skeletal muscles of both normal subjects and affected patients. Quantitative analysis of mRNA signals demonstrated no prominent reduction of dystrophin or beta-dystroglycan mRNA in DMD/BMD muscles. These results suggest that even mRNAs with deletions contain specific information that affects their localization, and the characteristic defect of dystrophin in DMD/BMD muscles seems to be caused mainly by the instability of dystrophin protein, as a post-transcriptional event.

Vitamin D3 elicits calcium response and activates blood monocyte-derived macrophages from patients with vitamin D dependent rickets type II.

We studied the effects of vitamin D3 metabolites on intracellular free Ca2+ concentration ([Ca2+]i) and the respiratory burst of monocyte-derived macrophages (MDM) from patients with vitamin D dependent rickets type II. Treatment of MDM from the patients and healthy donors with 1 nM 1,25(OH)2D3 produced a rapid elevation of [Ca2+]i and similarly primed both types of cells for enhanced capacity for O2- release with phorbol diester. These results suggest that macrophages may have distinct non-genomic pathways of vitamin D3, which partly explain the absence of immunodeficiency and the disappearance of rickets after treatment with vitamin D3 in the patients.

Effects of a time-varying strong magnetic field on transient increase in cytosolic free Ca2+ induced by bradykinin in cultured bovine adrenal chromaffin cells.

We tested the effects of exposure to a time-varying magnetic field changing between 0.07 and 1.7 T at an interval of 3 s on transient increase in intracellular Ca2+ stimulated by bradykinin in bovine adrenal chromaffin cells. Addition of bradykinin induced the increase in intracellular Ca2+ within a few minutes. The exposure to the magnetic field perfectly suppressed the increase in intracellular Ca2+ in Ca2+-free medium. The inhibition occurred for 15 min when the maximum magnetic flux density was more than 1.4 T. These results suggest that the exposure inhibits Ca2+ release from intracellular Ca2+ stores.

Combined quantitative cytophotometric method for staining indolyl and sulfhydryl groups and protein.

Sulfenylation with 2-nitrophenylsulfenyl chloride (NPS-Cl), which is specific for tryptophyl and cysteinyl residues in protein, was applied to quantitative histochemistry. By measurement of the absorbance values at 370 nm of sections stained with NPS-Cl, Beer-Lambert's law was found to hold for NPS staining. Treatment of NPS-stained sections with 2-mercaptoethanol (ME) (NPS-ME staining) resulted in sulfenylation of tryptophyl residues only. For determination of the amounts of tryptophyl and cysteinyl residues per unit of protein, protein staining with Coomassie Brilliant Blue (CB) was combined with NPS and NPS-ME staining. CB and NPS-CBB staining also followed Beer-Lambert's law. By measuring the absorbance values at 370 and 650 nm of doubly stained sections, the relative contents of tryptophyl and cysteinyl residues in various tissue proteins were calculated. This method will be useful for the investigation of changes in both protein amount and composition.

Regulation of lymphocyte proliferation by eosinophils via chymotrypsin-like protease activity and adhesion molecule interaction.

We investigated the regulatory mechanisms responsible for release of eosinophil cationic protein (ECP) from eosinophils activated by platelet-activating factor (PAF) and monitored intra-cellular pH (pHi) changes using a pH-sensitive fluorescent probe. We also explored the mechanisms by which eosinophils suppress T-lymphocyte proliferation induced by phytohaemagglutinin (PHA). In these experiments, a separated culture to investigate the ECP-mediated pathway and a coculture to identify the adhesion molecules involved in eosinophil-lymphocyte interactions were employed. Chymostatin (1x10(-6) M) inhibited ECP release by about 50% via stimulation by PAF or recombinant interleukin 5(rIL-5) plus IgG. PAF (1x10(-7) M) raised eosinophil pHi from 6.9 to 7.3 within 20 s and pretreatment of these cells with chymostatin (1x10(-6) M), but not with leupeptin or E64-d, completely prevented this increase. Calcium ionophore A23187 (1x10(-7) M) induced ECP release and raised pHi to within a range similar to that of PAF, however, chymostatin had no effect on either. Chymostatin reversed ECP-mediated suppression of PHA-induced T-lymphocyte proliferation in separated cultures, but not in cocultures. In coculture, eosinophils exhibited the same level of suppression of both CD4(+) and CD8(+) T-cell proliferation in response to PHA. Monoclonal antibodies against CD11a, CD18 and CD54, but not CD11b, restored eosinophil suppression of T-lymphocyte proliferation which was chymostatin-resistant in coculture. Eosinophils were unable to suppress the proliferative response to lymphocytes to anti-CD3 stimulation. In conclusion, chymostatin specifically inhibited both the eosinophil pHi increase and ECP release induced by PAF. Eosinophils regulate PHA-induced T-lymphocyte proliferation via the ECP-mediation associated with chymotrypsin-like protease activity. These cells also control interactions with lymphocyte between adhesion molecules, CD11a, CD18 and CD54.

Effect of serum depletion on centrosome overduplication and death of human pancreatic cancer cells after exposure to radiation.

The tumor microenvironment is one of the key factors affecting the cellular response to radiation; however, the influence of serum concentration on tumor radiosensitivity remains poorly understood. We recently discovered that gamma-irradiation of tumor cells causes centrosome overduplication, which may lead to lethal nuclear fragmentation through the establishment of multipolar mitotic spindles. In the present study, we investigated the effect of serum depletion on radiation-induced cell death in relation to the centrosome dynamics in human pancreatic cancer cells. Exposure of Capan-1 cells to gamma-irradiation resulted in a time-dependent increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death. Treatment of irradiated cells with serum depletion drastically accelerated centrosome overduplication and the formation of multipolar spindles, resulting in increased nuclear fragmentation and cell death. Cell cycle analysis of irradiated cultures revealed that the reduced serum level increased the population of cells arrested in the G2/M phase, which might be responsible for the abnormal centrosome accumulation. These findings suggest that serum concentration can influence radiation-induced cell killing through modulating cell cycle progression and possibly centrosome overduplication.

Kinetic analysis of endocytosis and intracellular fate of liposomes in single macrophages.

Endocytosis and the intracellular fate of liposomes in single mouse peritoneal macrophages were examined kinetically by fluorescence microphotometry. Liposomes labeled with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine or containing 8-amino-naphthalene-1,3,6-trisulfonate were promptly incorporated into macrophages on incubation at 37 degrees C, but fluorescence increase caused by hydrolysis of 4-methylumbelliferyl-beta-D-glucoside encapsulated in the liposomes was observed after 30 min of incubation. The fluorescences of calcein and 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS) in liposomes, which were respectively quenched statically due to high concentration and dynamically by a co-entrapped fluorescence quencher, p-xylene-bis-pyridinium bromide, also increased from 30 min after the start of liposome incorporation, indicating that macrophages require this period for intracellular delivery of liposomes from the cell surface to lysosomes. Measurement of the intraendosomal pH change in a single macrophage at 37 degrees C with liposomes containing a pH-sensitive fluorescent marker, HPTS, showed that the pH value decreased continuously to a constant value of 5.5 in 30-40 min after endocytosis, and this decrease was reversed on addition of NH4Cl, suggesting that acidification of endosomes is not a stepwise reaction and is coupled with delivery of liposomes. These fluorescence microphotometric systems using liposomes containing different fluorescent dyes should be useful for kinetic analyses of the endocytosis and intracellular fate of liposomes in various phagocytes.

Apolipoprotein E epsilon4 and tardive dyskinesia in a Japanese population.

The findings that free radicals play a causative role in the occurrence of tardive dyskinesia (TD) and that apolipoprotein E (ApoE) 4 has decreased anti-oxidant activity suggest a potential link between TD and ApoE alleles. We, therefore, examined ApoE allelic frequencies in schizophrenic subjects with TD and non-TD. Serum samples were obtained from 333 DSM IV-diagnosed schizophrenic patients and 191 controls in Japan. The presence of TD was evaluated by research diagnostic criteria for TD. ApoE phenotypes of the serum samples were determined by polyacrylamide gel isoelectricfocusing. A total of 62 TD subjects (31 males, 31 females) were identified among all patients examined. No significant differences in ApoE allelic frequency were found between TD and non-TD groups. ApoE epsilon4 allele frequency, however, was significantly lower in the female TD group than in the male TD group. These findings do not clearly demonstrate a certain association between TD and the epsilon4 allele, but may preliminarily reveal a difference in influence of this allele on the development of TD between males and females.

Apolipoprotein E epsilon2 allele and early onset schizophrenia.

To explore the role of apolipoprotein E (ApoE) in schizophrenia, we investigated ApoE phenotypes in a group of patients with schizophrenia. Serum samples were obtained from 122 schizophrenic patients and 126 controls in Japan and were examined using isoelectric focusing/immunoblotting. This experiment showed a trend toward a decreased frequency of ApoE epsilon4 in schizophrenia and no link between ApoE epsilon4 and familial schizophrenia or early onset schizophrenia. On the other hand, a decreased frequency of ApoE epsilon2 in early onset schizophrenia was detected. These results suggest that ApoE epsilon2 protects against early onset schizophrenia, and that ApoE epsilon4 is not involved in the development of schizophrenia in Japanese.

Regulations by cyclic nucleotides and phorbol ester of calcium efflux from cultured bovine adrenal chromaffin cells.

The effects of cyclic nucleotides and phorbol ester on Ca2+ efflux from cultured bovine adrenal chromaffin cells were examined. Dibutyryl cyclic AMP (DB-cAMP), forskolin (an activator of adenylate cyclase), dibutyryl cyclic GMP (DB-cGMP) and nitroprusside (an activator of guanylate cyclase) all stimulated 45Ca2+ efflux from the cells preloaded with 45Ca2+. These agents did not increase the intracellular free Ca2+ ([Ca2+]i) level. On the contrary, phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C) did not affect the efflux of 45Ca2+, but inhibited the increase in 45Ca2+ efflux caused by DB-cAMP, forskolin, DB-cGMP or nitroprusside. The 45Ca2+ effluxes stimulated by cyclic nucleotides, forskolin and nitroprusside were inhibited by deprivation of extracellular Na+ ([Na+]o). These results suggest that both cAMP- and cGMP-dependent protein kinases are involved in the stimulatory mechanism of [Na+]o dependent Ca2+ efflux, probably through acceleration of [Na+]o/[Ca2+]i exchange and that protein kinase C plays an inhibitory role in this mechanism.

Induction of inducible nitric oxide synthase by ginsenosides in cultured porcine endothelial cells.

Mechanism of Nitric oxide (NO) production by ginsenosides was investigated in cultured porcine endothelial cells. Beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) staining showed that the NO production was significantly enhanced by the presence of 40 microg/ml ginsenosides with 10 microM L-arginine after 12 h incubation. NO production was suppressed by addition of 0.5 microM Nomega-Nitro-L-arginine (L-NNA), an inhibitor of NO synthases (NOSs), to the incubation medium. In addition, the immunoreactive signals of inducible NOS (iNOS) were appeared in endothelial cells after 12-h incubation of ginsenosides, whereas the signals were not observed in non-treated cells. Our findings suggest that ginsenosides can enhance NO production by induction of iNOS in addition to its direct effect on endothelial cells by increasing intracellular Ca2+ concentration.

Adrenomedullin stimulates calcium efflux from adrenal chromaffin cells in culture: possible involvement of an Na+/Ca2+ exchange mechanism.

The effect of adrenomedullin, a hypotensive peptide, on Ca2+ efflux from cultured bovine adrenal chromaffin cells was examined. Adrenomedullin stimulated the efflux of 45Ca2+ from the cells in a concentration-dependent manner (10(-7)M - 3x10(-6)M). Adrenomedullin did not increase the intracellular free Ca2+ ([Ca2+]i) level and catecholamine secretion. The adrenomedullin-stimulated 45Ca2+ efflux was not inhibited by incubation with Ca2+-free medium, but was inhibited by incubation with Na+-free medium. These results indicate that adrenomedullin stimulates extracellular Na+-dependent 45Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably through its stimulatory effect on membrane Na+/Ca2+ exchange.

An incident involving blood sucking by a tick in a suburb in Japan.

We encountered a patient whose blood was sucked by Haemaphysalis longicornis in the suburb of a business city in Tokushima prefecture in Japan. The tick, which had been attached to the lower limb of the patient for one week, measured 10 mm in length. There were no notable objective or subjective findings after the complete extirpation of the tick. The area had not been known in recent history to be a habitat of ticks, and, thus, this case is of importance in terms of predicting future trends of tick-borne diseases in Japan.

Dynamic changes in the middle cerebral artery perfusion in normal full-term human fetuses in relation to the timing of behavioral state.

Our aim is to evaluate serial changes in normal full-term fetal cerebral circulation according to the behavioral states. Flow velocity waveforms in the middle cerebral artery and fetal heart rate (FHR) were well recorded in ten of 19 cases by pulsed Doppler ultrasonography and actocardiography over 45 min, respectively. Behavioral states were classified as resting or active phase by FHR patterns. Resistance indices (RIs) were calculated every 5 s as an average of two consecutive waveforms, and median RI was chosen in each 2-min segment. In order to evaluate changes in median RI from active-to-resting transition to resting-to-active one statistically, differences in RI between two 2-min segments were examined using Mann-Whitney U-test. As a result, median RI was decreased to the minimum one in the active-to-resting transition for 12 min in all cases: significant decrease to the 2-min segment with the minimum one (P<0.01). Various types of increase to the maximum median RI during resting phase followed the minimum one: significant increase from the 2-min segment with the minimum one to that with the maximum one in all cases (P<0.001 in eight cases, P<0.01 in two cases). Thereafter, median RI was decreased from the end of resting phase in the resting-to-active transition for 12 min in all cases: significant decrease from the last 2-min segment of resting phase in all cases (P<0.01). We reveal that fetal cerebral circulation changes dynamically in relation to the timing in each behavioral state.

Developmental characteristics in sustained fetal tachycardia in 30 to 41 weeks of gestation.

The aim of the present study is to reveal gestational age-related changes in sustained foetal tachycardia (SFT). 24 h fetal heart rate (FHR) recordings were made on 102 normal pregnant women in 30-41 weeks of gestation. SFT was defined as an increase for 20 or more beats per minute from the FHR-baseline persisting for more than 20 min. In the results, SFTs in 38-39 weeks started during night-time (1900-0700) more frequently than during day-time (0700-1900) (P < 0.01, by chi-square test). The rate and duration were 0% and 0 min in 30-31 weeks of gestation, and increased to 85% and 114 min in 40-41 weeks, respectively. Two critical points were detected by a piecewise linear regression analysis: in the rate between 34-35 weeks and 36-37 weeks, and in the duration between 38-39 to 40-41 weeks. We conclude that gestational age-related changes in SFT depend on the developmental stages.

Effects of time-varying electromagnetic fields on K+ (Rb+) fluxes and surface charge of HeLa cells.

The magnetic flux density was varied intermittently from 0.35 to 1.77T and from 0.07 to 1.54 or 1.77T by manual and automatic switchings, respectively, of the power source of an electromagnet. The durations of the "switching-on time" and "-off time" were varied but kept equal. An electric eddy current induced in the culture medium by changes in the magnetic flux density was simulated. When the durations were shorter than 10s, ouabain-sensitive Rb+ influx (active K+ influx) into cultured HeLa cells was significantly inhibited, but the ouabain-insensitive Rb+ influx (passive K+ influx) was not influenced significantly. Inhibition of active Rb+ influx increased with time during exposure for 2 h. Conversely, K+ efflux from the cells was significantly stimulated by the exposure. Microfluorometric examinations of cells loaded with the fluorescent pH indicator 4-heptadecyl-7-hydroxycoumarin (6 microM) and the membrane potential indicator diS-C3-(5) (1 microM) suggested increase in the negative charge on the cell surface during exposure. The observed changes in the K+ (Rb+) fluxes would be related to change in the electric properties of the cell surface caused by exposure to intermittent electromagnetic fields.

Radical forearm flap with vascularized tendons for hand reconstruction.

We treated six patients with soft-tissue defects involving the skin and extensor or flexor tendons using a cutaneotendinous radial forearm flap. There were five male patients and one female patient. Their ages at operation ranged from 22 to 65 years (mean 50 years). In one patient, the flexor pollicis longus tendon was reconstructed with a palmaris longus tendon graft. Four patients each had two extensor tendons reconstructed. This was performed either with the palmaris longus tendon folded in the center (n = 2) or with the palmaris longus and flexor carpi radialis tendons (n = 2). In the remaining patient, three extensor tendons were reconstructed with the palmaris longus and flexor carpi radialis tendons. All the flaps survived. Recovery of motor function has been satisfactory in all but one patient.

Separate histogenesis of combined hepatocellular and cholangiocellular carcinoma in two patients.

Double primary liver carcinomas, i.e. hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) are rare. Two patients in whom double primary liver carcinomas were surgically resected are described herein. Case 1: A 51-year-old Japanese man with chronic type B hepatitis underwent hepatectomy for primary HCC with intrahepatic metastasis. Case 2: A 67-year-old Japanese man with a history of rectal cancer and CCC underwent lateral hepatic segmentectomy for a suspected recurrence of intrahepatic CCC. Lack of direct contact between tumors, no evidence of histological transition and clearly different immunohistochemical staining for cytokeratin support a distinct histogenesis of the tumors in these two patients. The findings indicate that combined HCC and CCC can arise synchronously or metachronously as an intrahepatic double cancer.