Tristetraprolin (TTP) gene polymorphisms in patients with rheumatoid arthritis and healthy individuals

Tristetraprolin (TTP) is an intracellular protein that modulates the production of cytokines, including TNFα, by binding to and destabilizing the mRNAs of these cytokines. Therefore, differences in TTP gene expression may affect the severity of inflammatory diseases, such as rheumatoid arthritis (RA). We searched for polymorphisms in the human TTP gene and for this purpose, we sequenced the entire TTP gene in 20 Japanese individuals (ten with RA and ten healthy volunteers) and found one single nucleotide polymorphism (SNP) in the promoter region. We analyzed this SNP (A/G) by restriction fragment length polymorphism method in 155 RA patients and 100 control subjects. While the frequency of A allele in this SNP was similar in RA patients (74.5%) and controls (76.0%), the disease duration in RA patients with genotype GG was shorter than that of patients with genotypes AA/AG and RA patients with genotype GG had a higher probability of being treated with infliximab. We studied the difference in promoter activity between the two alleles by luciferase assay and found that the promoter activity of TTP promoter region with allele A was around two-fold higher than that with allele G. We conclude that this SNP in the promoter region of the TTP gene mildly affects promoter activity, and thus, may influence the disease activity of inflammatory disorders including RA.     

Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.     

Pharmacokinetics of ivermectin applied topically by whole‐body bathing method in healthy volunteers

As a novel administration method of ivermectin (IVM) for scabies treatment, we proposed a “whole‐body bathing method (WBBM)”. In this method, the patients would bathe themselves in a bathing fluid containing IVM at an effective concentration. Previously, we demonstrated that WBBM could deliver IVM to the skin but not to the plasma in rats. In the present study, to assess the clinical validity of the method an arm bathing examination (first trial) and a whole‐body bathing examination (second trial) were conducted in healthy volunteers. In both the first and second trials, after bathing in fluid containing IVM, the exposure in the stratum corneum was higher compared with that after taking IVM p.o. as reported previously. IVM was not detected in plasma at any sampling point after the whole‐body bathing in the second trial. Furthermore no serious adverse events were found. These results in both trials suggest that WBBM can deliver IVM to the human stratum corneum without systemic exposure or serious adverse effects in healthy volunteers, and at concentrations that would be adequate for scabies treatment.     

Clinical usefulness of sialyl SSEA-1 antigen as tumor marker for ovarian cancer as compared with CA125, CA19-9, TPA, IAP, CEA and ferritin

In order to determine the clinical significance of sialyl SSEA-1 antigen, we compared its usefulness as a tumor marker for ovarian cancer with simultaneously measured CA125, CA19-9, TPA, IAP, CEA and ferritin. The sialyl SSEA-1 antigen in serum was measured by radioimmunoassay with an "FH-6" Otsuka Kit. The immunohistochemical localization of sialyl SSEA-1 antigen in ovarian carcinoma tissues was determined by an immunoperoxidase method using FH-6 monoclonal antibody. Among fifty-one patients with ovarian cancer, the incidence of elevated serum levels was 54.9% with sialyl SSEA-1 antigen, 90.2% with CA125, 48.8% with CA19-9, 78.0% with TPA, 73.1% with IAP, 17.1% with CEA and 63.4% with ferritin. On the other hand, among the patients with uterine malignancies and gynecologic benign tumors, the incidence of elevated sialyl SSEA-1 antigen levels in serum was lower than that of other tumour markers. In the patients with ovarian cancer, the serum levels of sialyl SSEA-1 antigen increased in accordance with the advance of the clinical stage and were also correlated with the effect of therapy. In the examination of immunohistochemical localization of sialyl SSEA-1 antigen, a positive reaction occurred in 10 out of 30 ovarian carcinoma specimens. Intense staining appeared in the secretory materials, in the luminal surface of the glands, and in the cytoplasm of cells. Thus, sialyl SSEA-1 antigen appears to be a useful tumor marker for the diagnosis of ovarian cancer, especially when measured simultaneously with CA125, CA19-9, TPA, ferritin and IAP.     

Establishment and characterization of a new human cell-line (AMOC-2) derived from a serous adenocarcinoma of ovary

A new cell line, designated as AMOC-2, was established from a mouse graft of human ovarian serous adenocarcinoma, and has grown well without interruption for over 12 months. The cultured cells of the AMOC-2 line were spindle or polygonal in shape and showed a monolayer growth with a pavement-like arrangement and a slight tendency to pile up, thus lacking contact inhibition. This cell line has a doubling time of 17.4h, a saturation density of 9.17 X 10(4) cells/cm2, a plating efficiency of 36.2% and a mitotic coefficient of 6.2%. The chromosomal number of this cell line is distributed in the diploid range and three of the specific marker chromosomes were identified in the cells with 49 chromosomes. This cell line was able to be transplanted into the subcutis of nude mice and produced a poorly differentiated serous adenocarcinoma resembling the original tumor. These cells have maintained their tumorigenic capacity despite prolonged maintenance in culture and are now in the 60th passage.     

Collagen fibrils in nanophthalmic sclerae

PURPOSE: To examine collagen fibrils in 3 nanophthalmos sclerae and to compare them with normal control sclerae morphometrically. MATERIALS AND METHODS: Three cases of nanophthalmos associated with uveal effusion were studied. When sclerectomy was performed, scleral specimens were collected and fixed with 3% glutaraldehyde/2.5% paraformaldehyde. After epon-embedding and ultrathin sectioning, they were examined by transmission electron microscopy. Collagen fibrils from both nanophthalmos and normal control sclerae were compared in diameters and numbers per micron 2 areas. RESULTS: All scleral tissues from the three cases were associated with irregularly woven and unclear collagen bundles. Several abnormal findings, such as twisting or fraying, were also detected in a few collagen fibrils. The diameter and density of normal-appearing collagen fibrils that occupied most areas of nanophthalmos sclerae were the same as those from normal control sclerae morphometrically. CONCLUSIONS: Although nanophthalmos sclerae even with uveal effusion showed thick irregular collagen bundles and a few abnormal collagen fibrils, most collagen fibrils appeared the same as normal controls.     

Adenosquamous cell carcinoma arising from the papilla major

A 47-year-old man was admitted to hospital with complaint of general fatigue. Shortly before the admission a suspected obstructive jaundice was diagnosed at a local hospital. On admission, the physical examination was significant for jaundice; total bilirubin was 6.43 mg/dl. The tumor marker CA19-9 was 2056 U/ml. Endoscopic retrograde cholangiopancreatography (ERCP) was performed and showed dilatation of common bile duct and main pancreatic duct, accompanied with an endoscopic naso-biliary drainage (ENBD) in order to reduce the jaundice. The duodenoscopy showed enlarged and deformed papilla. Hypotonic duodenography showed a filling defect at the medial side of the second portion of the duodenum. Ultrasonography (US) showed a hyperechoic lesion, sized 15 mm in diameter, at the pancreas head with dilatation of biliary tract and main pancreatic duct. An abdominal enhanced CT scan showed a mass sized 15 mm at the lower edge of the common bile duct. A selective hepatic arteriography showed no special finding. We performed a pancreatoduodenectomy with dissection of the lymph nodes. The tumor, sized 22x15x20 mm, was white colored and solid on the papilla. Histopathological inspection of the specimen showed an adenosquamous cell carcinoma of the bile duct in the papilla. The tumor was found to infiltrate the neighboring pancreas and to contain metastasis in lymph nodes in the hepatoduodenal ligament, post pancreaticoduodenal and para-aortic lymph nodes. This is the first report on a case of adenosquamous carcinoma of the papilla major.     

Activation of calpain in cultured neurons overexpressing Alzheimer amyloid precursor protein

We have previously reported that overexpression of wild-type amyloid precursor protein (APP) in postmitotic neurons induces cleavage-dependent activation of caspase-3 both in vivo and in vitro. In this study, we investigated the mechanism underlying APP-induced caspase-3 activation using adenovirus-mediated gene transfer into postmitotic neurons derived from human embryonal carcinoma NT2 cells. Overexpression of wild-type APP significantly increased intracellular (45)Ca(2+) content prior to the activation of caspase-3 in NT2-derived neurons. Chelation of intracellular Ca(2+) markedly suppressed APP-induced activation of caspase-3. Furthermore, calpain, a Ca(2+)-dependent cysteine protease, was activated in neurons overexpressing APP as assessed by increased levels of calpain-cleaved alpha-fodrin and autolytic mu-calpain fragments. Neither calpain nor caspase-3 was activated in neurons expressing an APP mutant defective in the Abeta(1-20) domain. Calpain inhibitors almost completely suppressed APP-induced activation of neuronal caspase-3. E64d, a membrane permeable inhibitor of calpain, significantly suppressed APP-induced neuronal death. These results suggest that overexpression of wild-type APP activates calpain that mediates caspase-3 activation in postmitotic neurons.