Determination of plasma leukotrienes in antigen-induced bronchoconstrictive guinea pigs.

Convenient extraction and radioimmunoassay methods for measurement of leukotrienes C4 and D4 (LTC4 and LTD4) in biological fluids are described. LTC4 or LTD4 in plasma was extracted with acetonitrile, and the extract was washed with dichloromethane then adjusted to pH 3.5 or 6.0, respectively. Each leukotriene was partially purified by using a C18-bonded silica cartridge and quantitated by radioimmunoassay. Amounts of LTC4 and LTD4 in the range of 0.025-1.6 ng could be assayed in plasma. This procedure was employed to examine the increase in plasma LTC4 (0.249 +/- 0.036 ng/ml) and LTD4 (1.399 +/- 0.235 ng/ml) of guinea pigs during intravenous challenge-induced anaphylactic bronchoconstriction, and the suppression of the increase of bronchoconstriction and leukotrienes by the administration of 5-lipoxygenase inhibitors such as E6080 (6-hydroxy-2-(4-sulfamoylbenzyl-amino)- 4,5,7-trimethylbenzothiazole hydrochloride), AA861 (2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone ) and phenidone. On the other hand, LTC4 and LTD4 were not detected in plasma after an inhaled challenge, though significant bronchoconstriction was provoked. It was concluded that the present study validates a new technique for quantitating plasma leukotrienes on the basis of pH and a suitable method for evaluating the pharmacological efficacy of 5-lipoxygenase inhibitors.     

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.     

The cholecystokinin-induced Ca2+ shuttle from the inositol trisphosphate-sensitive and ATP-dependent pool, and initial pepsinogen release connected with cytoskeleton of the chief cell

In guinea pig chief cells, inositol 1,4,5-trisphosphate (IP3) caused release of Ca2+, which was accumulated by ATP, from an endoplasmic reticulum-enriched fraction in both the permeable system and the cell-free system. This was mimicked with the Ca2+ ionophores A23187 and ionomycin on a large scale since an IP3-sensitive Ca2+ pool might be a subset of the Ca2+ ionophore-sensitive Ca2+ pool. The permeable chief cells, but not the cell-free system, retained the ability to react to synthetic cholecystokinin octapeptide (CCK-OP) with Ca2+ release from an IP3-sensitive pool due to of the non-additive but constant effect in exerting Ca2+ release from the store(s) induced by the combination with IP3 and CCK-OP. The increase in the cytosolic free Ca2+ concentration of intact chief cells responding to CCK-OP or the Ca2+ ionophore, ionomycin, comprised two components, namely, that by the Ca2+ entry from the extracellular space, and that by the Ca2+ release from the intracellular space(s) (as measured by fura-2). When CCK-OP or ionomycin was added, there was a biphasic response of pepsinogen secretion. An initial but transient response reaching a peak in 5 min was followed by a sustained response reaching a peak in 30 min. The initial pepsinogen release was independent of medium Ca2+, whereas the sustained one was dependent on medium Ca2+. The results suggest that the intracellular Ca2+ release from the store(s), presumably endoplasmic reticulum, may trigger the initial pepsinogen release, whereas the sustained pepsinogen secretion may be caused by acting in concert with the initial response and external Ca2+ entry. On the other hand, the disruption of the microtubular-microfilamentous system by colchicine or cytochalasin D failed to cause the Ca2+ release evoked by either IP3, CCK-OP or Ca2+ ionophores and to cause the CCK-OP- or ionomycin-induced initial pepsinogen release. These findings suggest that the IP3-sensitive pool is the same Ca2+ store which is completely or partially sensitive to CCK-OP and Ca2+ ionophores, respectively, and that the assembly of the cytoskeletal system is involved in initial intracellular Ca2+ metabolism and the following initial pepsinogen release. The assembly of the cytoskeletal system may be an early event in mediating the CCK-OP-induced initial pepsinogen release, perhaps by causing the Ca2+ release from an IP3-sensitive pool of the chief cell. The translocation or attachment of the IP3-sensitive pool brought about by cytoskeletal system might be necessary to cause Ca2+ release after the cell stimulation with CCK-OP.     

Spacing and kinship in the Formosan squirrel living in different habitats

Summary Spacing and kinship of the Formosan squirrel, Callosciurus erythraeus thaiwanensis, were studied in two different habitats. One, native habitat in the woods of Kenting, southern Formosa, was rich in available food throughout the year and had several species of predators. The other, a site in Kamakura, central Japan where squirrels had been introduced, had relatively scanty food and few potential predators. 1. Home ranges among males and between sexes overlapped extensively in both habitats. 2. Females occupied exclusive home ranges in Kamakura but had small overlapping home ranges in Ken-ting. 3. Most males disappeared from their natal areas at 1 year old in both habitats (86% in Kamakura and 93% in Ken-ting), but less females disappeared (36% in Kamakura and 35% in Ken-ting). 4. In Kamakura, daughters settled adjacent to the mother or inherited the home range of the mother, but never shared the mother's home range. In Ken-ting, 35% of daughters shared the home range with their mothers. 5. Tolerance among female kin in Ken-ting was probably facilitated by the richness of available food throughout the year, and functioned to reduce predation risk via alarm calling and mobbing.     

Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families

Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan.     

Three main components in plasma proteases and their relation to the renin-angiotensin system

The relation of plasma renin activity (PRA) and plasma levels of angiotensin I (AI) and II (AII) to those of various proteases, including eight endopeptidases and four aminopeptidases, was investigated in 51 normal control subjects. The multivariate study using factor analysis showed that the plasma proteases can be classified into three main components: the aminopeptidase, the plasmin, and the kinin-kallikrein. PRA and AI were related almost exclusively to the aminopeptidase component, while the AII level was related not only to the same component but also to the kallikrein-kinin component. This kind of multivariate study may help in the elucidation of the role of proteases and bioactive peptides, such as angiotensin derivatives, in essential hypertension through a comparison of multivariate relationships in controls and patients.     

Functional and phenotypic analyses of interleukin 2-activated tumor-infiltrating lymphocytes

The tumor-infiltrating lymphocytes (TILs) were cultured with interleukin 2 (IL-2) to induce the activated killer cells possessing autologous tumor-killing activity, and analysed their cell surface phenotypes and assessed anti-tumor killing activity. Furthermore, the activated TILs were transferred into 7 patients adoptively resulting in complete remission in a patient with pancreatic cancer and partial remission in another patient with gastric cancer.The cytotoxic activities of activated TILs at 3 weeks-incubation was 72 ± 15, 42 ± 26, 27 ± 21 and 25 ± 15% against K562, Daudi, KATO-III and autologous tumor, respectively. The negative selection method, indicated that the killer cells recognizing autologous tumor cells consisted of CD4- or CD8-positive T lymphocytes and CD16- or CD56-positive natural killer cells. The activated TILs could not only lyse cultured tumor cell lines, but also autologous tumor cells.