Development of a high dynamic range spectroscopic system for observation of neutral hydrogen atom density distribution in Large Helical Device core plasma.

We report development of a high dynamic range spectroscopic system comprising a spectrometer with 30% throughput and a camera with a low-noise fast-readout complementary metal-oxide semiconductor sensor. The system achieves a 10(6) dynamic range (∼20 bit resolution) and an instrumental function approximated by a Voigt profile with Gauss and Lorentz widths of 31 and 0.31 pm, respectively, for 656 nm light. The application of the system for line profile observations of the Balmer-α emissions from high temperature plasmas generated in the Large Helical Device is also presented. In the observed line profiles, emissions are detected in far wings more than 1.0 nm away from the line center, equivalent to neutral hydrogen atom kinetic energies above 1 keV. We evaluate atom density distributions in the core plasma by analyzing the line profiles.

Engineered synthetic pathway for isopropanol production in Escherichia coli.

A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.

Suppression of TGF-beta signaling by conophylline via upregulation of c-Jun expression.

In the course of screening for inhibitors of transforming-growth factor-beta (TGF-beta) functions we found that conophylline, a vinca alkaloid, inhibited TGF-beta-induced apoptosis in rat hepatoma cells. Because conophylline also inhibited TGF-b-induced promoter activity in mink lung cells, we studied the mechanism of the inhibition in this cell line. Conophylline did not inhibit nuclear translocation of Smad2. Instead, we found that conophylline increased the expression of c-Jun, which had been earlier shown to interact with the corepressor TGIF to suppress the transcriptional activity dependent on Smad2. Conophylline attenuated the interaction between the Smad2 complex and p300 but enhanced that between the Smad2 complex and TGIF. In cells overexpressing c-Jun, suppression of promoter activity induced by TGF-beta and the enhancement of the association of the Smad2 complex with TGIF were also observed. Thus, our data suggest that inhibition of TGF-beta-induced promoter activity by conophylline can be attributed to its potency in modulating the interaction of downstream transcriptional factors via upregulation of c-Jun expression.

Design and development of a catalytic ribonucleoprotein.

Ribonucleoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahymena group I self-splicing intron RNA and two proteins (bacteriophage lambdaN and HIV Rev proteins) containing RNA-binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme.

A comparative study on two GNRA-tetraloop receptors: 11-nt and IC3 motifs.

Natural RNAs often contain terminal loops consisting of GNRA (N=A, G, C, U; R=A, G) and their receptors, which bind to the loops via long-range RNA-RNA interactions. Among several known receptors, two characteristic structural elements have been identified that are termed the 11-nt motif (CCUAAG-UAUGG) and IC3 motif (CCCUAAC-GAGGG). These two motifs that share a similar secondary structure have been shown to exhibit distinctively different binding specificities. The 11-nt motif recognizes a GAAA loop with highest specificity among the known receptors, whereas the IC3 motif distinguishes GAAA from other GNRA loops less stringently than any other receptors. To identify the elements in the receptors that determine the binding specificity, a series of chimeric receptors derived from the two motifs were prepared and their properties were examined. We identified characteristic base-pairs and a particular U residue in the receptors as such elements by means of a gel mobility shift assay that evaluates the degree of the tetraloop-receptor interaction. The relationship between the elements and the specificity is discussed together with a model that describes a possible evolutional linkage between the two receptors.

Synaptic architecture of glomeruli in lamina II of the chicken spinal cord, as revealed using ultrathin section and freeze fracture techniques.

Synaptic glomeruli in lamina II of the chicken dorsal horn were studied using the freeze fracture technique, and the results were compared with those obtained using the ultrathin section technique. Our findings using the freeze fracture technique were as follows. (1) On the presynaptic P-face of the central terminal, intramembrane particles (IMPs) were arranged circularly around a small dimple which was reported to be a synaptic vesicle attachment site. A distinct area with aggregated large IMPs was found on the postsynaptic E-face of some peripheral neuronal elements. (2) The area with small IMPs intermingled with several dimples and the area with aggregated large IMPs were present juxtaposed on the same central terminal P-face. The area with aggregated large IMPs indicates that the central terminal functions as a postsynaptic element; accordingly, the two areas represent a reciprocal synapse. (3) Distinct IMP aggregates were observed on the P-face of vesicle-containing dendrites which did not face the central terminal. (4) A fractured septate junction was revealed as numerous parallel-lined furrows on the E-face of the central terminal. The distribution of IMPs in the synaptic glomerulus supports the hypothesis that the synaptic glomerulus is the site of the local inhibitory feedback circuit for pain transmission.

Inhibition of nitric oxide synthase induction by 15-deoxyspergualin in a cultured macrophage cell line, J774A.1 [correction of J744A.1] activated with IFN-gamma and LPS.

Immunosuppressant 15-deoxyspergualin (DSG) inhibited induction of inducible nitric oxide synthase (iNOS) following stimulation with IFN-gamma and LPS in a cultured macrophage cell line, J774A.1 [corrected]. By DSG treatment NO2- accumulation in the medium was blocked, and cellular iNOS protein level decreased as shown by Western blotting. DSG didn't have any direct effect on iNOS activity. DSG was not used as a substrate of NOS in in vitro enzyme systems, and it was too weak an inhibitor of iNOS and cNOS to cause the inhibition of accumulation of NO2-. DSG did not scavenge NO spontaneously generated from NOR. Structure-activity relationships of analogs and decomposed elements showed that there is correlation between the inhibition of iNOS induction and immunosuppressive activity.

Rearrangements of actin cytoskeleton during infection with Escherichia coli O157 in macrophages.

The lamina propria of the large intestine is rich in macrophages, and they might be one of the first lines of the host defense in enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection. Although macrophages were infected with them, they can survive the EHEC O157 infection. We examined the structural rearrangements of the actin cytoskeleton during the microbial infection process. Macrophage actin filaments were rearranged in the following sequence; 1) disappearance of the actin filament bundles in the cytoplasm, 2) accumulation of actin filaments under the cell surface, and 3) construction of actin networks underlying the endosome membrane. Before infection, actin filaments were distributed under the cell surface and in bundles located in the macrophage cytoplasm. Within 2 min, infection caused a rapid and marked loss of the actin filament bundles that had run parallel to the long axis of the cell. Concomitant with the loss, actin filaments became more markedly distributed under the cell surface. In the formation of the endosome, new networks of actin filaments were constructed below the phagosome membrane. The networks contained a large amount of actin as well as a fodrin-like immunoreactivity. The thickness of the networks reached about 400 nm under the phagosome membrane. The actin networks disappeared again after the bacterial digestion. The results of this study showed that actin filaments undergo three major rearrangements of the actin filaments during the infection in macrophages, and suggested that the third rearrangement is mediated by actin-binding proteins, such as a fodrin-like molecules. These morphological changes in macrophages were not clear after infection with other strains of Escherichia coli.

Exocytotic secretion of toxins from macrophages infected with Escherichia coli O157.

This study examined whether macrophages are involved in the development of pathogenicity in Shiga-like toxin (SLT)-producing enterohemorrhagic Escherichia coil (EHEC) O157:H7. Macrophages were infected with the bacteria, after which the macrophage culture medium showed a clear increase in toxicity in rats in vivo as well as in rat aortic endothelial cells in vitro. The increased toxicity resulted mainly from a rapid increase in the concentrations of SLT type I (SLT-I) and type II (SLT-II) and partly from an increase in concentrations of the proinflammatory cytokines, tumor necrosis factor alpha (TNFalpha) and interleukin-1 (IL-1), in the culture medium. Most of the EHEC O157 added to the macrophage culture were quickly incorporated to form phagosomes, which then fused with lysosomes to become phagolysosomes. During this intracellular digestion process, the EHEC O157 remained alive for about 15 min, and continued synthesizing and secreting the toxins SLT-1 and SLT-II. The bacteria were then killed and digested in the phagolysosomes with significant amounts of the toxins retained. Subsequently, the contents of the phagolysosomes were exocytotically secreted from the macrophage cell membrane into the surrounding culture medium. Such a sequence of events in macrophages may occur in vivo, suggesting the active involvement of macrophages in the rapid increase in pathogenicity, such as seen in the onset of hemolytic-uremic syndrome (HUS) in patients infected with EHEC O157. The exocytotic secretion is considered to be one of the most basic cellular functions in macrophages.

Sex Differences in Polymorphism and Expression of AAT-1 in the Hiroshima Population of Buergeria buergeri (Anura, Rhacophoridae).

Buergeria buergeri is female heterozygous in sex determination; chromosome pair No. 7 in this species is a pair of sex chromosomes of the ZZ/ZW type. Genetic analysis of AAT-1 variants was carried out to elucidate the mode of inheritance of this locus by starch-gel electrophoresis using field-caught females and males and their offspring produced by artificial crossings. The results showed that the AAT-1 locus is sex-linked and that alleles are expressed on the Z chromosome, but not the W chromosome. It is evident that the AAT-1 gene of female offspring is hemizygous and that the allele present is on the Z chromosome, which is derived from the male parent.

Large dorsal horn neurons which receive inputs from numerous substance P-like immunoreactive axon terminals in the laminae I and II of the chicken spinal cord.

Large neurons outlined with numerous substance P (SP)-like immunoreactive (LI) boutons were detected immunocytochemically in the dorsal horn of the chicken spinal cord at the light microscopic level. The cervical enlargement was mainly used for observations. By electron microscopy, asymmetrical synapses were observed between the SP-LI axon terminals and the soma and dendrites of the large neurons. Cell bodies of the large neurons were mostly localized in the lamina I and the region lateral to the lamina I. Some of the cell bodies were also located in the lamina II. Their dendrites extended in the lamina I, in the region lateral to the lamina I, and deeply in the lamina II. In the lamina II, dendrites of these neurons formed synapses with SP-containing central terminals in synaptic glomeruli known to originate from primary afferents. The findings suggest that these large neurons receive nociceptive information directly from primary afferents. In the light of previous investigations, these neurons are considered to be pain-transmitting long ascending tract neurons.

Induction of flat morphology in K-ras-transformed fibroblasts by lycorine, an alkaloid isolated from the tropical plant Eucharis grandiflora.

In the course of screening for Ras function inhibitors from plant extracts, we isolated lycorine from a chloroform extract of Eucharis grandiflora leaves. Lycorine induced flat morphology in K-ras-NRK cells after treatment for 2-3 days, whereas its morphological effect on NRK cells was weaker. Lycorine was found to inhibit protein synthesis specifically in cultured K-ras-NRK cells. It also lowered the cellular amount of Ras in 2-3 days.

Expression of p21(waf-1/cip-1) is significantly induced in the livers of LEC rats with chronic liver injury.

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21(waf-1/ciP-1) and p27, cyclin-dependent kinase inhibitors, by northern blot analysis. The expression of p21(waf-1/cip-1 ) in the LEC rat liver was 3-fold higher than that of age-matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p21(waf-1/cip-1) in the nuclear matrix fraction of the LEC rat liver. Immunohistochemically, p21(waf-1/cip-1) was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.

[Bilateral non-Hodgkin's lymphoma of the adrenal glands with adrenal insufficiency].

A 66-year-old female presented with anorexia, fatigue, skin pigmentation, weight loss and low grade fever. Imaging studies demonstrated bilateral bulky masses confined to the adrenal glands. Ultrasonography guided needle biopsy of the mass showed findings of diffuse large B-sell lymphoma. Low levels of serum cortisol, urinary 17-OHCS and 17-KS, a high level of ACTH and a non-reactive pattern on the rapid ACTH test led to a diagnosis of Addison's disease. Only a partial regression was achieved by the first chemotherapy. She died due to disease progression, while the next course of chemotherapy had been postponed because of interstitial pneumonitis due to methotrexate.

Interaction between substance P-immunoreactive central terminals and gamma-aminobutyric acid-immunoreactive elements in synaptic glomeruli in the lamina II of the chicken spinal cord.

We investigated the interaction between gamma-aminobutyric acid (GABA)-immunoreactive (IR) elements and substance P (SP)-IR central terminals in synaptic glomeruli in lamina II of the chicken spinal cord in order to ascertain how pain information is modulated in the spinal dorsal horn. We combined the peroxidase-antiperoxidase (PAP) technique and the protein A-gold (PAG) technique to observe the synaptic relationship between these two components. At the light microscopic level, we observed both GABA-IR and SP-IR elements in the lamina II. GABA-IR elements were also observed in the lamina III. At the electron microscopic level, the following three GABA-IR elements formed synapses with the SP-IR central terminals in synaptic glomeruli: (1) elements which appeared to be axon terminals containing tightly-packed pleomorphic clear vesicles; (2) elements which appeared to be vesicle-containing dendrites with loosely-packed clear and dense-cored vesicles (DCVs); and (3) dendrites without synaptic vesicles. The first type of element was always presynaptic to the SP-IR central terminal. The second type was postsynaptic, presynaptic or in some cases reciprocal to the SP-IR central terminals. The third type was postsynaptic to the SP-IR central terminal. These results suggest that the SP-containing primary afferents activate GABA-containing dendrites and that the SP-containing primary afferents are inhibited presynaptically by GABA-containing neurons through axo-axonic and dendro-axonic synapses.

In vivo responsiveness of thyroid glands to TSH in normal and novel 'growth-retarded' mice: transient elevation in normal mice and impairment in 'growth-retarded' mice.

The in vivo responsiveness of thyroid glands to TSH at various ages in novel 'growth-retarded' (grt/grt) mice derived from Snell's dwarf (DW/J) mice and in their normal counterparts were analysed by determining serum T4 concentrations before and after the administration of exogenous TSH. The serum T4 concentration in normal mice increased in response to TSH at 2, 4 and 12 weeks of age but not at 1 week of age. A transient augmentation of such thyroidal responsiveness to TSH was apparent in normal mice at 2 weeks of age, when the serum T4 level exhibits a peak and the pubertal growth of mice starts. In contrast to normal mice, at any age examined from 2 to 12 weeks after birth, exogenous TSH did not influence serum T4 concentrations in the grt/grt mice at all. On the other hand, serum TSH concentrations in young grt/grt mice were highly elevated compared with those in normal mice and they were normalized by a 2-3 week's treatment with T3. Morphological studies demonstrated degenerated thyroid glands in the grt/grt mice. These results suggest that the severe hypothyroidism and consequent growth retardation in growth-retarded mice are due to impairment of the thyroid glands of the mutant mice in producing and/or secreting thyroid hormones in response to TSH.

A novel hypothyroid 'growth-retarded' mouse derived from Snell's dwarf mouse.

This paper describes a novel mutant mouse that has been spontaneously derived from the Snell's dwarf (DW/J) mouse. It was named the 'growth-retarded mouse' because of a characteristic growth pause followed by the delayed onset of pubertal growth. The onset of the increase in pituitary GH content that normally occurs concomitant with pubertal growth was also delayed in the growth-retarded mice. The serum concentration of thyroxine was very low in these mice from the neonatal period through adulthood, and a supplement of tri-iodothyronine was effective in shortening the growth pause and commencing the suppressed pubertal growth. Histological and immunohistochemical studies revealed that the anterior pituitary gland of the growth-retarded mouse contains clustered unusual chromophobic cells which are not reactive to various antisera against anterior pituitary hormones and the gland becomes enlarged with age. Breeding data indicated that these characteristics of the mice show an autosomal recessive inheritance and the gene responsible was designated as 'grm'. Partial linkage analysis utilizing microsatellite polymorphism demonstrated that the grm gene does not identify with the lit or hyt genes. Based on comparison of the hormonal status and growth pattern between growth-retarded, dwarf and normal mice, we have suggested the existence of a mutual interaction, possibly positive feedback regulation, between the pituitary and thyroid glands, that develops or matures the hormonal network which is responsible for rapid somatic growth and metabolic changes at puberty in mice.