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The release of radiocesium (r-Cs) into natural aqueous systems is of concern because of its extended solubility as an alkaline metal ion and its facile incorporation into living beings. A technique for the selective separation of Cs from an aqueous matrix using dual solid-phase extraction (SPE) systems in a series is proposed in this paper. The SPEs equipped with chelates (Nobias Chelate-PA1 and Nobias Chelate-PB1), an ion-exchange resin (Nobias Ion SC-1), or macrocycles (MetaSEP AnaLig Cs-01 and MetaSEP AnaLig Cs-02) were evaluated in terms of selectivity and retention/recovery behavior toward Cs and other potentially competing ions (Li, Na, K, Rb, Ba, Ca, Mg, and Sr). The simulated solution of 133 Cs, a chemical analog of r-Cs, was used to optimize the separation process. Operating parameters such as pH (3-13), flow rate (0.2-5.0 mL min -1 ), and elution behavior (HCl, 0.1-5.0 mol L - 1 ) were optimized to ensure maximum removal of Cs from the aqueous matrices. The dual SPE system comprised Nobias Chelate-PB1 that minimized the competing impact of ions, while selective Cs retention was attained with MetaSEP AnaLig Cs-02. The proposed process was verified using real r-Cs-contaminated water from Fukushima, Japan, to observe the quantitative separation and preconcentration of r-Cs from the complex matrices., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N 2 O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO 2 - ) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cytoglobin (Cygb) is a member of the hexacoordinated globin protein family and is expressed ubiquitously in rat and human tissues. Although Cygb is reportedly upregulated under hypoxic conditions both in vivo and in vitro, suggesting a physiological function to protect cells under hypoxic/ischemic conditions by scavenging reactive oxygen species or by signal transduction, the mechanisms associated with this function have not been fully elucidated. Recent studies comparing Cygbs among several species suggest that mammalian Cygbs show a distinctly longer C-terminal domain potentially involved in unique physiological functions. In this study, we prepared human Cygb mutants (ΔC, ΔN, and ΔNC) with either one or both terminal domains truncated and investigated the enzymatic functions and structural features by spectroscopic methods. Evaluation of the superoxide-scavenging activity between Cygb variants showed that the ΔC and ΔNC mutants exhibited slightly higher activity involving superoxide scavenging as compared with wild-type Cygb. Subsequent experiments involving ligand titration, flash photolysis, and resonance Raman spectroscopic studies suggested that the truncation of the C- and N-terminal domains resulted in less effective to dissociation constants and binding rates for carbon monoxide, respectively. Furthermore, structural stability was assessed by guanidine hydrochloride and revealed that the C-terminal domain might play a vital role in improving structure, whereas the N-terminal domain did not exert a similar effect. These findings indicated that long terminal domains could be important not only in regulating enzymatic activity but also for structural stability, and that the domains might be relevant to other hypothesized physiological functions for Cygb., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A relatively rapid and precise method is presented for the determination of lead in aqueous matrix. The method consists of analyte quantitation using the liquid electrode plasma-optical emission spectrometry (LEP-OES) coupled with selective separation/preconcentration by solid-phase extraction (SPE). The impact of operating variables on the retention of lead in SPEs such as pH, flow rate of the sample solution; type, volume, flow rate of the eluent; and matrix effects were investigated. Selective SPE-separation/preconcentration minimized the interfering effect due to manganese in solution and limitations in lead-detection in low-concentration samples by LEP-OES. The LEP-OES operating parameters such as the electrical conductivity of sample solution; applied voltage; on-time, off-time, pulse count for applied voltage; number of measurements; and matrix effects have also been optimized to obtain a distinct peak for the lead at λ max =405.8nm. The limit of detection (3σ) and the limit of quantification (10σ) for lead determination using the technique were found as 1.9 and 6.5ng mL -1 , respectively. The precision, as relative standard deviation, was lower than 5% at 0.1μg mL -1 Pb, and the preconcentration factor was found to be 187. The proposed method was applied to the analysis of lead contents in the natural aqueous matrix (recovery rate:>95%). The method accuracy was verified using certified reference material of wastewaters: SPS-WW1 and ERM-CA713. The results from LEP-OES were in good agreement with inductively coupled plasma optical emission spectrometry measurements of the same samples. The application of the method is rapid (≤5min, without preconcentration) with a reliable detection limit at trace levels., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cytoglobin (Cgb) was discovered a decade ago and is a fourth member of the group of hexacoordinated globin-folded proteins. Although some crystal structures have been reported and several functions have been proposed for Cgb, its physiological role remains uncertain. In this study, we measured cyanide binding to the ferric state of the wild-type (WT) Cgb, and found that the binding consisted of multiple steps. These results indicated that Cgb may be comprised of several forms, and the presence of monomers, dimers, and tetramers was subsequently confirmed by SDS-PAGE. Remarkably, each species contained two distinguishable forms, and, in the monomer, analyses of alternative cysteine states suggested the presence of an intramolecular disulfide bond (monomer SS form) and a structure with unpaired thiol groups (monomer SH form). These confirmed that forms were separated by gel-exclusion chromatography, and that the cyanide binding of the separated fractions was again measured; they showed different affinities for cyanide, with the monomer fraction showing the highest affinity. In addition, the ferrous state in each fraction showed distinct carbon monoxide (CO)-binding properties, and the affinities for cyanide and CO suggested a linear correlation. Furthermore, we also prepared several variants involving the two cysteine residues. The C38S and C83S variants showed a binding affinity for cyanide similar to the value for the monomer SH form, and hence the fraction with the highest affinity for exogenous ligands was designated as a monomer SS form. We concluded that polymerization could be a mechanism that triggers the exertion of various physiological functions of this protein and that an appropriate disulfide bond between the two cysteine residues was critical for regulating the binding affinity of Cgb, which can act as a ROS scavenger, for exogenous ligands., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Indium is a rare metal that is mostly consumed as indium tin oxide (ITO) in the fabrication process of liquid crystal display (LCD) panels. The spent LCD panels, termed as LCD-waste hereafter, is an increasing contributor of electronic waste burden worldwide and can be an impending secondary source of indium. The present work reports a new technique for the reclamation of indium from the unground LCD-waste using aminopolycarboxylate chelants (APCs) as the solvent in a hyperbaric environment and at a high-temperature. Microwave irradiation was used to create the desired system conditions, and a substantial abstraction of indium (≥80%) from the LCD-waste with the APCs (EDTA or NTA) was attained in the acidic pH region (up to pH 5) at the temperature of ≥120 °C and the pressure of ~50 bar. The unique point of the reported process is the almost quantitative recovery of indium from the LCD-waste that ensured via the combination of the reaction facilitatory effect of microwave exposure and the metal extraction capability of APCs. A method for the selective isolation of indium from the extractant solution and recycle of the chelant in solution is also described., (Copyright © 2013 Elsevier B.V. All rights reserved.)

TNF has been reported to induce caspase-independent necroptosis in the presence of Z-VAD-fmk, a pan-caspase inhibitor. We examined whether necroptosis was induced by caspase inhibitors other than Z-VAD-fmk. TNF-induced necroptosis was detected in the presence of Z-DEVD-fmk, which is commonly used as a caspase-3-specific inhibitor, but not in the presence of Z-Asp-CH2-DCB, which was reported to be a pan-caspase inhibitor. TNF-induced caspase-3 activity was completely inhibited by Z-VAD-fmk, Z-DEVD-fmk, or Z-Asp-CH2-DCB. Although TNF-induced proteolytic activation of procaspase-3 was completely prevented by Z-VAD-fmk or Z-DEVD-fmk, the partial proteolysis of procaspase-3 was induced in the presence of Z-Asp-CH2-DCB. Furthermore, although TNF-induced proteolytic activation of procaspase-8 was completely inhibited by Z-VAD-fmk or Z-DEVD-fmk, the partial proteolysis of procaspase-8 to the p43/41 intermediate and p18 active fragment was detected in the presence of Z-Asp-CH2-DCB. The cleavage of RIP1, which plays a crucial role in TNF-induced necroptosis and is cleaved by caspase-8, was completely inhibited by Z-VAD-fmk or Z-DEVD-fmk, whereas the partial degradation of RIP1 was detected in the presence of Z-Asp-CH2-DCB. These results suggest that the partial activation of caspase-8 in the presence of Z-Asp-CH2-DCB may suppress TNF-induced necroptosis via the cleavage of RIP1, and also suggest that Z-Asp-CH2-DCB, but not Z-DEVD-fmk, may be used as a caspase-3-specific inhibitor in cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

•A seminoma developed in a patient with androgen insensitivity syndrome.•The patient had a de novo androgen receptor mutation.•Proper management of AIS, including appropriate genetic counseling, is necessary.

Background: Autoimmune pancreatitis is categorized as an IgG4-related autoimmune disease, mostly associated with serological alterations, however characteristics of autoimmune pancreatitis based on serum markers have not been fully evaluated., Methods: We evaluated demographics, symptoms, imaging and therapeutic outcome in 27 cases of autoimmune pancreatitis stratified by serum IgG4 level., Results: Twenty patients (74%) had elevated serum IgG4 and 7 (26%) had normal IgG4 levels. Compared to patients with normal serum IgG4 levels, patients with elevated IgG4 had higher incidence of jaundice at onset (14.3% vs. 80%, respectively; P=0.002), more frequent diffuse pancreatic enlargement at imaging (14.3% vs. 60%, respectively; P=0.04), significantly higher 18F-2-fluoro-2-deoxy-d-glucose uptake of pancreatic lesions (SUV max: 4.0 vs. 5.7, respectively; P=0.02), more frequent extrapancreatic lesions (42.9% vs. 85%, respectively; P=0.03). Response to steroids was recognized regardless of serum IgG4 level, however maintenance therapy was required more frequently amongst patients with elevated compared to normal IgG4 (85.7% vs. 33.3%, respectively; P=0.04)., Conclusions: Clinical features of autoimmune pancreatitis are different based on level of serum IgG4. Further studies are needed to clarify if normal serum IgG4 cases are a precursor of active type 1 or type 2 autoimmune pancreatitis., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Apoptotic cell death eventually results in secondary necrotic cell death, whereas caspase-independent primary necrotic cell death has been reported and its mechanism involving RIP1 and RIP3 has been recently elucidated. Dual staining with fluorescent Annexin V and propidium iodide (PI) has been used to discriminate apoptotic and necrotic cell death, in which Annexin V-positive/PI-negative staining is regarded as apoptosis and PI-positive staining as necrosis. Here we demonstrate that primary necrotic cells unexpectedly show Annexin V-positive/PI-negative staining before they become PI-positive, and that primary necrotic and apoptotic Annexin V-positive/PI-negative cells can be discriminated by necrostatin-1, an inhibitor of primary necrosis by inhibition of RIP1., (Copyright © 2011 Elsevier Inc. All rights reserved.)

We recently reported that Bax activation occurs downstream of caspase activation in Triton X-100 (TX)-induced apoptosis. Here, Bak was found to be activated in TX-induced apoptosis. Although z-VAD-fmk completely suppressed Bax activation, it only partially attenuated TX-induced Bak activation. Moreover, activation of both Bak and Bax was detected in apoptosis induced by deoxycholate, a physiological detergent in bile. z-VAD-fmk completely suppressed deoxycholate-induced Bak as well as Bax activation. Furthermore, Bak siRNA attenuated TX- but not deoxycholate-induced caspase activation. These results suggest that Bak activation may occur upstream of caspase activation in TX- but not deoxycholate-induced apoptosis and that the mechanism of TX-induced apoptosis may differ from that of deoxycholate-induced apoptosis at least with regard to the role for Bak.

We previously showed the enhancement of survival of retinal ganglion cells (RGCs) by electrical stimulation (ES) of the optic nerve (ON) stump in adult rats. To elucidate the mechanisms underlying the survival enhancement, we determined whether the neuroprotective effect of ES is affected by the following parameters: stimulation time, frequency of current pulses and starting of ES. ES for 10min immediately after ON transection was not effective in increasing the number of surviving RGCs 7 days after the transection, but that for 30min was effective. ES at 20Hz was the most effective, when applied just after axotomy. When the starting of ES to the ON was shifted either 3h after or 4h before the axotomy, the neuroprotective effect of ES was not observed. These results suggest that the electrical activation of RGCs and/or the transected ON interfere with early events after axotomy that leads to RGC death.

Phospholipases are essential enzymes in cellular signalling processes such as cellular differentiation, proliferation and apoptosis. Based on its high degree of homology with sequences of prokaryote SMases, a type of Mg(2+)-dependent PLC (nSMase-1) was recently discovered which displayed strong redox dependence for activity in vitro [F. Rodrigues-Lima, A.C. Fensome, M. Josephs, J. Evans, R.J. Veldman, M. Katan (2000), J. Biol. Chem. 275 (36) 28316-28325]. The aim of this work was to test the hypothesis that glutathione could be a natural regulator of nSMase-1 activity ex vivo. We studied how altering glutathione levels and redox ratio modulate nSMase-1 activity in a HEK293 cell line that ectopically overexpressed the nSMase-1 gene. Diminishing total glutathione with BSO without altering significantly the GSH/GSSG ratio did not affect nSMase-1 activity. Treatment of cells with diamide produced a transient decrease of total glutathione and a sharp, but also transient, decrease of the GSH/GSSG ratio. Under these conditions, nSMase-1 activity was temporarily activated and then returned to normal levels. Simultaneous treatment with BSO and diamide that resulted in permanent decreases of total glutathione and GSH/GSSG redox ratio produced a sustained activation of nSMase-1 activity. Taken together, these data indicate that altering the GSH/GSSG ratio by increasing GSSG or decreasing GSH levels, but not the total concentration of glutathione, modulates nSMase-1 activity. Our findings are the first evidence supporting the ex vivo regulation of nSMase-1 through a redox glutathione-dependent mechanism.

Ceramide, the backbone of sphingolipids, has been reported to be involved in various cellular responses including apoptosis. We recently established and characterized a C2-ceramide-resistant HL-60 subline designated HL-CR. HL-CR cells were resistant to not only ceramide but also anti-cancer drugs including daunorubicin, etoposide, and cytosine arabinoside. To elucidate the mechanisms by which HL-CR cells became resistant to various apoptosis-inducing stimuli, the levels of Bcl-2 family proteins, which play crucial roles in drug-induced apoptosis, were compared between HL-CR and parental HL-60 cells. Among Bcl-2 family members, Bax, a pro-apoptotic Bcl-2 family protein, was highly expressed in HL-60 but was hardly detected in HL-CR cells. Transient transfection of bax-expressing plasmid, but not the vector alone, induced apoptosis in HL-CR cells. These results suggest that reduced expression of Bax might play a role in resistance to various apoptosis-inducing stimuli in HL-CR cells.

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.

Antisense activity in living cells has been thought to occur via a mechanism involving both DNA-mediated hybridization arrest of target mRNA and RNase H-mediated mRNA digestion. Therefore an ideal antisense agent should be permeable to the cell and possess capacities (1) to form a thermally stable duplex in vivo with its target, (2) to discriminate between mRNAs with different degrees of complementarity, and (3) to form antisense/RNA complexes that are susceptible to RNase H hydrolysis. A trisamine-modified deoxyuridine derivative of a novel phosphorothioate DNA 15-mer that meets all these criteria is described here. Compared with the unmodified phosphorothioate oligomer, the phosphorothioate derivative exhibits a higher antisense activity as well as reduced cytotoxicity in cells infected with HIV-1. Our data suggest that the melting temperature (T(m)) between antisense DNA and the target mRNA is not only one of the factors contributing to this derivative's improved antisense activity. Also important are an enhanced ability to discriminate between sequences and an increased susceptibility of the DNA/mRNA complex to RNase H hydrolysis. These results will be useful in designing more active, clinically useful antisense drugs.

2'-Deoxyuridine derivatives bearing a substituent at the C5-position, which has a different chain length and a different functional group (methyl or amino), were synthesized and incorporated into oligodeoxyribonucleotides. The effect of the substituent groups in the major groove on the stability of the duplexes was investigated by UV melting experiments. It was found that the stabilization of these duplexes by a terminal amino group depended on the length of a linker arm.

Novel yttrium-90 (90Y)-labeled phosphorothioate antisense oligonucleotides were designed as a potential targeted radionuclide therapeutic agent for malignant tumors. A 15-mer phosphorothioate antisense oligonucleotide, which was complementary to the translation start region of the N-myc oncogene mRNA, was conjugated with isothiocyanobenzyl ethylenediamine tetraacetic acid (SCN-Bn-EDTA), via a C-5-substituted deoxyuridine that had replaced a thymine in the oligonucleotide, and was then labeled with 90Y-acetate. Following purification, the radiochemical purity of the 90-Y-Bn-EDTA-phosphorothioate antisense oligonucleotides was estimated by 2.0% agarose gel electrophoresis, and the specific hybridization of 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide to a phosphorodiester sense oligonucleotide was investigated by 20% polyacrylamide gel electrophoresis in a cell-free system. Radiochemical purity was 98.7 +/- 0.4% at 72 h after labeling and 90.3 +/- 0.9% after 72-h incubation with human normal serum. The 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide hybridized specifically to a complementary phosphorodiester sense oligonucleotide. In conclusion, phosphorothioate antisense oligonucleotides can be labeled stably with 90Y using SCN-Bn-EDTA without loss of hybridization properties.

The structure of DNA in a DNA-protein complex was studied by means of fluorescence resonance energy transfer (FRET) method. Oligonucleotide phosphorothioates were labeled with fluorescein and eosin to obtain a donor- and acceptor-labeled DNA. The formation of a complex of the DNA with PAP1(70), which is a DNA binding site fragment derived from transcription regulatory protein, PAP1, of fission yeast, was confirmed by gel retardation analysis and fluorescence measurements. FRET of the donor- and acceptor-labeled DNA with and without PAP1(70) indicated that the DNA in the complex was bent about 26 degrees toward the protein-binding surface.

We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LoVo, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase delta. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these four cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.

Using horseradish peroxidase (HRP) tracing technique, we were able to confirm the critical age in hamsters as reported previously (SO et al., 1981). Thus, following transection of the retinal fibers at the brachium of the superior colliculus (BSC) on postnatal-day 4 (P4) or later, no retinocollicular projections were observed in the adult stage. However, the retinal fibers were observed to reinnervate the superior colliculus (SC) if the BSC was cut on P3 or earlier. Physiological recording showed a close to normal retinocollicular map following a BSC damage on P0. Although retinal fibers did not reinnervate the SC following a BSC cut on or after P4, they could be observed to grow along a membrane over the damaged site. Bridging the site of BSC damage in adult hamsters using a segment of peripheral nerve (PN), retinal fibers labelled with WGA-HRP were observed to reinnervate the SC along the PN graft and visual evoked responses could be recorded in the SC showing the PN graft is effective in restoring damaged central visual pathways in adult mammals.

We previously reported that the K562 cell line K562YO expressed a high level of the c-kit gene. In this study, we analyzed the mechanism of this expression and investigated the effects of the serine/threonine kinases such as protein kinase C (PKC) and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent kinase (PKA) on it. The half-life of the c-kit mRNA in K562YO cells was greater than 10 hours, compared with 2 hours in the original K562 cells, which expressed a very low level of c-kit mRNA. This prolonged half-life can contribute to the high level of c-kit expression in K562YO cells. Cycloheximide (CHX), a protein synthesis inhibitor, caused increases in c-kit mRNA levels in K562YO cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), by which PKC was activated at first and downregulated in a late phase, gradually decreased c-kit mRNA in K562YO cells until 9 hours and then returned to the control level 24 hours after treatment. TPA also rapidly decreased c-kit protein level on the membranes. In whole cells, c-kit protein was also decreased 6 hours after incubation with TPA. Calphostin C, a light-dependent PKC inhibitor, decreased c-kit mRNA levels within 30 minutes in a light-dependent manner. It also decreased c-kit protein in whole cells 2 hours after the addition. However, it increased the amount of c-kit protein on the cell surfaces. Dibutyryl cyclic AMP (dbc-AMP) increased c-kit mRNA as well as c-kit protein on membranes and in whole cells. Run-on transcriptional assay suggested that the agent (dbc-AMP) enhanced the transcription rate of the gene. These results suggest that c-kit protein on the membranes is downregulated by PKC activation and upregulated by PKC inhibition. In the whole cell lysate, c-kit proteins are decreased by PKC inhibition through downregulation of mRNA. On the other hand, the elevation of an intracellular cAMP level causes upregulation of both the mRNA and c-kit protein on membranes and in whole cells through enhanced transcription. Thus, c-kit gene expression is apparently modulated by PKC and PKA.

2',5'-Oligoadenylate and 2',5'-oligoadenylate phosphodiesterase activity were detected in the human plasma and serum by sensitive radioimmuno assays. The phosphodiesterase in the serum degraded 20 nM of added 2',5'-oligoadenylate in less than 1 hr. Addition of EDTA in the blood sample inhibited the phosphodiesterase activity completely and allowed the measurement of low levels of 2',5'-oligoadenylate. The concentration in the plasma from healty people was in the range of 0.03 to 0.3 nM.

In anesthetized and immobilized Japanese monkeys (Macaca fuscata), intraretinal conduction velocities of the ganglion cell axons were measured. The field potentials elicited by optic chiasm shocks consisted of fast and slow components with estimated conduction velocities of 1.19 and 0.72 m/s in recordings from the optic nerve fiber layer, and 1.65 and 1.00 m/s in recordings from the ganglion cell layer. Single cell recordings verified that the time course of the fast component corresponded to the antidromic spike latencies of Y-like cells, whereas that of the slow component covered the latency range of both X-like and W-like cells. In an electron microscopic study of the cross-sections of the intraretinal optic nerve fiber bundles, the axon diameter histograms of large samples (n = 3000-6000) all showed a unimodal distribution with a sharp peak at 0.3-0.6 micron and a long tail extending to 2-3 micron. The mean diameter was largest in the ventral and nasal bundles, smallest in the papillomacular bundle and intermediate in the dorsal, upper arcuate and lower arcuate bundles. However, diameter histograms of a small number of regional axons (n = 255-300) showed a broad tail distinct from the peak at 0.3-0.6 micron, enabling us to segregate a group of larger axons from the medium-sized to small axons. From such regional axon diameter histograms we estimated the mean relative occurrences of the larger axons (7.1-11.3%) and their mean diameters (0.9-1.3 micron). We further applied this relative frequency to the unimodal distribution of the histograms with larger samples in the upper and lower arcuate bundles and estimated the mean axon diameter of the large axons (1.1 micron) and that of the medium-sized to small axons (slightly below 0.5 micron). Finally, in studying the relation between axon diameter and conduction velocity in the two arcuate fiber bundles, we found it to be somewhat different from that previously reported for the cat retina.