Increased DNA methyltransferase 1 (DNMT1) protein expression in precancerous conditions and ductal carcinomas of the pancreas

Aberrant DNA methylation has been shown to play an important role during multistage carcinogenesis in various human organs. The aim of the present study was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression during pancreatic carcinogenesis. Immunohistochemical analysis of DNMT1 in 48 peripheral pancreatic duct epithelia showing no remarkable histological findings without an inflammatory background (DE), 54 peripheral pancreatic duct epithelia with an inflammatory background (DEI), 188 pancreatic intraepithelial neoplasias (PanIN), and 220 areas of invasive ductal carcinoma from surgical specimens resected from 100 patients, was carried out. The average incidence of DNMT1 immunoreactivity increased progressively from DE to DEI (P = 0.003), from DE and DEI to PanIN (P < 0.0001), among PanIN with different grades of dysplasia (from PanIN I to PanIN II, P = 0.0012), from PanIN to invasive ductal carcinomas (P < 0.0001) and among invasive ductal carcinomas with different grades of histological differentiation (from well or moderately to poorly differentiated adenocarcinomas, P < 0.0001). High-level DNMT1 protein expression in invasive ductal carcinomas was correlated significantly with an advanced t category (P = 0.0224) and an advanced stage (P = 0.0294). Moreover, patients with invasive ductal carcinomas showing high-level DNMT1 protein expression had a poorer outcome (P = 0.0469). These data suggest that increased DNMT1 protein expression participates in multistage pancreatic carcinogenesis from the precancerous stage to malignant progression of ductal carcinomas and may be a biological predictor of poor prognosis.     

Intrathecal or dietary glycine inhibits bladder and urethral activity in rats with spinal cord injury

PURPOSE: We examined the influence of intrathecal or dietary glycine on bladder and urethral activity in rats with spinal cord injury. MATERIALS AND METHODS: A total of 20 female Sprague-Dawley rats were used 4 weeks after lower thoracic spinal cord injury. The rats were divided into standard and 1% glycine diet groups. In the standard diet group isovolumetric cystometry and urethral pressure measurement were performed before and after intrathecal injection of glycine. In the 1% glycine diet group bladder and urethral activity were compared with control recordings in the standard diet group. RESULTS: In the standard diet group intrathecal injection of glycine prolonged the interval and decreased the amplitude of bladder contractions, decreased baseline urethral pressure and altered urethral activity during bladder contraction from a pattern of detrusor-sphincter dyssynergia to detrusor-sphincter synergy at 100 mug glycine. In the 1% glycine diet group the interval and amplitude of bladder contractions were prolonged and decreased, respectively, compared with those in the standard diet group. Baseline urethral pressure was lower than in the standard diet group even after intrathecal injection of 100 mug glycine. Urethral pressure did not change during bladder contraction and it was the same as baseline pressure. Residual urine volume was lower than in the standard diet group. CONCLUSIONS: Intrathecal or dietary glycine inhibits bladder and urethral activity, and improves detrusor hyperreflexia and detrusor-sphincter dyssynergia.     

Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer's disease

Citrullinated proteins are the products of a posttranslational process in which arginine residues undergo modification into citrulline residues when catalyzed by peptidylarginine deiminases (PADs) in a calcium ion-dependent manner. In our previous report, PAD2 expressed mainly in the rat cerebrum became activated early in the neurodegenerative process. To elucidate the involvement of protein citrullination in human neuronal degeneration, we examined whether citrullinated proteins are produced during Alzheimer's disease (AD). By Western blot analysis with antimodified citrulline antibody, citrullinated proteins of varied molecular weights were detected in hippocampal tissues from patients with AD but not normal humans. Two of the citrullinated proteins were identified as vimentin and glial fibrillary acidic protein (GFAP) by using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Interestingly, PAD2 was detected in hippocampal extracts from AD and normal brains, but the amount of PAD2 in the AD tissue was markedly greater. Histochemical analysis revealed citrullinated proteins throughout the hippocampus, especially in the dentate gyrus and stratum radiatum of CA1 and CA2 areas. However, no citrullinated proteins were detected in the normal hippocampus. PAD2 immunoreactivity was also ubiquitous throughout both the AD and the normal hippocampal areas. PAD2 enrichment coincided well with citrullinated protein positivity. Double immunofluorescence staining revealed that citrullinated protein- and PAD2-positive cells also coincided with GFAP-positive cells, but not all GFAP-positive cells were positive for PAD2. As with GFAP, which is an astrocyte-specific marker protein, PAD2 is distributed mainly in astrocytes. These collective results, the abnormal accumulation of citrullinated proteins and abnormal activation of PAD2 in hippocampi of patients with AD, strongly suggest that PAD has an important role in the onset and progression of AD and that citrullinated proteins may become a useful marker for human neurodegenerative diseases.     

Rectal distention inhibits bladder activity via glycinergic and GABAergic mechanisms in rats

PURPOSE: We examined the influence of rectal distention on the spinobulbospinal micturition reflex and the mechanism underlying the inhibition of bladder contraction. MATERIALS AND METHODS: A total of 22 female Sprague-Dawley rats were used in this study. Using urethane anesthesia isovolumetric cystometry was performed before and after distention of the rectum by inflation of a rectal balloon (0 to 3 cm3), followed by the intrathecal injection of strychnine (a glycine receptor antagonist, 0.001 to 10 microg) and/or bicuculline (a gamma-aminobutyric acid(A) receptor antagonist, 0.001 to 1 microg) at the lumbosacral level of the spinal cord. RESULTS: Rectal distention (1.5 to 3.0 cm3) prolonged the interval, decreased the amplitude and shortened the duration of bladder contraction and finally almost abolished bladder activity. After intrathecal injection of strychnine or bicuculline in animals with inhibition of the bladder by rectal distention the interval and duration of bladder contraction returned to baseline but amplitude only recovered to 47% to 54% of the control level. However, simultaneous intrathecal injection of strychnine and bicuculline (0.001 microg each) restored amplitude to the control level. There were no differences between strychnine and bicuculline with respect to their effects on the interval, amplitude and duration of bladder contraction. CONCLUSIONS: An inhibitory rectovesical reflex exists in the lumbosacral cord of rats. The afferent limb of the spinobulbospinal micturition reflex pathway may be additionally and redundantly inhibited by glycinergic and GABAergic mechanisms, while the efferent limb of this pathway may be synergistically inhibited by these mechanisms.     

The AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes, which encode functionally redundant kinesins, are essential for cytokinesis in Arabidopsis

Cytokinesis is the critical step during which daughter cells are separated. We showed previously that a protein complex that consists of NACK1 (and NACK2) kinesin-like protein and NPK1 MAPKKK and its substrate NQK1 MAPKK are required for progression of cytokinesis in Nicotiana tabacum. The genome of Arabidopsis thaliana encodes homologues of NACK1 and NACK2, namely, AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2, respectively. Loss-of-function mutations in AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 result in the occasional failure of somatic and male-meiotic cytokinesis, respectively. However, it is likely that these genes function redundantly to some extent in somatic tissues and female gametogenesis. We describe the phenotypes of Arabidopsis plants that have mutations in both the AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes. These phenotypes suggest that the two genes are essential during both male and female gametogenesis. Female gametes with atnack1 atnack2 double mutations failed to cellularize and to generate a central cell, synergids and the egg cells. Male gametes with atnack1 atnack2 mutations were also not transmitted to the next generation. The AtNACK1/HINKEL and STUD/TETRASPORE/AtNACK2 genes for kinesin-like proteins have overlapping functions that are essential for gametogenetic cytokinesis. They appear to be essential components of a MAP kinase cascade that promotes cytokinesis of plant cells in both gametophytic (haploid) and sporophytic (diploid) proliferation.     

Efficacy of docetaxel as a second-line chemotherapy for thymic carcinoma

Thymic carcinoma is a rare and aggressive tumor, and the efficacy of second-line chemotherapy is still unclear. Here, we reported a case of thymic carcinoma that responded well to the administration of docetaxel alone as a second-line chemotherapy. A 64-year-old woman was diagnosed with thymic carcinoma (squamous cell type) with bone metastasis, and she, therefore, received nedaplatin combined with etoposide and ifosfamide. She responded partially, after which she received irradiation for bone metastasis. Two months after chemotherapy, the thymic carcinoma exhibited gradual regrowth and she experienced shoulder pain. We treated this with docetaxel alone (60 mg/m2 every 4 weeks). After three courses of docetaxel, we observed a partial response and her shoulder pain disappeared. This case demonstrated that docetaxel is effective as a second-line chemotherapy for thymic carcinoma.     

Comparison of the Hershberger assay and androgen receptor binding assay of twelve chemicals

We performed the Hershberger assay of 12 chemicals based on the OECD draft protocol. The chemicals tested by the Hershberger assay were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, diethylstilbestrol, 17beta-estradiol, tamoxifen, 5alpha-dihydrotestosterone, dichlorodiphenyldichloroethane, cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, atrazine, and spironolactone. Phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, and phthalic acid di-n-propyl ester are phthalates; diethylstilbestrol and 17beta-estradiol are estrogenic chemicals; tamoxifen is partial estrogen receptor antagonist with mainly estrogenic properties; 5alpha-dihydrotestosterone is an androgen derivatives; dichlorodiphenyldichloroethane is a reference androgen antagonistic chemical; cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, and spironolactone have an androgenic steroid structure and are known as androgen antagonistic chemicals; and atrazine is a reference endocrine disruptor. We also subjected these chemicals to the receptor binding assay for androgen. A clear androgen agonistic effect was detected in 5alpha-dihydrotestosterone, and an androgen antagonistic effect was observed in five chemicals: cyproterone acetate, spironolactone, 6alpha-methyl-17alpha-hydroxy-progesterone, phthalic acid di-n-amyl ester, and dichlorodiphenyldichloroethane. By contrast, diethylstilbestrol, 17beta-estradiol, tamoxifen, 5alpha-dihydrotestosterone, dichlorodiphenyldichloroethane, cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, and spironolactone were positive in the receptor binding assay for androgen. Three estrogenic chemicals, diethylstilbestrol, 17beta-estradiol, and tamoxifen, were negative in the Hershberger assay with receptor binding affinity. On the other hand, the Hershberger assays of three phthalates were performed at the same dosages, and the results showed androgen antagonistic affinity only in the assay of phthalic acid di-n-amyl ester without receptor binding affinity.