Radio-transparent multi-layer insulation for radiowave receivers.

In the field of radiowave detection, enlarging the receiver aperture to enhance the amount of light detected is essential for greater scientific achievements. One challenge in using radio transmittable apertures is keeping the detectors cool. This is because transparency to thermal radiation above the radio frequency range increases the thermal load. In shielding from thermal radiation, a general strategy is to install thermal filters in the light path between aperture and detectors. However, there is difficulty in fabricating metal mesh filters of large diameters. It is also difficult to maintain large diameter absorptive-type filters in cold because of their limited thermal conductance. A technology that maintains cold conditions while allowing larger apertures has been long-awaited. We propose radio-transparent multi-layer insulation (RT-MLI) composed from a set of stacked insulating layers. The insulator is transparent to radio frequencies, but not transparent to infrared radiation. The basic idea for cooling is similar to conventional multi-layer insulation. It leads to a reduction in thermal radiation while maintaining a uniform surface temperature. The advantage of this technique over other filter types is that no thermal links are required. As insulator material, we used foamed polystyrene; its low index of refraction makes an anti-reflection coating unnecessary. We measured the basic performance of RT-MLI to confirm that thermal loads are lowered with more layers. We also confirmed that our RT-MLI has high transmittance to radiowaves, but blocks infrared radiation. For example, RT-MLI with 12 layers has a transmittance greater than 95% (lower than 1%) below 200 GHz (above 4 THz). We demonstrated its effects in a system with absorptive-type filters, where aperture diameters were 200 mm. Low temperatures were successfully maintained for the filters. We conclude that this technology significantly enhances the cooling of radiowave receivers, and is particularly suitable for large-aperture systems. This technology is expected to be applicable to various fields, including radio astronomy, geo-environmental assessment, and radar systems.

Celecoxib upregulates endoplasmic reticulum chaperones that inhibit celecoxib-induced apoptosis in human gastric cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in cancer cells and this effect is involved in their antitumor activity. We recently demonstrated that NSAIDs upregulate GRP78, an endoplasmic reticulum (ER) chaperone, in gastric mucosal cells in primary culture. In the present study, induction of ER chaperones by NSAIDs and the effect of those chaperones on NSAID-induced apoptosis were examined in human gastric carcinoma cells. Celecoxib, an NSAID, upregulated ER chaperones (GRP78 and its cochaperones ERdj3 and ERdj4) but also C/EBP homologous transcription factor (CHOP), a transcription factor involved in apoptosis. Celecoxib also upregulated GRP78 in xenograft tumors, accompanying with the suppression of tumor growth in nude mice. Celecoxib caused phosphorylation of eukaryotic translation initiation factor 2 kinase (PERK) and eukaryotic initiation factor-2alpha (eIF2alpha) and production of activating transcription factor (ATF)4 mRNA. Suppression of ATF4 expression by small interfering RNA (siRNA) partially inhibited the celecoxib-dependent upregulation of GRP78. Celecoxib increased the intracellular Ca2+ concentration, while 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, an intracellular Ca2+ chelator, inhibited the upregulation of GRP78 and ATF4. These results suggest that the Ca2+-dependent activation of the PERK-eIF2alpha-ATF4 pathway is involved in the upregulation of ER chaperones by celecoxib. Overexpression of GRP78 partially suppressed the apoptosis and induction of CHOP in the presence of celecoxib and this suppression was stimulated by coexpression of either ERdj3 or ERdj4. On the other hand, suppression of GRP78 expression by siRNA drastically stimulated cellular apoptosis and production of CHOP in the presence of celecoxib. These results show that upregulation of ER chaperones by celecoxib protects cancer cells from celecoxib-induced apoptosis, thus may decrease the potential antitumor activity of celecoxib.

Rapid adsorption pretreatment with submicrometre powdered activated carbon particles before microfiltration.

Manufacturer-supplied powdered activated carbon (PAC) was ground to produce submicrometre particles (0.8 and 0.6 m median diameter) for use as an adsorbent before microfiltration (MF) for drinking water treatment. Batch tests revealed that the microground PAC adsorbed natural organic matter (NOM) much more rapidly and had a higher adsorptive capacity than ordinary PAC. The water samples pretreated with the submicrometre PAC were subjected to MF, and the results of experiments with different PAC contact times revealed that a 1 min retention time was sufficient for adsorptive removal of NOM. The use of submicrometre PAC permitted not only shorter PAC contact times but also a 75% reduction in dose.

Endoplasmic reticulum stress response is involved in nonsteroidal anti-inflammatory drug-induced apoptosis.

Apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAID-induced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guinea-pig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.

Interaction of metallopyrazoliumylporphyrins with calf thymus DNA.

The interaction of transition metal complexes of cationic porphyrins bearing five membered rings, meso-tetrakis(1,2-dimethylpyrazolium-4-yl)porphyrin (MPzP, M=Mn(III), Ni(II), Cu(II) or Zn(II)), with calf thymus DNA (ctDNA) has been studied. Metalloporphyrins NiPzP and CuPzP are intercalated into the 5'GC3' step of ctDNA. MnPzP is bound edge-on at the 5'TA3' step of the minor groove of ctDNA, while ZnPzP is bound face-on at the 5'TA3' step of the major groove of ctDNA. The binding constants of the metalloporphyrins to ctDNA range from 1.05x10(5) to 2.66x10(6) M(-1) and are comparable to those of other reported cationic porphyrins. The binding process of the metallopyrazoliumylporphyrins to ctDNA is endothermic and entropically driven. These results have revealed that the kind of central metal ions of metalloporphyrins influences the binding characteristics of the porphyrin to DNA.

Molecular mechanism for functional interaction between DnaA protein and acidic phospholipids: identification of important amino acids.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be reactivated from the ADP-bound form to its ATP-bound form through stimulation of ADP release by acidic phospholipids such as cardiolipin. We previously reported that two potential amphipathic helices (Lys-327 to Ile-344 and Asp-357 to Val-374) of DnaA protein are involved in the functional interaction between DnaA and cardiolipin. In relation to one of these helices (Asp-357 to Val-374), we demonstrated that basic amino acids in the helix, especially Lys-372, are vital for this interaction. In this study, we have identified an amino acid in the second potential amphipathic helix (Lys-327 to Ile-344), which would also appear to be involved in the interaction. We constructed three mutant dnaA genes with a single mutation (dnaAR328E, dnaAR334E, and dnaAR342E) and examined the function of the mutant proteins. DnaAR328E, but not DnaAR334E and DnaAR342E, was found to be more resistant to inhibition of its ATP binding activity by cardiolipin than the wild-type protein. The stimulation of ADP release from DnaAR328E by cardiolipin was also weaker than that observed with the other mutants and the wild-type protein. These results suggest that Arg-328 of DnaA protein is involved in the functional interaction of this protein with acidic phospholipids. We propose that acidic phospholipids bind to two basic amino acid residues (Arg-328 and Lys-372) of DnaA protein and change the higher order structure of its ATP-binding pocket, which in turn stimulates the release of ADP from the protein.

Fluorescent molecular sensory system based on bis pyrene-modified gamma-cyclodextrin dimer for steroids and endocrine disruptors.

A gamma-cyclodextrin dimer modified with two pyrene moieties, 6-(2-pyrenebutylate-aminoethyl)pyrenebutylate-amino-6-deoxy-bis-gamma-cyclodextrin, has been synthesized in the presence of N,N'-dicyclohexycarbodiimide from gamma-cyciodextrin dimer linked with ethylenediamine at an upper rim. The sensing ability and binding property of the titled dimer were investigated for bile acids and endocrine disruptors. This cyclodextrin dimer showed both monomer and excimer fluorescence; the guest-induced emissions were observed as increases or decreases depending on the guest. The values deltaI(m)/I0(m) and deltaI(ex)/I0(ex), where I0(m) and I(m) are fluorescence intensities of monomer emission in the absence and presence of a guest and I0(ex) and I(ex) are those of excimer emission and deltaI(m) and deltaI(ex) were I(m) - I0(m) and I(ex) - I0(ex), respectively, were used as a parameter of sensitivity. This host exhibited highly sensitive molecular recognition ability for bile acids and endocrine disruptors, in which the sensing parameters obtained from monomer emission were plus or minus values, whereas the parameters obtained as excimer emission were minus ones. The behavior of the appended moieties of the host during a host-guest complexation was studied by induced circular dichroism (ICD) and fluorescence spectra. The ICD intensities of the titled dimer were decreased upon an addition of a guest. The guest-induced variations in the fluorescence and ICD intensity suggest that the appended moieties move by altering the spatial relationship in the hydrophobic space between two linked cyclodextrins.

Suppression of temperature-sensitivity of a dnaA46 mutant by excessive DNA supercoiling.

We report here that the high-temperature sensitivity of a dnaA46 mutant was suppressed by addition of high concentrations of NaCl into the culture medium. This suppression was also observed with other high-temperature-sensitive dnaA mutants, except dnaA167 and dnaA508 mutants, which have mutations in the N-terminal region of DnaA protein. Since high concentrations of NaCl in the medium increased negative DNA supercoiling in a dnaA46 mutant, we hypothesized that the increase in DNA supercoiling is involved in the suppression of the temperature-sensitivity of the dnaA46 mutant by high concentrations of NaCl. This hypothesis was supported by in vitro and in vivo results as follows. A low DNA replication activity of purified DnaA46 protein at high temperatures was increased in line with an increase in DNA supercoiling of template DNA. The dnaA46 mutant showed higher sensitivity to nalidixic acid, a DNA-relaxing drug, than did the wild-type cells under the conditions of high temperatures and high concentrations of NaCl.

Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be regulated through its binding to acidic phospholipids, such as cardiolipin. In our previous paper (Hase, M., Yoshimi, T., Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T. (1998) J. Biol. Chem. 273, 28651-28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg(360), Arg(364), and Lys(372)) were mutated to acidic amino acids showed a decreased ability to interact with cardiolipin in vitro, suggesting that DnaA protein binds to cardiolipin through an ionic interaction. In this study, we construct three mutant dnaA genes each with a single mutation and examined the function of the mutant proteins in vitro and in vivo. All mutant proteins maintained activities for DNA replication and ATP binding. A mutant protein in which Lys(372) was mutated to Glu showed the weakest interaction with cardiolipin among these three mutant proteins. Thus, Lys(372) seems to play an important role in the interaction between DnaA protein and acidic phospholipids. Plasmid complementation analyses revealed that all these mutant proteins, including DnaA431 could function as an initiator for chromosomal DNA replication in vivo.

Role of the amino-terminal region of the DnaA protein in opening of the duplex DNA at the oriC region.

We report in this paper that the amino acid residues Ile-26 and Leu-40 of the DnaA protein are essential for the DNA replication activity in vitro. Lines of evidence to support this conclusion are as follows. Variants of the DnaA protein containing either an Ile-26-Ser or Leu-40-Ser replacement were unable to support oriC DNA replication in vitro. Though the mutant DnaA proteins retained the capability to bind oriC DNA, they were unable to open the duplex DNA at oriC. Based on these and other results, we conclude that the N-terminal region of the DnaA protein is involved in the oligomerization of this protein, an essential step for the duplex opening activity at oriC.

Expression pattern of a newly recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion.

Leukocyte cell-derived chemotoxin 2 (LECT2) is a recently isolated protein and has been shown to be synthesized by human hepatocytes. All hepatocytes show diffuse immunostaining for LECT2 within the cytoplasm. In the present study, an attempt was made to clarify the expression pattern of LECT2 in nine cases of low-grade malignant hepatocellular carcinoma (LGM-HCC) and five cases of advanced HCC and 19 cases of premalignant lesion, termed atypical hyperplasia (AH), using the indirect immunoperoxidase technique. Variable spotty to coarsely diffuse staining in the majority of cells, a mixture of positively staining and negatively staining areas, and essentially negative staining was observed within the cellular cytoplasm of AH, LGM-HCC and advanced HCC, respectively. The expression of LECT2 became weaker with the progression of multistep hepatocarcinogenesis. The data clearly demonstrate that LECT2 becomes essentially negative in full-blown HCC cells and that the histological distinction between AH and LGM-HCC is valid. It also seems likely that LECT2 is related to hepatocyte growth.

Involvement of Arg-328, Arg-334 and Arg-342 of DnaA protein in the functional interaction with acidic phospholipids.

We reported previously that three basic amino acids (Arg-360, Arg-364 and Lys-372) of DnaA protein are essential for its functional interaction with cardiolipin. In this study, we examined the effect of mutation of some basic amino acids in a potential amphipathic helix (from Lys-327 to Ile-345) of DnaA protein on this interaction. ATP binding to the mutant DnaA protein, in which Arg-328, Arg-334 and Arg-342 were changed to acidic amino acids, was less inhibited by cardiolipin than that of the wild-type protein, as was the case for mutant DnaA protein with mutations of Arg-360, Arg-364 and Lys-372. A mutant DnaA protein with mutations of all six basic amino acids showed the most resistance to the inhibition of ATP binding by cardiolipin. These results suggest that Arg-328, Arg-334 and Arg-342, like Arg-360, Arg-364 and Lys-372, are also involved in the functional interaction between DnaA protein and acidic phospholipids.

Apoptosis of the intestinal principal cells of Xenopus larvae exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In our previous work using paraffin-embedded sections, we determined that Xenopus larvae exposed to 200 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 5 days from shortly after fertilization to the early larval stage showed a shortening of the digestive tract and a loss of mucosal epithelium cells due to exfoliation into the gut cavity. In the current study, we ultrastructurally examined the mucosal epithelium of the gut of TCDD-exposed Xenopus larvae 12 days after fertilization. Exfoliated cell structures at the villus tip and in the lumen were equipped with a microvillus portion and occasionally had terminal web-like structures seen by ultramicroscopy. As these exfoliated structures had nuclear fragments with condensed chromatin, they were considered to be apoptotic bodies derived from the principal cells of the epithelium. In addition, many membrane-bound cell fragments-identified as apoptotic bodies derived from the principal cells based on their ultrastructural features-were observed at the basal side of the mucosal epithelium. These apoptotic bodies were phagocytized and digested chiefly by the neighboring intact principal cells. No such cells and/or cell fragments showing apoptotic features were observed in the controls. Our observations indicate that marked apoptosis occurs in the intestinal principal cells of TCDD-exposed larvae, which may result in the shortening of the gut.

Site-directed mutational analysis for the membrane binding of DnaA protein. Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli.

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

Hepatitis C virus is frequently coinfected with serum marker-negative hepatitis B virus: probable replication promotion of the former by the latter as demonstrated by in vitro cotransfection.

Patients with hepatitis C have been reported occasionally to be coinfected with serum marker-negative (silent) hepatitis B virus (HBV). The frequency and significance of such coinfection were investigated. Thirty patients with hepatitis C virus (HCV) infections (10 acute, 10 chronic, 10 cirrhotic) were selected randomly; the acute cases were without serum hepatitis B surface antigen (HBsAg) and anti-hepatitis B core IgM, and the chronic cases were without HBsAg. A nested polymerase chain reaction for the X open reading frame was used to amplify HBV DNA in serum, and immunoperoxidase staining was carried out on liver biopsy specimens. Nucleotide sequencing was carried out to characterize the amplified HBV DNAs. In order to clarify the possibility that the silent HBV mutant promotes HCV replication in the liver, the full-length HCV RNA and the cloned silent HBV DNA dimer were cotransfected into an established cell line, HuH-7, and the amount of secreted HCV RNA was quantified serially. The target HBV DNA was amplified in 26 (86.7%) of the 30 patients. Subsequent direct nucleotide sequencing in 9 selected patients revealed an 8-nucleotide deletion, characteristic of a silent HBV mutant. Immunostaining revealed hepatitis B surface antigen in 15 (50.0%). Cotransfected silent HBV DNA augmented the secretion of HCV RNA by up to 5-fold in comparison with HCV RNA transfection alone. In conclusion, HCV is coinfected frequently with the silent HBV mutant and the latter probably promotes the replication of the former in the liver.

Inhibition of thymidine transport in dnaA mutants of Escherichia coli.

DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli. We report here our evidence that thymidine transport across cytoplasmic membranes in temperature-sensitive dnaA mutants is greatly decreased at a permissive temperature for growth of the mutants. Complementation analysis with a plasmid containing the wild type dnaA gene and P1 phage-mediated transduction confirmed that mutations in the dnaA gene were responsible for the phenotype. A low level of nucleoside transport in the dnaA mutant was specific for thymidine; transport activities for other nucleosides were much the same as those in wild type cells. Membrane vesicles prepared from the dnaA mutant showed much the same activity of thymidine transport as did those from the wild type cells. No significant difference in the activity of thymidine kinase, which is known to facilitate thymidine transport, was seen between the mutant and the wild type cells. An increase in the pool of dTTP, a negative regulator for thymidine kinase, was observed in the dnaA mutant. Based on these observations, we suggest that inhibition of thymidine transport in dnaA mutants is caused by increases in the dTTP pool.

Apoptotic cell death of erythrocytes in Xenopus larvae exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Xenopus embryos were exposed to 200 ppb 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 5 days from the 2- to 8-cell stage of cleavage to the early larval stage. Larvae that developed generalized edema were collected at 7 days after the end of TCDD exposure for light and electron microscopic studies. Erythrocytes in the peripheral blood of the edematous larvae were examined. Between 0.3 and 33.9% of identifiable erythrocytes of exposed larvae had dilated perinuclear cisternae. Furthermore, some had extremely condensed nuclear chromatin usually coalesced against 1 pole of the nuclear membrane and overall compacted cytoplasm. The erythrocytes showing nuclear condensation were phagocytosed by macrophages. These features are typical of cells undergoing apoptosis. Anemia is 1 symptom of TCDD toxicity in various animal species, including mammals. In this study, we demonstrate that TCDD induces apoptotic cell death in circulating erythrocytes of Xenopus larvae, which may be 1 cause of anemia in this species.