Improvement of Renal Function by Long-Term Sustained Eculizumab Treatment in a Patient with Paroxysmal Nocturnal Hemoglobinuria.

Chronic kidney disease (CKD) is one of the major manifestations of paroxysmal nocturnal hemoglobinuria (PNH). CKD in PNH is induced mainly by intravascular hemolysis of PNH-affected red blood cells (RBC) missing the glycosylphosphatidylinositol-anchored proteins with complement-regulatory activities, CD55 and CD59. CKD develops by heme absorption in the proximal tubules resulting in the interstitial deposition of iron in the kidneys. We administered eculizumab to a patient with PNH, who was one of 29 patients enrolled in the AEGIS clinical trial, an open-label study of eculizumab in Japan. The patient was complicated by stage 3 CKD with impaired estimated glomerular filtration rate (eGFR), at grade G3b, and had obvious proteinuria (2-3+, 1-2 g/day). In a two-year extension to the 12-week AEGIS study, eGFR improved significantly, and the eGFR has since been maintained at grade G2 without proteinuria by sustained eculizumab treatment (>6 years). Renal function improved and maintained by long-term sustained eculizumab treatment, presumably by clearance of iron from the kidney as well as inhibition of the production of anaphylatoxin C5a, even in advanced stages of CKD, is one of the benefits of eculizumab treatment in PNH.

Validation of cleaning evaluation of surgical instruments with RFID tags attached based on cleaning appraisal judgment guidelines.

In medical institutions, the threat of infection is closely focused, in particular, inspections regarding surgical site infections (SSI) are carried out. In this study, development of the application of Radio frequency identification (RFID) tags for surgical instrument has been promoted. It enables traceability and individual management of surgical instruments. An experiment was carried out following the cleaning Appraisal guidelines, which contaminated surgical instruments, and using the washer-disinfector (WD) as the main cleaner for surgical instruments with developed RFID tags attached to them. As a result, all of the instruments with RFID tags, the amount of residual protein was less than the recommended acceptable level of 100µg. If WD is used correctly, a sufficient cleaning effect can be expected. From this result, it became evident that the secondary infection risk is low from surgical instrument with RFID tags attached.

Loss-of-function of a ubiquitin-related modifier promotes the mobilization of the active MITE mPing.

Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.

Hyperproduction of 3,4-dihydroxyphenyl-L-alanine (L-Dopa) using Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR.

In the last few decades, enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using tyrosine phenol-lyase (Tpl) has been industrialized. This method has an intrinsic problem of tyrosine contamination because Tpl is synthesized under tyrosine-induced conditions. Herein, we constructed a hyper-L-dopa-producing strain by exploiting a mutant TyrR, an activator of tpl. The highest productivity was obtained for the strain grown under non-induced conditions. It was 30-fold higher than that obtained for tyrosine-induced wild-type cells.

STAT3 signaling is induced by intercellular adhesion in squamous cell carcinoma cells.

The signal transducer and activator of transcription-3 (STAT3) frequently activated during tumor progression has been linked to enhanced cell growth. In squamous cell carcinoma of the head and neck (HNSCC), STAT3 signaling has been shown to inhibit apoptosis and induce a more aggressive phenotype through the activation of specific signaling pathways. In the present study, we have examined the potential mechanism by which cell-cell contact initiates STAT3 activation. Using a panel of HNSCC cell lines, Ca(+2)-dependent cell-cell adhesion and adherens junction formation in multicellular aggregates triggered phosphorylation of STAT3-Y705 and STAT1-Y701. This intercellular adhesion-induced STAT3 activation was mediated by JAK and Src signaling and partially by EGFR signaling. In addition, immunolocalization studies revealed initial formation of phosphorylated STAT3-Y705 at nascent E-cadherin cell junctions with eventual translocation to the nucleus in cell aggregates. Adhesion-mediated STAT activation in monolayer and cell aggregate cultures required functional E-cadherin. These results indicate that, in HNSCC cells, cadherin-mediated intercellular adhesion induces STAT signaling that may modulate cell survival and resistance to apoptosis during tumor progression.

Dystroglycan loss disrupts polarity and beta-casein induction in mammary epithelial cells by perturbing laminin anchoring.

Precise contact between epithelial cells and their underlying basement membrane is crucial to the maintenance of tissue architecture and function. To understand the role that the laminin receptor dystroglycan (DG) plays in these processes, we assayed cell responses to laminin-111 following conditional ablation of DG gene (Dag1) expression in cultured mammary epithelial cells. Strikingly, DG loss disrupted laminin-111-induced polarity and beta-casein production, and abolished laminin assembly at the step of laminin binding to the cell surface. Dystroglycan re-expression restored these deficiencies. Investigations of the mechanism revealed that DG cytoplasmic sequences were not necessary for laminin assembly and signaling, and only when the entire mucin domain of extracellular DG was deleted did laminin assembly not occur. These results demonstrate that DG is essential as a laminin-111 co-receptor in mammary epithelial cells that functions by mediating laminin anchoring to the cell surface, a process that allows laminin polymerization, tissue polarity and beta-casein induction. The observed loss of laminin-111 assembly and signaling in Dag1(-/-) mammary epithelial cells provides insights into the signaling changes occurring in breast carcinomas and other cancers, where the binding function of DG to laminin is frequently defective.

The yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a novel glutathione importer with an ATP-binding cassette.

Glutathione protects cells and organisms from oxygen species and peroxides and is indispensable for aerobically living organisms. Moreover, it acts against xenobiotics and drugs by the formation and excretion of glutathione S conjugates. In this study, we show that the yliA, -B, -C, and -D genes of Escherichia coli K-12 encode a glutathione transporter with the ATP-binding cassette. The transporter imports extracellular glutathione into the cytoplasm in an ATP-dependent manner. This transporter, along with gamma-glutamyltranspeptidase, has an important role in E. coli growth with glutathione as a sole sulfur source.

Effects of hormones targeting nuclear receptors on transcriptional regulation of the growth hormone gene in the MtT/S rat somatotrope cell line.

We have examined the effects of nuclear receptor hormones such as glucocorticoid, gonadal steroid hormones, thyroid hormone and retinoids on the transcriptional regulation of the 5'-promoter activity of growth hormone (GH) gene using the MtT/S rat pure somatotrope cell line or MtT/SGL, a subclone of MtT/S in which the rat GH gene 5'-promoter (1.7 Kb)-luciferase fusion gene was stably incorporated. RT-PCR analyses revealed that receptors for all the hormones except androgen receptor were expressed in the cell line. Triiodothyronine (T(3), 10 nM) transiently but significantly stimulated the promoter activity of GH gene, whereas retinoic acids (9-cis and all-trans, both 1 microM) showed sustained stimulation. There were no additive effects among the T(3), all-trans, and9-cis retinoic acids. Synthetic glucocorticoid hormone dexamethasone (100 nM) showed an inhibitory effect but, interestingly, significantly enhanced T(3)-stimulated GH promoter activity during long-term incubation. Among the gonadal steroid hormones tested, estradiol and estriol had significant stimulatory effects, and deletion analysis showed that the estrogen effect was maintained with the shortest construct examined (-150 to +6, +1 denotes the transcription start site). These results suggest that thyroid hormone and retinoids stimulate the transcription of GH gene, probably through a common response element, whereas glucocorticoid has both negative and positive effects on GH expression, depending on the combination with other hormones and the time of exposure. Estrogens also have direct stimulatory effects through the proximal promoter region of GH gene.

Basic and clinical studies on quantitative analysis of lymph node micrometastasis in oral cancer.

Metastasis to cervical lymph nodes (LN) is significantly associated with the outcome of patients with oral cancer. To provide a useful method for the detection of micrometastases, we analyzed 115 LNs from 10 patients with oral cancer using real-time quantitative polymerase chain reaction (PCR) based on the expression of squamous cell carcinoma antigen (SCCA) and cytokeratin 13 (CK13). The sensitivity and quantification of this method were assessed by means of limited dilution of cultured oral cancer cells and a model of cervical LN-metastasis established by inoculating green fluorescent protein (GFP)-expressing cells into the tongue of nude mice. In both investigations, a few cancer cells were detected by real-time quantitative PCR, but not by conventional reverse transcription-PCR (RT-PCR). SCCA mRNA was detected at high levels in metastatic LNs. In contrast, 26 of the 30 control cervical LNs did not express the gene at all, and the rest showed fairly low levels. Of 108 histologically metastasis-negative LNs, 19 (17.6%) expressed SCCA mRNA levels higher than the cut-off value (1.0: mean expression of control LNs + 2SD). CK13 mRNA is not a suitable marker for the real-time PCR since it was detected frequently even in the control LNs. These findings suggest that genetic diagnosis by real-time quantitative PCR based on SCCA mRNA expression may be clinically useful for detecting occult tumor cells in cervical LNs.