In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme-labeled antigen method.

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme-labeled antigen method was applied to detecting plasma cells producing P. gingivalis-specific antibodies in biopsied gingival tissue of periodontitis. N-terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro and Lys-hgp) were prepared by the cell-free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme-labeled antigen method on paraformaldehyde-fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme-labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.

Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system.

Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates.

Decomposition of carbon dioxide by metals during gamma irradiation.

We have proposed a technique to enhance the decomposition of carbon dioxide by gamma irradiation. This is possible by putting metal components into CO2 gas to promote the conversion of gamma rays to lower-energy electrons through Compton, photoelectron and cascading electron knock-on events in metals. Numerical simulations using the EGS code indicated that the number of lower-energy electrons ejected from metals into CO2 gas increases with increasing Z number and/or density of the metals; this was supported by the experimental results, i.e., the CO2-containing metals with a higher Z number exhibited a greater efficiency for production of CO. In addition, production of CO could be enhanced by carefully controlling the volume and surface area of metal components as well as the proximity to adjacent metal components. These experimental results successfully demonstrated that modification of the kinds of metal components and metal structures can control the energy and the number of electrons ejected from the metals and can lead to enhancement of production of CO from CO2.

Transmembrane serine protease TADG-15 (ST14/Matriptase/MT-SP1): expression and prognostic value in ovarian cancer.

Tumour-associated differentially expressed gene-15 (TADG-15/ST14/matriptase/MT-SP1) is a novel member of the transmembrane serine proteases. Previous studies indicated that TADG-15 is overexpressed in ovarian tumours; however, relationships between expression of TADG-15 and clinical characteristics of ovarian cancer remain unclear. The purpose of this study was to examine TADG-15 expression in ovarian cancers and determine any associations with clinicopathological characteristics or patient survival. Immunohistochemical study revealed that TADG-15 was expressed in 50 (56.2%) of 89 ovarian carcinomas, whereas it was not detected in normal ovaries. TADG-15 expression was significantly more common in patients with early stage disease compared with patients with advanced stage diseases (namely, stage I, 24 out of 33: 72.7%; stage II/III/IV, 26 out of 56: 46.4%; P=0.0157). Kaplan-Meier survival curves demonstrated that patients with TADG-15-positive tumours have had substantially longer survival (P=0.0480). The mean value of relative TADG-15 mRNA expression ratio was significantly higher in stage I tumours than in stage II/III/IV tumours (P=0.0053). Increased expression of TADG-15 is frequently detected in early stage cancers, with expression level downregulated during progression of disease. TADG-15 is associated with early stage ovarian cancer and longer patient survival; therefore, it may be a favourable prognostic marker for this malignancy.

Correlation between spontaneous feeding behavior and neuropeptide Y profile in the third ventricular cerebrospinal fluid of goats.

Feeding behavior is regulated by neural signals in the hypothalamus, but secretory activities of these signals in vivo and their relationship with spontaneous feeding remain to be solved. In the present study, we investigated the correlation between neuropeptide Y (NPY) and somatostatin (SRIF) profiles in cerebrospinal fluid (CSF) and spontaneous feeding behavior in goats. CSF samples were collected every 15 min for 8 h from the third ventricle and feeding behavior was observed throughout the experimental period. The spontaneous feeding behavior, the mean duration of which was 58 min, occurred with an interval of 146 min. NPY in the CSF fluctuated in an episodic fashion with a 145 min interval. Each NPY episode was followed by spontaneous feeding with a time lag of 24 min. SRIF levels in CSF changed more frequently in a pulsatile manner and were related to neither NPY profiles nor feeding behavior. These results suggest that NPY, but not SRIF, is a physiological signal to drive feeding in goats.

Recurrence of invasive cervical carcinoma more than 5 years after initial therapy.

OBJECTIVE: To estimate the probability of and risk factors for the recurrence of invasive cervical carcinoma over 5 years after initial therapy. METHODS: Patients (n = 827) with invasive cervical carcinoma were treated and received follow-up care for up to 29 years. Late recurrence was defined as recurrence more than 5 years after initial therapy. The probability of late recurrence was evaluated in terms of clinical stage, histologic type, and type of initial therapy. RESULTS: Late recurrence was seen in 21 of 569 patients who had survived 5 years (3.7%). Recurrence rates were 1.8% (six of 331) in stage I, 5.2% (eight of 154) in stage II, 8.6% (seven of 81) in stage III, and 0% (none of three) in stage IV. The probability of late recurrence in patients with stage I disease was significantly lower than that in stage II and stage III diseases (stage I compared with stage II, P = .038, stage I compared with stage III, P = .002). Late recurrence occurred in 21 (3.8%) of 547 cases of squamous cell carcinoma, whereas no late recurrences were found in 22 cases of adenocarcinoma. The late recurrence rate in patients who received radiation (7.1%, 17 of 241) was significantly higher than that in patients who received surgery (1.2%, four of 328; P = .001). CONCLUSION: Patients with uterine cervical squamous cell carcinoma, especially those with stage II or stage III diseases who received radiation therapy as initial treatment, warrant annual follow-up care beyond the standard 5 years after initial therapy.

Cyclin E mRNA overexpression in epithelial ovarian cancers: inverse correlation with p53 protein accumulation.

OBJECTIVE: We investigated the relationship between cyclin E mRNA overexpression and p53 protein accumulation in epithelial ovarian cancers. METHODS: mRNA was isolated and cDNA was prepared from 36 epithelial ovarian tumors (three adenomas, three low malignant potential tumors, and 30 carcinomas), and six normal ovaries. The cyclin E mRNA expression levels relative to an internal control, beta-tubulin, were determined by semiquantitative polymerase chain reaction (PCR). Cyclin E and p53 protein expression in ovarian cancer tissues were examined by immunohistochemistry using the same series of samples. Fisher exact test of significance and an unpaired t test were used for statistical analysis. RESULTS: Considerable levels of cyclin E mRNA were detected in all normal ovaries and ovarian tumor samples examined by semiquantitative PCR amplification. mRNA levels of cyclin E were significantly higher in nine of 30 (30%) ovarian cancers compared with those in normal ovaries. The immunohistochemical expression of cyclin E protein was confirmed in the nuclei of tumor cells in 13 of 30 (43%) ovarian cancers. p53 protein accumulation was detected in 12 of 30 (40%) ovarian cancers examined. There was a significant inverse correlation between cyclin E mRNA overexpression and p53 protein accumulation (P <.01, Fisher exact test). CONCLUSIONS: Cyclin E mRNA overexpression frequently occurs in ovarian cancers without p53 protein accumulation. Cyclin E might have an important effect on the development of a limited number of ovarian cancers.

Underexpression of cyclin-dependent kinase inhibitor p27 is associated with poor prognosis in serous ovarian carcinomas.

Reduced expression of a cyclin-dependent kinase inhibitor, p27, has been reported to be associated with poor prognosis in several human cancers. The aim of this study was to investigate the potential role of p27 in ovarian cancer development and progression. Immunohistochemical expression of p27 was determined using 117 epithelial ovarian tumor tissues and 8 normal ovaries. p27 mRNA expression was examined by semi-quantitative PCR amplification using 26 ovarian cancer samples. Nuclear staining of p27 was commonly observed in the normal ovarian surface epithelium and the epithelial cells of germinal inclusion cysts. Positive p27 staining rates were significantly higher in serous adenomas (p=0.006) and in serous LMP tumors (p=0.013) than that in serous carcinomas (Fisher's exact test). In serous ovarian cancers, positive p27 staining rate was significantly higher in early stage (stage1/2) than that in advanced stage (stage 3/4) diseases (p=0.030, Fisher's exact test). Log-rank testing showed that negative p27 expression significantly correlates with poor survival in serous ovarian cancer patients (p=0.041). Considerable levels of p27 mRNA were detected in all ovarian cancer samples examined. These results suggest that the underexpression of p27 caused by post-translational mechanism may contribute to the development and progression and result in poor prognosis of serous ovarian cancers.

Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals.

The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10(-9) to 10(-5) U/nucleus, the amount of 5x10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI-Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.

A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes.

Current cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.

Construction of an efficient expression vector for coupled transcription/translation in a wheat germ cell-free system.

Using the expression vector, pEU, which we have constructed, highly efficient in vitro protein synthesis can be achieved: The system works for 150 hours and without further template addition once the reaction has started, yielding 5 mg of enzymatically active protein in a 1 ml reaction.

A new class of enzyme acting on damaged ribosomes: ribosomal RNA apurinic site specific lyase found in wheat germ.

A new enzyme, which we named ribosomal RNA apurinic site specific lyase (RALyase), is described. The protein was found in wheat embryos and has a molecular weight of 50 625 Da. The enzyme specifically cleaves the phosphodiester bond at the 3' side of the apurinic site introduced by ribosome-inactivating proteins into the sarcin/ricin domain of 28S rRNA. The 3' and 5' ends of wheat 28S rRNA at the cleavage site are 5'-GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA-3', demonstrating that the enzyme catalyzes a beta-elimination reaction. The substrate specificity of the enzyme is extremely high: it acts only at the apurinic site in the sarcin/ricin domain of intact ribosomes, not on deproteinized rRNA or DNA containing apurinic sites. The amino acid sequences of five endopeptidase LysC-liberated peptides from the purified enzyme were determined and used to obtain a cDNA sequence. The open reading frame encodes a protein of 456 amino acids, and a homology search revealed a related rice protein. Similar enzyme activities were also found in other plants that express ribosome-inactivating proteins. We believe that RALyase is part of a complex self-defense mechanism.

Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.

Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.

Molecular cloning of cDNA for equine follistatin and its gene expression in the reproductive tissues of the mare.

A cDNA clone encoding equine follistatin was isolated from an equine ovarian cDNA library. Out of 1.2 x 10(5) independent clones screened, one positive clone was isolated and its cDNA sequence determined. The isolated clone, named EQ-FS-1, contained a complete open reading frame encoding 344 amino acid residues. The similarity of its deduced amino acid sequence to these of other mammalian species was greater than 95%. Although its expression level varied among the tissues examined, follistatin mRNA was detected in the equine uteroplacental tissues, follicles and corpora lutea by Northern blot analysis. In situ hybridization revealed that the expression of follistatin mRNA in the equine follicle was restricted exclusively to granulosa cells. When the expression pattern of follistatin mRNA in the equine uteroplacental tissues from mid- to late-pregnancy was examined, it was shown that its expression level tended to decrease after mid-pregnancy. These results suggest that follistatin acts in the reproductive tissues of the mare in maintaining pregnancy.

Mechanism of ribosome RNA apurinic site specific lyase.

A new enzyme, which we named ribosome RNA apurinic site specific lyase (RALyase), has been characterized. The enzyme specifically cleaves a phosphodiester bond at the apurinic site in the sarcin/ricin domain of 28S rRNA in ribosomes. The cut ends of wheat 28S rRNA were determined as 5'---GUACG-alpha-hydroxy-alpha, beta-unsaturated aldehyde and pGAGGA---3' for the 3' fragment, demonstrating that the enzyme catalyzes the beta-elimination reaction.

Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices.

To clarify the molecular structure of the integration sites of transgenes, we used particle bombardment to examine the DNA sequences of transgene loci. Three transgenic Arabidopsis lines gave a single Southern hybridization band with a selectable gene as the probe. Junction regions flanked by the transgenes were cloned by the inverse polymerase chain reaction method, and the characteristics of the DNA sequences of the 10 junction regions were investigated. All but two of these were AT-rich sequences bearing motifs characteristic of a scaffold/matrix-attachment region (S/MAR). Calculations showed that seven of them should have a propensity for curvature. An assay of in-vitro binding to tobacco nuclear matrices showed that all the junction regions bound to nuclear matrices and that the two input DNAs did not bind. The 12 chromosome/transgene (CT) junctions in these three transgene loci were investigated. Cleavage sites for topoisomerase I were found at 10 of the 12, near the junction point. The other two junctions had sites within 6bp of the junction point. The sequence near one terminal of the transgene in the transgene loci was compared with that near the other terminal. Short, direct repeats consisting of 4-6bp were present within 10bp of the junction points in the sequence. We speculate that the transgene introduced by particle bombardment is delivered on AT-rich S/MAR that has a propensity for curvature, and then a nucleotide near the short, direct repeat on the transgene is joined near the cleavage sites on the genome for topoisomerase I.

Equine inhibin/activin beta A-subunit mRNA is expressed in the endometrial gland, but not in the trophoblast, during pregnancy.

The expression of both inhibin alpha- and inhibin/activin beta A-subunit mRNA was examined in equine uteroplacental tissues collected during pregnancy (days 90 to 300). Northern blot analysis revealed that 5 transcripts (7.0, 4.1, 3.4, 2.6, 1.5 kb) of beta A-subunit were present, and the most abundantly expressed transcript was the 1.5 kb one. Relatively high levels of the 1.5 kb transcript were seen in the second trimester of pregnancy compared to what was found in the third trimester. To identify the tissue localization of beta A-subunit mRNA, in situ hybridization was performed, and the positive signal was observed exclusively in the endometrial glands, but not in the fetal placental tissue (trophoblast) at days 150, 210, and 300 of pregnancy. On the other hand, inhibin alpha-subunit transcript could not be detected at any stage of pregnancy examined either by Northern blot analysis or in situ hybridization. Although the factor(s) regulating the gene expression of beta A-subunit in this equine tissue is currently unknown, these results suggest that activin, but not inhibin, is predominantly produced in the endometrial glands of the pregnant mare, and thus produced activin may play a paracrine or endocrine role during pregnancy in this species.

Expression and cellular localization of inhibin alpha-subunit mRNA in equine fetal gonads.

The expression of inhibin alpha-subunit mRNA in equine fetal gonads during pregnancy (Days 90 to 300) was examined by means of Northern blot analysis. In all samples examined, a single species of transcript was detected at the size of 1.5 kb. A digoxigenin-labeled antisense cRNA probe specific to equine inhibin alpha-subunit was synthesized and in situ hybridization analysis to locate the inhibin alpha-subunit mRNA positive cells was performed using frozen tissue sections of equine fetal ovary (day 150 of pregnancy) and equine fetal testis (day 180 of pregnancy). In the fetal ovary, positive cells were seen throughout the interstitial area but did not show any particular localization. In the fetal testis, on the other hand, the antisense cRNA hybridized almost exclusively to the interstitial cells surrounding developing seminiferous cords and Sertoli cells within the cords. Positive signals were also detected in a limited number of the interstitial cells located away from the cords. These results suggest that in equine fetal gonads, inhibin and/or inhibin alpha-subunit related molecules such as the monomeric form are produced and these molecules may have a paracrine/autocrine role within the gonads.