Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16

Summary A physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 by inverted repeat containing trnI-CAU and the 3 end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.     

Neurotensin receptors on the rat liver plasma membranes

Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT.     

Supersensitivity to substance P after dorsal root section.

Two to three weeks after unilateral dorsal root section, specific [³H] substance P binding in synaptic membranes from rabbit spinal cord increased significantly compared with that in intact side, while non-specific [³H] substance P binding was unchanged. Scatchard analysis indicates that the development of such supersensitivity is due to an increase in a number of receptor sites, not to an increase in a receptor affinity. These results strongly support the notion that substance P exerts a transmitter role for primary afferent fibers.     

Weakened Intracellular Zn<Superscript>2+</Superscript>-Buffering in the Aged Dentate Gyrus and Its Involvement in Erasure of Maintained LTP

Memory is lost by the increased influx of extracellular Zn²⁺ into neurons. It is possible that intracellular Zn²⁺ dynamics is modified even at non-zincergic medial perforant pathway-dentate granule cell synapses along with aging and that vulnerability to the modification is linked to age-related cognitive decline. To examine these possibilities, vulnerability of long-term potentiation (LTP) maintenance, which underlies memory retention, to modification of synaptic Zn²⁺ dynamics was compared between young and aged rats. The influx of extracellular Zn²⁺ into dentate granule cells was increased in aged rats after injection of high K⁺ into the dentate gyrus, but not in young rats. This increase impaired maintained LTP in aged rats. However, the impairment was rescued by co-injection of CaEDTA, an extracellular Zn²⁺ chelator, or CNQX, an AMPA receptor antagonist, which suppressed the Zn²⁺ influx. Maintained LTP was also impaired in aged rats after injection of ZnAF-2DA into the dentate gyrus that chelates intracellular Zn²⁺, but not in young rats. Interestingly, the capacity of chelating intracellular Zn²⁺ with intracellular ZnAF-2 was almost lost in the aged dentate gyrus 2 h after injection of ZnAF-2DA into the dentate gyrus, suggesting that intracellular Zn²⁺-buffering is weakened in the aged dentate gyrus, compared to the young dentate gyrus. In the dentate gyrus of aged rats, maintained LTP is more vulnerable to modification of intracellular Zn²⁺ dynamics than in young rats, probably due to weakened intracellular Zn²⁺-buffering.     

Low-temperature culturing improves survival rate of tissue-engineered cardiac cell sheets

Assembling three-dimensional (3D) tissues from single cells necessitates the use of various advanced technological methods because higher-density tissues require numerous complex capillary structures to supply sufficient oxygen and nutrients. Accordingly, creating healthy culture conditions to support 3D cardiac tissues requires an appropriate balance between the supplied nutrients and cell metabolism. The objective of this study was to develop a simple and efficient method for low-temperature cultivation (< 37 °C) that decreases cell metabolism for facilitating the buildup of 3D cardiac tissues. We created 3D cardiac tissues using cell sheet technology and analyzed the viability of the cardiac cells in low-temperature environments. To determine a method that would allow thicker 3D tissues to survive, we investigated the cardiac tissue viability under low-temperature culture processes at 20–33.5 °C and compared it with the viability under the standard culture process at 37 °C. Our results indicated that the standard culture process at 37 °C was unable to support higher-density myocardial tissue; however, low-temperature culture conditions maintained dense myocardial tissue and prevascularization. To investigate the efficiency of transplantation, layered cell sheets produced by the low-temperature culture process were also transplanted under the skin of nude rats. Cardiac tissue cultured at 30 °C developed denser prevascular networks than the tissue cultured at the standard temperature. Our novel findings indicate that the low-temperature process is effective for fabricating 3D tissues from high-functioning cells such as heart cells. This method should make major contributions to future clinical applications and to the field of organ engineering.     

A real-time quantitative PCR detection method for pork, chicken, beef, mutton, and horseflesh in foods

A rapid real-time quantitative PCR method to detect trace amounts of pork, chicken, beef, mutton, and horseflesh in foods was developed. The primers and TaqMan MGB (minor groove binder) probes were designed on the gene encoding cytochrome b for the specific detection of each species. The limit of quantification of this method was found to be 100 fg/microl of each mitochondrial DNA in 10 ng/microl of the wheat mitochondrial DNA matrix. The calculated R(2) values of the standard curves for the five species ranged between 0.994 and 0.999. This method would be particularly useful in the detection of hidden meat mince in processed foods, which would verify food labeling and gain consumers' trust.     

Control of Boolean networks: hardness results and algorithms for tree structured networks

Finding control strategies of cells is a challenging and important problem in the post-genomic era. This paper considers theoretical aspects of the control problem using the Boolean network (BN), which is a simplified model of genetic networks. It is shown that finding a control strategy leading to the desired global state is computationally intractable (NP-hard) in general. Furthermore, this hardness result is extended for BNs with considerably restricted network structures. These results justify existing exponential time algorithms for finding control strategies for probabilistic Boolean networks (PBNs). On the other hand, this paper shows that the control problem can be solved in polynomial time if the network has a tree structure. Then, this algorithm is extended for the case where the network has a few loops and the number of time steps is small. Though this paper focuses on theoretical aspects, biological implications of the theoretical results are also discussed.     

Cellular localization of a putative Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger 3 during ontogeny in the pronephros and mesonephros of the Japanese black salamander (<Emphasis Type="Italic">Hynobius nigrescens</Emphasis> Stejneger)

The cloning of cDNA and an examination of the tissue distribution of Na⁺/H⁺ exchanger 3 (NHE3) were carried out in the Japanese black salamander, Hynobius nigrescens. The cellular localization of Hynobius NHE3 was examined by in situ hybridization and immunohistochemistry during ontogeny in the nephron of the pronephros and mesonephros of the salamander. The partial amino acid sequence of Hynobius NHE3 was 81% and 72% identical to rat NHE3 and stingray NHE3, respectively. Hynobius NHE3 mRNA and protein were exclusively expressed along the late portion of the distal tubule to the anterior part of the pronephric duct of premetamorphic larvae (IY stages 43–50). NHE3 mRNA was expressed in the pronephros but not in the external gills in the larvae at the digit differentiation stage (IY stage 50). In the adult, mRNA was strongly expressed in the mesonephros but not in the ventral and dorsal skin. In juvenile and adult specimens, NHE3 immunoreactivity was observed at the apical membrane of the initial parts of the distal tubules of the mesonephric kidney. Immunohistochemical and in situ hybridization studies suggested that Na⁺ absorption coupled with H⁺ secretion via NHE3 occurred in the distal nephron of the pronephros and mesonephros. This is the first study to indicate NHE3 expression during ontogeny in amphibians. This work was supported in part by a research grant (a priority project in Science Faculty) from the University of Toyama to M.U.     

Laser impact on marine planktonic diatoms: an experimental study using a flow cytometry system

A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum. Three flow speeds, i.e. 9, 18 and 27 ml min-1 and three laser fluences, i.e. 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study. The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls. Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species). A maximum mortality of 0.77 log10 (about 83%) for C. gracilis and 0.68 log10 (about 78%) for S. costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence. The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C. gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S. costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2. In general, mortality increased with an increase in the laser fluence. The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures. The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water.