A calcium-activated cation current by an alternatively spliced form of Trp3 in the heart.

To investigate a cDNA encoding cation current, we isolated an alternatively spliced form of a rat Trp3, designated Trp3sv. Trp3sv encodes 736 amino acids with a unique N terminus and six transmembrane segments. Expression of the cRNA in Xenopus oocytes was successfully performed. The cation selective current appeared after the addition of ionomycin or induced by prolonged depolarization but not by hyperpolarization. This induction was not observed by a treatment with thapsigargin, phorbol ester, or ATP. Na(+), K(+), tetraethylammonium, and divalent cations were permeable, while N-methylglucamine and chloride were nominally impermeable ions. The currents were not inhibited by flufenamate ruthenium red but nonspecifically by 2 mm Gd(3+). Northern as well as Western blot suggested lower levels of the expression observed in some organs, while reverse transcriptase-polymerase chain reaction suggested that it widely spread among various organs. Therefore, we may conclude that N-terminal spliced valiant of Trp3, Trp3sv, encodes a calcium-activated cation channel in various organs.

Absorption of calcium, magnesium, phosphorus, iron and zinc in growing male rats fed diets containing either phytate-free soybean protein or soybean protein isolate or casein.

The effect of dietary phytate-free soybean protein (PFS) on intestinal mineral absorption and retention was examined in growing male rats using a three-day mineral balance technique. The rats were fed diets containing PFS, soybean protein isolate (SPI) or casein at a 20% level for 5 wk. Total calcium (Ca), magnesium (Mg), phosphorus (P), iron (Fe) and zinc (Zn) contents in diets were adjusted to 0.35, 0.05, 0.7, 0.0035 and 0.003%, respectively, by supplementation of the diet with their salts. Mineral absorption and retention ratios in rats fed the PFS diet were significantly higher than those in rats fed either the SPI or casein diet. These results suggest that PFS may be a promising dietary protein source for improving the mineral bioavailability in humans.

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels.

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.

Cloning of a stretch-inhibitable nonselective cation channel.

A homologue of the capsaicin receptor-nonselective cation channel was cloned from the rat kidney to investigate a mechanosensitive channel. We found this channel to be inactivated by membrane stretch and have designated it stretch-inactivated channel (SIC). SIC encodes a 563-amino acid protein with putative six transmembrane segments. The cDNA was expressed in mammalian cells, and electophysiological studies were performed. SIC-induced large cation currents were found to be regulated by cell volume, with currents being stimulated by cell shrinkage and inhibited by cell swelling. Single channel analysis showed a conductance of 250 pS with cation permeability (PCl/PNa < 0.1), and the channel possessed some of the characteristics of a stretch-inactivated channel in that it was permeable to calcium, sensitive to membrane stretch, and blocked by Gd3+. Therefore, we cloned one of the mechanosensitive cation channels of mammals, which is considered to regulate Ca2+ influx in response to mechanical stress on the cell membrane.

Measurement of reverse transcriptase of feline immunodeficiency virus by poly A-linked colorimetric assay.

The method of the poly A-linked colorimetric reverse transcriptase assay (PAC-RTA) was developed and evaluated for the measurement of Mg(2+)-dependent reverse transcriptase (RT) activity of feline immunodeficiency virus (FIV). PAC-RTA was first evaluated for the detection of RT activity in the culture supernatant of FIV Petaluma strain. The detection limit of RT activity by PAC-RTA was about 10-fold better than that by the conventional non-radioisotopic RT assay kit. Then, PAC-RTA was evaluated for the indication of FIV isolation from cats naturally infected with FIV. FIV was isolated from peripheral blood mononuclear cells of 9 FIV-seropositive cats. The time course appearance of RT activity measured by PAC-RTA corresponded with the analysis of FIV antigen expression by indirect immunofluorescence. Finally, PAC-RTA evaluated the drug susceptibility of FIV. MYA-1 cells (feline T-lymphoblastoid cells) were infected with FIV and were cultured in the presence of various concentrations of anti-human immunodeficiency virus agents such as azidothymidine (AZT) or dextran sulfate. An inverse relationship between the RT activities and the concentrations of these agents in the culture supernatant was confirmed by PAC-RTA. PAC-RTA is easy to perform without using radioactive materials, and one plate can handle 96 samples at one time. By monitoring the RT activity, this assay is a useful method for FIV studies such as viral replication and drug susceptibility.

Successful treatment of a rectal anastomotic stenosis by transanal endoscopic microsurgery (TEM) using the contact Nd:YAG laser.

We report the advantage of employing transanal endoscopic microsurgery (TEM) using the contact Nd:YAG laser for the treatment of a rectal anastomotic stenosis. A 72-year-old woman was admitted to our hospital with a postoperative rectal anastomotic stenosis. Twenty months prior to admission, the patient underwent a low anterior resection for the treatment of the rectal cancer using an EEA stapling device. A barium enema and colonoscopy revealed a rectal stenosis, 0.8-cm diameter. This stenosis was at the anastomotic site, approximately 4.0 from the dental line. An endoscopic treatment was performed transanally using the contact Nd:YAG laser. The stenotic rectal wall was fulgurated or vaporized completely. There were no intraoperative or postoperative complications. We concluded that TEM appears to be a safe and minimally invasive procedure. Furthermore, the contact Nd:YAG laser is very effective in treating the gastrointestinal stenotic area. To our knowledge, this is the first successful report of this novel procedure.

[Investigation of AZT susceptibility of sequential HIV-1 isolates from patients treated with ddI after long-term therapy with AZT].

AZT susceptibility of sequential 12 HIV-1 isolates was investigated by culture method. These isolate were obtained from three patients who had received AZT for at least 12 months and switched to ddI after they appeared to deteriorate clinically. Cultures of isolate from a patient before therapy with AZT or ddI did not show cytopathic effect (CPE) in the presence of 0.1 microM or 1.0 microM AZT. Culture of 2 isolates from two patients treated with AZT more than 6 months before switched to ddI therapy showed CPE in the presence of AZT and HIV-1 p24 antigen was detected by ELISA in the supernatants. Culture of 6 of 7 isolates from three patients treated with ddI after long-term therapy with AZT also showed CPE in the presence of 1.0 microM AZT and HIV-1 p24 antigen was detected. These results demonstrated that AZT-resistant variant was still a dominant population in the isolate from patients after 11 to 13 months of discontinuing AZT therapy.

[Polarized light irradiation near the stellate ganglion in a patient with Raynaud's sign].

Polarized light irradiation near the stellate ganglion was performed in a 55-year-old female with Raynaud's sign. She was suffering from cold and numb pain in bilateral fingers for 1 year. Stellate ganglion block and low reactive-level laser therapy near the stellate ganglion were not sufficient to relieve this symptom. Polarized light irradiation near the stellate ganglion induced a sting stimulation and warm sensation in her hands. Thermograms revealed a remarkable increase in temperature of her hands. The results imply that polarized light irradiation near the stellate ganglion increases blood flow of forearms and relieves Raynaud's sign.

[A successful treatment of patient with paraquat poisoning].

A 60-year-old man was admitted 1 hour after ingesting approximately 40 ml of Gramoxon (paraquat) in an attempt to commit suicide. Gastrointestinal lavage, compulsory urination, and direct hemoperfusion (DHP) were immediately performed to remove the paraquat. He was then administered Vitamin E and high dose methylprednisolone therapy to prevent lung injury. Chest roentgenogram on the 3rd day showed ground glass appearance in the outer zone of bilateral lung fields. Chest CT scans on the 9th day revealed a faint high density area in the same area, suggesting interstitial change. The shadows gradually improved and had disappeared on the 35th day after admission. Serum superoxide dismutase (SOD) activity, measured by electron spin resonance spectroscopy, was undetectably low until the 22nd day, and then increased into the normal range. It was suggested that SOD was consumed to neutralize the paraquat toxicity. Serum SOD activity changed according to the paraquat activity, and was a useful indicator of paraquat toxic activity. He was discharged without any symptoms 50 days after admission.

[The effect of orthography and imagery on recall of stimulus items which were read aloud].

The purpose of this study was to compare Kanji items with Kana ones in terms of the free recall performance, and to investigate the effect of orthography and imagery on memory. Two experiments were conducted in incidental learning paradigm, in which subjects were instructed to read aloud each stimulus item. In Experiment 1, an immediate recall test was used. Number of correct free recall for low imagery items was larger in Kanji than in Kana, and this result agreed with Yokoyama and Imai (1989). In Experiment 2, 30-s delay was inserted between a reading aloud task and a recall test. The recall performance indicated superiority of Kanji items in high imagery items. These results were interpreted that the recall process was different between in Kanji items and in Kana ones.

[The effect of orthographic difference between kanji and kana word on incidental memory].

Two experiments were designed to examine the effect of orthographic difference between Kanji and Kana word on memory. The experiments were carried out in incidental memory paradigms, where high and low imagery words without any homonyms were used as stimuli. In Exp. I, subjects were instructed to perform a phonological orienting task, then they were forced to take an unexpected memory test. Number of correct free recall for low imagery words was larger in Kanji than in Kana. In Exp. II, however, where subjects were instructed to perform a semantic orienting task, this effect was disappeared. These results were interpreted in terms of semantic encoding.

Blood pressure control during eye surgery under local anesthesia. Actual status of blood pressure and experience with a new control method using intravenous nitroglycerin.

Blood pressure during eye surgery under local anesthesia was measured in 186 cases treated in our department. 58 cases showed an increase in blood pressure before the operation in spite of premedication; in 32 of these cases there was a 40% rise in blood pressure. Moreover, 38 cases showed at least one ECG abnormality just before the operation in spite of no past history of heart disorders. To control the blood pressure in these cases, intravenous injection of nitroglycerin (Millisrol) was performed continuously and immediately stabilized the blood pressure.

The effect of vitamin E deficiency on some erythrocyte membrane properties.

The erythrocytes from the vitamin E-deficient rats showed no changes in comparison with the erythrocytes from the vitamin E-supplemented rats in lipid peroxides, membrane fluidity, lipid composition, and activities of defensive enzymes against the lipid peroxide formation. Once erythrocytes from vitamin E-deficient rats were exposed to H2O2, the erythrocyte membrane fluidity was significantly reduced, which was presumed to be accompanied by an increase in the hemolysis and lipid peroxide formation. Large decreases in arachidonic acid and phospholipid were also observed. These findings suggest that the reduced membrane fluidity is due to the loss of phospholipid from the erythrocytes, and raises the possibility that the reduced fluidity might contribute to the peroxidative hemolysis of the vitamin E-deficient rat erythrocytes.

Demonstration of the immunogenicity of hepatitis B core antigen in a hepatitis B e antigen polypeptide (P19).

The core of Dane particles, the presently accepted hepatitis B virus nucleocapsid, contains two polypeptides (P19 and P45) with the antigenicity of hepatitis B e antigen (HBeAg). The antigenicity of hepatitis B core antigen (HBcAg) was not detectable in either of them by the conventional in vitro assay methods, despite the fact that both of these polypeptides were derived from the core of Dane particles. When a rabbit had been immunized with the purified preparation of P19 emulsified in complete Freund's adjuvant, however, humoral antibody against HBcAg was produced in addition to the antibody against HBeAg. Amino acid analysis of P19 disclosed a high content of arginine (12.9%), leucine (11.9%), serine (10.3%) and proline (10.2%). The amino acid composition of P19 was found to be strikingly similar to the composition of the 183 amino acid sequence deduced from the sequence of hepatitis B virus DNA which has been presumed to be encoding HBcAg. We conclude that both HBeAg and HBcAg are antigenic determinants borne by the major polypeptide (P19) constituting the core of Dane particles.