Serum soluble CD26 levels: diagnostic efficiency for atopic dermatitis, cutaneous T-cell lymphoma and psoriasis in combination with serum thymus and activation-regulated chemokine levels.

BACKGROUND: CD26 is a multifunctional type II transmembrane glycoprotein, which also exists as a secreted isoform, soluble CD26 (sCD26). The CD26 expression on circulating T cells is decreased in some skin diseases such as cutaneous T-cell lymphoma (CTCL) and psoriasis. It remains to be determined whether sCD26 can be used as a marker of skin diseases or not. OBJECTIVE: To investigate utility of sCD26 as a diagnostic marker of skin diseases in combination with thymus and activation-regulated chemokine (TARC). METHODS: Serum sCD26 levels were measured using enzyme-linked immunosorbent assay in 130 participants including 32 patients with atopic dermatitis (AD); 45 patients with CTCL; 26 patients with psoriasis; and 27 healthy controls. RESULTS: Serum sCD26 levels in patients with CTCL and psoriasis (162.1 ± 80.2 ng/mL and 125.4 ± 82.1 ng/mL respectively) were significantly lower than those of healthy controls (392.6 ± 198.7 ng/mL; P < 0.01 and 0.01 respectively). In patients with CTCL, serum sCD26 levels of patients with advanced stage were 135.0 ± 51.5 ng/mL and they were significantly lower than those with early stage (193.1 ± 96.0 ng/mL; P < 0.05). When we used serum sCD26 and TARC levels for diagnostic criteria, sensitivity, specificity, positive predictive value and negative predictive value for AD, CTCL and psoriasis were 65.2-73.7%, 81.4-97.6%, 65.2-94.4%, and 81.4-88.9% respectively. CONCLUSION: Serum sCD26 levels, combined with serum TARC levels, are helpful in diagnosis of AD, CTCL and psoriasis.

Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesis.

The requirement of Ni2+ and Co2+ addition on methanogenic activity and the coenzymes involved in methanogenesis were investigated in anaerobic continuous cultivation with synthetic wastewater using acetate as the sole carbon source. Addition of Ni2+ and Co2+ to the synthetic wastewater drastically increased the maximum dilution rate of the cultivation. The concentrations of coenzymes F430 and corrinoids in the biomass increased to 0.62 micromol-Ni/g-VSS and 0.67 micromol-Co/g-VSS, respectively with the increase of the dilution rate. Methanogenic activity of the culture broth also increased with an increase of dilution rate. However, without addition of Ni2+ and Co2+, F430 and corrinoids were not detected in the biomass and methanogenic activity was only a trace level at a dilution rate of 0.025 d(-1). When the amounts of Ni2+ and Co2+ added at a dilution rate of 0.6 d(-1) were lowered in steps, the concentrations of F430 and corrinoids in the biomass and methanogenic activity decreased with decreasing amounts of Ni2+ and Co2+ added. These results suggest that Ni2+ and Co2+ were required for the methane-producing reactions via increases of coenzymes F430 and corrinoids.

decapentaplegic overexpression affects Drosophila wing and leg imaginal disc development and wingless expression.

We have used the GAL4-UAS expression system to increase the level of expression of the Drosophila gene decapentaplegic (dpp) in a pattern approximating its normal pattern in leg and wing imaginal discs. Intermediate increases of dpp expression have little effect in wing discs but high levels of dpp overexpression lead to reduction of the scutellum and duplication of posterior wing structures. In leg discs intermediate increases cause supernumerary outgrowths of ventral leg structures in the anterior-ventral region. Greater increases of dpp expression cause the loss of ventral leg structures with the concomitant fusion of left and right dorsal forelegs. The defects observed in both legs and wings appear to arise through dose-dependent effects of dpp on wingless (wg) expression. A high level of dpp overexpression in the wing disc causes reduction of wg expression in the presumptive scutellar region, consistent with the subsequent reduction of the scutellum. An intermediate increase of dpp expression in leg discs induces the expansion of wg expression into the ventral outgrowths. At higher dpp expression levels, ventral wg expression in leg discs is eliminated, consistent with the loss of ventral leg cuticle. In the leg disc end knob and in the wing margin primordium, where wg and dpp cooperate in producing distal outgrowth, dpp overexpression has no detectable effect either on patterning or on wg expression. We propose that a critical role for dpp in other regions of the leg and wing discs is to reduce or block the expression of wg. This role of dpp is supported by the observation that ectopic wg expression is detected in imaginal discs where dpp signaling is compromised by lowering the activity of one of its receptors, tkv. This antagonism between dpp and wg expression may be critical to assigning only one disc region as the distal organizer.

Activation of glutathione transferase P gene by lead requires glutathione transferase P enhancer I.

Glutathione transferase P (GST-P) is specifically induced in rat liver and kidney by lead cation. The increase of GST-P mRNA after lead administration is blocked by actinomycin D, suggesting that GST-P production by lead is regulated at the transcriptional level. To further determine which part of the flanking region of the GST-P gene has the lead-responsive cis-element in vivo, we utilized transgenic rats with five different constructs having GST-P and/or chloramphenicol acetyl-transferase coding sequence. We studied the effect of lead on these transgenic rats and on transfected NRK (normal rat kidney) cells and found that GST-P induction by lead is indeed regulated at the transcriptional level and that the GST-P enhancer I (GPEI) enhancer is an essential cis-element required for the activation of the GST-P gene by lead. GPEI consists of two AP-1 (c-Jun/c-Fos heterodimer) site-like sequences that are palindromically arranged and can bind AP-1, c-jun mRNA in the liver increased after lead administration and GST-P, and c-Jun had patchy expression in the same hepatocytes 24 h after lead exposure. These results suggest that activation of the GST-P gene by lead is mediated in major part by enhancer GPEI and that AP-1 may be involved at least partially. GPEI has been shown to have essential sequence information for the trans-activation of the GST-P gene during chemical hepatocarcinogenesis of the rat (Morimura, S., Suzuki, T., Hochi, S., Yuki, A., Nomura, K., Kitagawa, T., Nagatsu, I., Imagawa, M., and Muramatsu, M. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2065-2068; Suzuki, T., Imagawa, M., Hirabayashi, M., Yuki, A., Hisatake, K., Nomura, K., Kitagawa, T., and Muramatsu, M. (1995) Cancer Res. 55, 2651-2655). The present study establishes that the same enhancer element does operate in the activation of the GST-P gene by lead regardless of the trans-activators involved.

Trans-activation of glutathione transferase P gene during chemical hepatocarcinogenesis of the rat.

Glutathione transferase P (GST-P; glutathione transferase, EC 2.5.1.18) is known to be specifically expressed at high levels in precancerous lesions and in hepatocellular carcinomas from a very early phase of chemically induced hepatocarcinogenesis in the rat. The almost invariable occurrence of this phenotype in these lesions strongly suggests a mechanism by which GST-P gene is activated together with a crucial transforming gene of liver cells. To distinguish the two alternative possibilities--either the GST-P gene is coactivated with a closely located transforming gene by a cis mechanism or it is activated in trans by a common trans-acting factor--we carried out carcinogenesis experiments using transgenic rats harboring the bacterial chloramphenicol acetyltransferase reporter gene ligated to the upstream regulatory sequence of the GST-P gene. In each of three independent lines tested, liver foci and nodules produced by chemical carcinogens (Solt-Farber procedure) were found to express high levels of chloramphenicol acetyltransferase activity, indicating clearly that the GST-P gene is activated by a trans mechanism during hepatocarcinogenesis.

The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression.

The ftsH gene is essential for cell viability in Escherichia coli. We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene. It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain. The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala. Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively. Possible implications of these observations are discussed.

Chromosomal structure of the halophilic archaebacterium Halobacterium salinarium.

The chromosomal structure of the extremely halophilic archaebacterium Halobacterium salinarium was examined. Sheared chromosomes prepared from the bacteria in the late exponential phase were separated into two peaks (peaks I and II) by sucrose gradient centrifugation, suggesting that the chromosomes consist of two parts differing in quality. The UV spectra of peaks I and II resembled those of DNA and eukaryotic chromatin, respectively. Electron microscopic observations revealed that the major component of peak I was protein-free DNA, while the major components of peak II were rugged thick fibers with a diameter of 17 to 20 nm. The rugged fibers basically consisted of bacterial nucleosome-like structures composed of DNA and protein, as demonstrated in experiments with proteinase and nuclease digestion. Whole-mount electron microscopic observations of the chromosomes directly spread onto a water surface revealed a configuration in which the above-described regions were localized on a continuous DNA fiber. From these results it is concluded that the H. salinarium chromosome is composed of regions of protein-free DNA and DNA associated with nucleosome-like structures. Peaks I and II were predominant in the early exponential phase and stationary phase, respectively; therefore, the transition of the chromosome structure between non-protein-associated and protein-associated forms seems to be related to the bacterial growth phase.

Complex regulation of a tumor marker expression. Enhancer and silencer of the GST-P gene.

Glutathione transferase P (GST-P) is expressed at high levels in precancerous lesions and hepatocellular carcinomas from a very early stage of chemically-induced hepatocarcinogenesis in the rat. To explore the molecular mechanisms of its specific activation, we are investigating the regulation mechanisms of the GST-P gene expression. By using gene technology, we have identified a strong enhancer, GPEI, at 2.5 Kb and a silencer region at about 400 bp upstream from the transcription start site. GPEI has a palindromic structure composed of two TPA-responsive element (TRE)-like sequences and binds at least three proteins including AP-1 (c-jun/c-fos). The silencer is composed of several sequences resembling each other and binds at least three proteins including SF-B/LAP/LIP. To determine whether the GST-P gene is activated together with a putative hepato-oncogene because they are located close to each other (cis-mechanism), or because they share a trans-acting factor that can activate both genes simultaneously (trans-mechanism), transgenic rats were produced with GST-P control region connected to the CAT reporter. The results unequivocally demonstrate that GST-P gene is activated position-independently by a trans-mechanism.

Analysis of glutathione transferase P gene regulation with liver cells in primary culture.

Glutathione transferase P (GST-P) gene is specifically and highly activated during rat chemical hepatocarcinogenesis. We have previously cloned the GST-P gene and have identified putative regulatory regions. To further explore regulatory mechanisms, deletion constructs of the GST-P gene fused to the chloramphenicol acetyltransferase (CAT) structural gene were introduced into primary cultured rat hepatocytes by electroporation, and their activity was determined. The expression of the GST-P-CAT fusion gene is quite low in these cells as compared to that in both a rat fibroblast cell line, 3Y1 cells, and a rat hepatoma cell line, dRLh84. The presence of the strong enhancer GPEI did not elicit any enhancing activity at its original position, or when it was located 3' of the CAT gene, although this element does enhance CAT activity significantly when located adjacent to the promoter. Cotransfection of neither c-jun nor c-fos expression vector, nor both vectors, could enhance the CAT activity, even though GPEI consists of two phorbol ester response element-like sites. Furthermore, the expression of jun family gene was not correlated with GST-P gene expression either in primary cultured hepatocytes or in hepatoma cell lines.

Transformation of mammalian cells by luteoskyrin.

The transforming activity of luteoskyrin (LS), a bis-anthraquinoid mycotoxin produced by Penicillium islandicum Sopp., and a hepatocarcinogen in rodents, was examined by an in vitro transformation assay using mouse embryonal Balb/3T3 A31-1-1 cells. The results revealed that LS induced type III foci at 0.5 micrograms/ml, and that the cells selected from these foci by soft-agar cloning grew with a high saturation density. Thus, it was confirmed that LS not only induces hepatic tumours in laboratory animals, but also transforms in vitro cultured mammalian cells. The tumorigenicity of the transformants obtained was confirmed by transplantation into nude mice and by image analysis with IIIIn. A transfection assay, using calcium phosphate co-precipitation, demonstrated that the DNA of the cloned cells transformed NIH3T3 cells. Northern blot also revealed transcriptional activation of c-myc and c-Ha-ras oncogenes. The possible participation of LS-derived hydroxy radicals in the formation of genetic lesions was discussed.

SF-B that binds to a negative element in glutathione transferase P gene is similar or identical to trans-activator LAP/IL6-DBP.

We have previously identified a silencer (negative enhancer) in glutathione transferase P (GST-P) gene which is strongly and specifically induced during hepatocarcinogenesis of the rat. At least three trans-acting factors bind to multiple cis-elements located in this silencer. One of these factors, SF-B (Silencer Factor B) specifically binds to GPS1 (GST-P Silencer 1) and has been cloned by a Southwestern protocol. Analysis of DNA and deduced amino acid sequence reveals that SF-B clone is most likely identical to an IL-6 inducible trans-activator LAP/IL6-DBP. Binding efficiency of SF-B to GPS1 is indistinguishable from that to IL6-responsive element found in C-reactive protein gene. The possibility that SF-B/LAP/IL6-DBP functions as a dual positive and negative regulator is discussed.

Structure and function of the ftsH gene in Escherichia coli.

The ftsH mutant Y16 shows thermosensitive filamentation with reduced amounts of penicillin-binding protein 3 (PBP3) (Ferreira et al., 1987). Genetic analysis, however, showed that the lethality of the ftsH mutation was not due to a lack of PBP3 activity alone. The ftsH gene was cloned and sequenced and the FtsH protein was deduced to be a membrane protein of 70.7 kDa which has an ATP-binding domain. Highly significant homology of amino acid sequence was observed between FtsH protein and two eukaryotic proteins, yeast Saccharomyces cerevisiae Sec 18p and its mammalian homologue NSF, which are involved in protein transport pathways. This suggests that FtsH protein may act for translocation of specific proteins including PBP3 and at least one other additional protein essential for cell growth. Suppressor mutants of Y16, which were able to grow at 42 degrees C, were isolated, and the suppressor mutations (sfh) were mapped to 16 min. A wild type chromosomal fragment able to complement the sfh mutations was cloned. We also identified another gene (ftsJ) affecting cell division in the region upstream of the ftsH gene.

Modulation of c-myc gene expression by extracellular stimuli in rat hepatoma cells.

Modulation of c-myc gene expression by extracellular stimuli in H4IIE rat hepatoma cells was investigated by Northern blot analysis. Treatment of these cells with phorbol 12-O-tetradecanoate 13-acetate (TPA), insulin and concanavalin A (Con A) resulted in transient accumulation of c-myc transcripts within 2 hours. The induction of c-myc mRNA was dose dependent with similar responses for all three agents. The maximally induced c-myc mRNA levels varied from 5- to 15-fold of the control. Treatment with cycloheximide (10 micrograms/ml) and H7, a protein kinase C inhibitor (20 microM), inhibited this induction, suggesting that c-myc induction by these agents requires protein synthesis and protein kinase C activation.

Establishment and characterization of cell lines (Kagura-1 and Kagura-2) from aflatoxin B1-induced rat hepatoma.

Two hepatoma cell lines designated Kagura-1 and Kagura-2 were established from rat hepatocellular carcinomas induced by aflatoxin B1, and have been propagated for over two years. Both cell lines grew as monolayered sheets with a population doubling time of about 20 h. Chromosome counts of Kagura-1 cells ranged from 34 to 45 with a modal number of 40, while that of Kagura-2 cells ranged widely from 40 to 130 with a modal number of 65. Subcutaneous inoculation of cultured cells of both these lines into nude mice resulted in tumor formation. The histopathological appearances of the induced tumors were similar to those of the original tumors. Kagura-1 and Kagura-2 cell lines express at least two tumor markers, glutathione-S-transferase P and gamma-glutamyl transpeptidase; the level of c-myc messenger ribonucleic acid was also highly elevated.

Mechanism of specific expression of glutathione transferase P gene during hepatocarcinogenesis.

The mechanism of specific expression of glutathione transferase P gene during hepatocarcinogenesis of the rat has been investigated by cloning the gene and determining the upstream regulatory sequences. Two enhancers and a silencer are located within 3 kb upstream of the promoter. The stronger enhancer designated GPEI has two TPA (12-O-tetradecanoyl phorbol 13-acetate)-response element (TRE)-like sequences arranged in a palindrome at a 3 base pairs spacing. This special combination was found to form a very strong enhancer which could act efficiently even in F9 cells where the collagenase enhancer with a singlet TRE cannot work due to the low c-jun content. Whether this structure is operating with a very low concentration of c-jun/c-fos heterodimer or with any other proteins remains to be determined. These findings suggest that new and more efficient enhancers evolve by a combination of basic enhancer elements. The silencer region consists of several sequences that can bind specific protein(s) and works cooperatively.

Expression of the c-Ha-ras and c-myc genes in aflatoxin B1-induced hepatocellular carcinomas.

Expression and activation of several c-oncogenes in seven hepatocellular carcinomas from seven separate rats treated with aflatoxin B1 (AFB1) were examined by Northern and Southern blot analyses. Both c-Ha-ras and c-myc transcripts were elevated at high levels in all hepatomas. Moreover, in one of them, T2-1 hepatoma, the c-myc gene was amplified only in a tumor part of liver without significant rearrangement. N-ras specific transcripts were not elevated in these hepatomas. The present data suggest that the consistently increased expression or deregulation of the c-myc and c-Ha-ras genes may play an important role in the development of hepatomas induced by AFB1.