Nitrogen-flow in the rotifer Brachionus rotundiformis and its significance in mass cultures

The nitrogen budget in the rotifer Brachionus rotundiformis wasmeasured by the stable-isotope technique. The budget was estimatedusing the difference in the turnover time between egestion andexcretion. The rotifer was fed on the algae Nannochloropsiswhich was labeled with ¹⁵N as a tracer. The turnover time ofegestion and excretion were 20 min and 2.5 hours, respectively. Where77% of the ingested nitrogen was egested, and of the assimilated23%, 18% were devoted to growth and 5% to excretion.As for the unassimilated nitrogen egested as faeces, it recycled tothe rotifer through bacteriovory. When the algae provided as foodwere almost fully consumed, bacteriovory became dominant. Thethreshold occurred when the concentration of algae in the culture wasbetween 1.5 and 0.5 million cells of Nannochloropsis per ml. Ina chemostat operated with un-limited food condition, bacterialnitrogen corresponding to 20% of algal feeding, was consumed by therotifer.In a semi-continuous mass culture where food condition was limited,bacteriovory was more effective in supporting the rotiferreproduction. It contributed to the extremely high nitrogen recoveryfrom the provided foods (algae and oil-yeast) to the harvestedrotifers. The rapid and large nitrogen outflow from rotifersaccelerated the propagation of edible bacteria and can explain thestrange paradox observed in the culture; daily supply of foods didnot cover the sum of growth and excretion.It is not too exaggerated to state that the rotifer mass culture issupported by bacteria. The future strategy for maintenance of masscultures should consider this aspect.     

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-l -cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [³H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na⁺-containing Tyrode's buffer, SNC-stimulated [³H]NA release was inhibited 30% by the coaddition of l -leucine. In the Na⁺-free, choline-containing buffer, SNC-stimulated [³H]NA release, which was similar to that in the Na⁺-containing buffer, was inhibited markedly by l -leucine, l -alanine, l -methionine, l -phenylalanine, and l -tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l -leucine was stronger than that of d -leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [³H]NA release in the Na⁺-free buffer. Uptake of l -[³H]leucine into the slices in the Na⁺-free buffer was inhibited by SNC, BCH, and l -phenylalanine, but not by l -lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l -leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na⁺-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.     

Clonal expansion of superantigen-reactive T cells is resistant to FK506 in mice with AIDS.

Subcutaneous injection of FK506 (10 mg/kg of body weight) completely blocked the clonal expansion of staphylococcal enterotoxin A (SEA)-reactive T cells in healthy (control) mice after SEA injection but did not disturb it in mice with murine AIDS (MAIDS) caused by infection with LP-BM5 murine leukemia virus. MAIDS mice are characterized by utilization of a FK506-insensitive pathway for clonal expansion of superantigen-reactive T cells.     

ET-1-induced bronchoconstriction is mediated via ETB receptor in mice

Nagase, Takahide, Tomoko Aoki, Teruaki Oka, YoshinosukeFukuchi, and Yasuyoshi Ouchi. ET-1-induced bronchoconstriction ismediated via ET_B receptor in mice.J. Appl. Physiol. 83(1): 46-51, 1997.Endothelin (ET)-1 is one of the most potent agonists of airwaysmooth muscle and can act via two different ET receptor subtypes, i.e.,ET_A andET_B. To determine the effects ofET-1 on in vivo pulmonary function and which ET receptors are involved in murine lungs, we investigated 1)the effects of ET and sarafotoxin S6c (S6c), a selectiveET_B agonist, on pulmonary functionand 2) the effects of BQ-123 andBQ-788, specific ET_A- andET_B-receptor antagonists, onET-1-induced bronchoconstriction. ICR mice were anesthetized and mechanically ventilated (frequency = 2.5 Hz, tidalvolume = 8 ml/kg, positive end-expiratory pressure = 3 cmH₂O). Intravenous ET-1, ET-2,and ET-3 increased lung resistance similarly and equipotently, whereasS6c elicited a greater degree of bronchoconstriction. Mice were thenpretreated with saline (Sal), BQ-123 (0.2, 1, and 5 mg/kg), or BQ-788(0.2, 1, and 5 mg/kg) before administration of ET-1(10⁷ mol/kg iv). No dose ofBQ-123 blocked ET-1-induced constriction, whereas pretreatment witheach dose of BQ-788 significantly inhibited ET-1-induced responses.There were significant differences in morphometrically assessed airwayconstriction between Sal and BQ-788 and between BQ-123 and BQ-788,whereas no significant difference was observed between Sal and BQ-123.There were no significant morphometric differences in the airway wallarea among the three groups. These observations suggest that theET_B- but notET_A-receptor subtype may mediatethe changes in murine pulmonary function in response to ET-1. Inaddition, the ET_B-receptorantagonist reduces ET-1-induced airway narrowing by affecting airwaysmooth muscle contraction in mice.

     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Phylogenetic relationships of someMicrosporum andArthroderma species inferred from mitochondrial DNA analysis

Thirty-eight strains of 12Microsporum and 10Arthroderma (Nannizzia) species were investigated by analysis of mitochondrial DNA with 6 restriction enzymes, and classified into 13 genetic groups. The phylogenetic tree of the 13 groups thus established was constructed. On the tree,M. audouinii, M. langeronii, M. rivalieri, M. distortum, M. equinum, M. ferrugineum andA. otae comprise one genetic group and are suggested to be the same species.A. gypseum, A. fulvum, M. duboisii, M. ripariae, A. incurvatum, A. persicolor andA. obtusum are clustered on one of five boughs of the tree indicating their close relation.A. racemosum andA. cajetani are also closely related.     

Entrainment of the circadian locomotor activity rhythm in the Japanese newt by melatonin injections

We examined whether melatonin can act as a synchronizing agent within the circadian system of amphibians by testing the ability of melatonin injections to entrain the circadian locomotor activity rhythm of a newt (Cynops pyrrhogaster). Under constant darkness, all newts (13 cases) showing the free-running rhythms were subcutaneously injected with 10 g melatonin at the same time every other day for at least 30 days. Subsequently, they were injected with vehicle (1% ethanolic saline) instead of melatonin for at least another 30 days. In 10 of the 13 newts, the locomotor activity rhythms could be entrained to a period of 24 h by melatonin injections but not by vehicle injections. During the entrained steady-state, the active phase of an activity-rest cycle preceded the time of melatonin injections as previously reported in other diurnal species. These results suggest that the endogenous circadian rhythm of melatonin concentration may be involved in synchronizing circadian oscillator(s) within the newt's circadian system.     

Characterization of Methanosarcina mazeii TMA isolated from a paddy field soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H₂–CO₂, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6^T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.