Regulation of CD8+ T cell development by thymus-specific proteasomes

Proteasomes are responsible for generating peptides presented by the class I major histocompatibility complex (MHC) molecules of the immune system. Here, we report the identification of a previously unrecognized catalytic subunit called beta5t. beta5t is expressed exclusively in cortical thymic epithelial cells, which are responsible for the positive selection of developing thymocytes. Although the chymotrypsin-like activity of proteasomes is considered to be important for the production of peptides with high affinities for MHC class I clefts, incorporation of beta5t into proteasomes in place of beta5 or beta5i selectively reduces this activity. We also found that beta5t-deficient mice displayed defective development of CD8(+) T cells in the thymus. Our results suggest a key role for beta5t in generating the MHC class I-restricted CD8(+) T cell repertoire during thymic selection.     

Identification of currently cultivated Porphyra species by PCR-RFLP analysis

ABSTRACT:   Porphyra yezoensis and P. tenera are the representative species of the marine crop Porphyra (nori) in Japan. Since the two species are extremely similar to each other in morphology, nori breeders have tentatively classified many strains of cultivated Porphyra into the two species without strict species identification. In order to facilitate rapid and reliable identification of the many strains of Porphyra currently cultivated in various Japanese regions, 24 conchocelis strains were examined by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis using the plastid RuBisCo spacer and the nuclear internal transcribed spacer regions. From the results, all the strains were identified as P. yezoensis f.  narawaensis , and P. tenera was not detected. Hence, it was confirmed that most of the Porphyra strains currently cultivated in Japan are P. yezoensis f.  narawaensis , and that intensive selective breeding has led to a reduced genetic diversity in the stock used for nori cultivation.     

Trophic structure of a macroarthropod litter food web in managed coniferous forest stands: a stable isotope analysis with δN and δC

We studied the composition of a litter detrital community in a temperate coniferous forest using stable isotopes of nitrogen and carbon. Samples of mineral soil, bulk litter material, macroarthropods and understory plants were collected from ten experimental forest stands. Half of the stands were previously thinned 17–42 years ago, the other half served as controls. Values of δ¹⁵N and δ¹³C were based on the analysis of almost 500 individuals of at least 22 species in 11 arthropod families. The isotopic analysis showed a significant increase in δ¹⁵N and δ¹³C values with soil depth. Isotopic signatures of macroarthropods ranged from −26.51‰ to −20.52‰ for δ¹³C and −2.85‰ to 5.10‰ for δ¹⁵N. All consumers showed levels of ¹³C enrichment substantially higher than those of primary producers and litter. Predators were generally significantly more ¹⁵N enriched than detritivores and herbivores, but their δ¹³C levels were similar to those of primary consumers. Our data indicate that this community consists of at least 2–3 trophic levels with a considerable amount of variation in the ¹⁵N enrichment among detritivores and predators. We suggest that the spread of δ¹⁵N values of predators likely reflects the diversity of potential prey among detritivores and a varying degree of intraguild predation among different species. Our findings generally agree closely with the results of similar studies from other forest litter communities. Thinning did not appear to influence the overall isotopic composition of the detrital food web. Extensive omnivory and intraguild predation among litter consumers may buffer long-term effects of thinning on the trophic structure of these species-rich communities.     

Effects of urea and trimethylamine-N-oxide on ATPase of requiem shark myofibril and its constituents

ABSTRACT: The effects of trimethylamine- N -oxide (TMAO) on the urea-resistibility of requiem shark myofibrils were investigated, using Ca²⁺- and Mg²⁺-ATPase activities as a parameter. Both activities were hardly changed or activated up to 0.6 M urea. In contrast, the two activities both decreased to less than 50% in the presence of TMAO up to 0.5 M. When measured at a 2 : 1 molar ratio of urea and TMAO, Ca²⁺- and Mg²⁺-ATPase activities were similar to those in the presence of TMAO alone, indicating that TMAO reduced the urea-resistibility of myofibrils. Myosin, the most abundant protein in myofibrils, from requiem shark exhibited the effects of urea and TMAO on its Ca²⁺-ATPase activity, which was primarily similar to those of myofibrils. However, Ca²⁺-ATPase activities in the coexistence of urea and TMAO for actomyosin reconstituted from requiem shark myosin and chicken F-actin were approximately average of those measured independently in the presence of either urea or TMAO alone. Carp myofibrils, reconstituted actomyosin and myosin, which were used as teleost references, all showed a tendency in the effects of urea and TMAO on Ca²⁺-ATPase activities that was similar to those of requiem shark counterparts.     

Effects of 17beta-estradiol on in vitro maturation of pig oocytes in protein-free medium

The present study examined the effects of 17 beta-estradiol (E(2)) on in vitro maturation and subsequent in vitro fertilization of pig oocytes matured with or without cumulus cells. When E(2) (10 ng/ml) was added to the protein-free maturation medium, the proportions of cumulus-enclosed oocytes that underwent germinal vesicle breakdown and reached metaphase II were significantly reduced (P<0.05), and cumulus expansion was also significantly inhibited (P<0.05) compared with the control (no E(2) added). Although oocytes matured in the presence of E(2) were penetrated by sperm in vitro at the same level as the control, the incidences of male pronuclear (MPN) formation and activated oocytes were significantly lower (P<0.05) than the control. These inhibitory effects of E(2) were prevented when the medium was supplemented with E(2) together with its antagonist, ICI 182,780 (1 microg/ml), although the presence of the antagonist alone in the medium had no effect on the maturation and fertilization in vitro of oocytes. In cumulus-free oocytes, E(2) had no effect on nuclear maturation and penetration in vitro, but low MPN formation was observed in oocytes matured in the presence and absence of E(2). When cumulus-enclosed oocytes were cultured in the presence of progesterone (P(4); 600 ng/ml) alone or together with E(2), no significant differences in nuclear maturation, cumulus expansion or penetration in vitro were observed compared with control oocytes. The concentration of P(4) in maturation medium was significantly (P<0.01) lower when cumulus-enclosed oocytes were cultured for 44 h in the medium with E(2) than in medium without E(2). These results indicate that E(2) inhibits both nuclear and cytoplasmic maturation of cumulus-enclosed pig oocytes, and that this inhibition can be prevented by an E(2) antagonist or P(4). This E(2) inhibition may occur indirectly via the cumulus cells and inhibition of P(4) synthesis.     

Reproduction of acute bracken poisoning in a calf with ptaquiloside, a bracken constituent

Acute bracken fern toxicity in a calf was reproduced with ptaquiloside, a norsesquiterpene glucoside, isolated from the boiling water extract of bracken fern. Ptaquiloside was dissolved in 500 ml of saline and administered by drench at increasing dosages for six days out of every seven for the following periods: 400 mg/day for 24 days, 800 mg/day for 14 days and 1600 mg/day for four days. Neutrophilic granulocytes began to decrease markedly around 50 days after the start of the experiment, and granulocytopenia continued for a further 35 days until the autopsy, despite the discontinuance of ptaquiloside administration. Thrombocytes showed a relatively slow depression and reached 1 X 10(5)/mm3 at the lowest level. The calf was autopsied 86 days after the start of administration of ptaquiloside. Sternal bone marrow was found to be mostly replaced with fat marrow and only small foci of erythropoietic cells and a small number of megakaryocytes remained.     

Mechanisms responsible for reduced cardiotoxicity of mitoxantrone compared to doxorubicin examined in isolated guinea-pig heart preparations

We reported previously that doxorubicin, an anticancer agent that has an anthracycline structure, alters Ca2+ releasing and uptake mechanisms in the sarcoplasmic reticulum of myocardial cells. These effects of doxorubicin are apparently related to its cardiotoxicity. Mitoxantrone is a similar anticancer agent with an anthracenedion structure that has been shown to be significantly less cardiotoxic. In the present study, the effects of mitoxantrone on the functions of the sarcoplasmic reticulum were examined in isolated muscle preparations obtained from the guinea-pig heart. In electrically-stimulated left atrial muscle preparations, incubation in vitro for 4 hr with 30 or 100 microM mitoxantrone significantly prolonged the time to the peak of twitch tension, markedly increased the developed tension observed at lower stimulation frequencies, thereby attenuating the slope of positive force-frequency relationships, and increased the postrest contraction observed after a 60-sec quiescent period. In myocytes isolated from ventricular muscles, 30 microM mitoxantrone increased the peak and the size of intracellular Ca2+ concentrations ([Ca2+] i), and prolonged the time to peak [Ca2+]i. In skinned muscle fiber preparations obtained from the left ventricular muscle, 30 muM mitoxantrone significantly increased the caffeine-induced contraction without affecting the Ca2+ sensitivity of contractile proteins. These results suggest that mitoxantrone enhances Ca2+ release from the sarcoplasmic reticulum in isolated atrial muscle preparations obtained from the guinea-pig heart. Apparent enhancement of the sarcoplasmic reticulum functions, in contrast to anthracyclines that has been shown to suppress these functions, seems to explain the relative lack of marked cardiotoxicity of mitoxantrone.