Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli

Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, in E. coli produced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the epsilon-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfish Aequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems

Retroperitoneoscopic resection of a congenital adrenal teratoma in an infant

AbstractWe present a case of a 4-month-old boy with a congenital adrenal teratoma. A right suprarenal cystic mass was detected before birth. Ultrasonography at the age of one day showed a 20-mm suprarenal mass. No blood flow was observed in the mass. It decreased in size over the first ten days after birth, but subsequently increased in size during four months of follow-up. Imaging examinations showed that the mass changed from cystic to solid with areas of adipose tissue. Complete resection of the mass was performed retroperitoneoscopically with preservation of the right adrenal gland. Pathological examination showed a mature teratoma with no immature or malignant elements. There were no postoperative complications. With the growing use of ultrasonography during pregnancy, increasing numbers of neonates present for management of suprarenal masses. In the majority of cases, the mass is a congenital cystic neuroblastoma or adrenal hemorrhagic pseudocyst. Although congenital adrenal teratoma is extremely rare, it should be considered in the differential diagnosis of a neonatal suprarenal mass

Laryngotracheal separation for a type 4 laryngotracheoesophageal cleft with multiple significant malformations

AbstractLaryngotracheoesophageal cleft (LTEC) is an extremely rare congenital malformation that is difficult to treat. We present a female patient with type 4 LTEC associated with multiple significant malformations. She suffered from a severe asthma attack due to aspiration of saliva, and it was determined that she should undergo surgery for associated congenital heart disease as early as possible to ensure long-term survival. Therefore, we performed laryngotracheal separation with an end-tracheostomy to completely and immediately prevent aspiration. She was then able to undergo radical surgery for her congenital heart disease. She recovered well postoperatively, and long-term survival is expected

Giant Retroperitoneal Mucinous Tumor Supportively Diagnosed as a Dedifferentiated Liposarcoma by Fluorescence In Situ Hybridization of MDM2 Gene

Surgical resection was performed on a 47-year-old woman for a retroperitoneal mass that weighed 8.5 kg. Histological examination revealed a myxoid sarcomatous tumor. Because diagnosis could not be determined by immunohistochemistry, attention was focused on MDM2 (murine double minute) gene amplification by fluorescence in situ hybridization (FISH) analysis. The tumor was finally determined to be a dedifferentiated liposarcoma. We experienced a case of a giant retroperitoneal dedifferentiated liposarcoma. FISH analysis was useful for the diagnosis and determination of the therapeutic strategy

Mineralocorticoid receptor stimulation induces urinary storage dysfunction via upregulation of epithelial sodium channel expression in the rat urinary bladder epithelium

AbstractWe aimed to evaluate mineralocorticoid receptor (MR) expression in rat bladder and the physiological role of the MR-epithelial sodium channel (ENaC) pathway in controlling bladder function in 10–12-week-old, male Sprague–Dawley rats. First, we examined the mRNA expression of MR and localization of MR and ENaC-α proteins in the urinary bladder. MR mRNA expression was observed in untreated-rat urinary bladders, and MR and ENaC-α proteins were localized in the epithelium. Next, rats were treated with vehicle (controls) or fludrocortisone (an MR agonist) for 3 days, and ENaC-α protein expression levels and bladder function were evaluated on day 4. ENaC-α protein expression was significantly higher in fludrocortisone-treated rats than in controls. In addition, cystometry was performed during intravesical infusion of saline and amiloride (an ENaC inhibitor). While intercontraction intervals (ICIs) during saline infusion were significantly shorter in the fludrocortisone group than in the controls, infusion of amiloride normalized the ICIs in the fludrocortisone group. However, no intra- or inter-group differences in maximum intravesical pressure were observed. Taken together, MR protein is localized in the rat urinary bladder epithelium, and may regulate ENaC expression and bladder afferent input. The MR-ENaC pathway may be a therapeutic target for ameliorating storage symptoms

Cancer activity and bleeding events post-PCI

Purpose : Limited data exist about clinically relevant bleeding events related to antiplatelet therapy after percutaneous coronary intervention (PCI) in cancer patients. We investigated the risk factors for clinically relevant bleeding events in patients with cancer after PCI with stent implantation. Patients and Methods : Patients with solid cancer subjected to first PCI were divided into active (n = 45) and non-active cancer groups (n = 44). The active group included non-operable patients on treatment or with metastasis ; the non-active included those already subjected to or for whom radical surgery was planned within 3 months after the index PCI. Results : During a median follow-up of 2.2 years, 11 bleeding events occurred, with only one occurring in the non-active cancer group. Half of them occurred during the dual-antiplatelet therapy (DAPT) period, and the rest occurred during single-antiplatelet therapy (SAPT) period. Kaplan-Meier analysis showed significantly more bleeding events in the active cancer group (p = 0.010). Multivariate Cox regression hazard analysis revealed cancer activity as a significant independent risk factor for bleeding (p = 0.023) ; but not for three-point major adverse cardiovascular events. Conclusion : Clinically relevant bleeding risk after PCI was significantly lower in non-active cancer. Active cancer group had clinically relevant bleeding during both DAPT and SAPT periods

Mechanical tension-stress induces expression of bone morphogenetic protein (BMP)-2 and BMP-4, but not BMP-6, BMP-7, and GDF-5 mRNA, during distraction osteogenesis. J Bone Miner Res

ABSTRACT Bone lengthening with osteotomy and gradual distraction was achieved in 57 rats, and the effect of mechanical tension-stress on gene expression of bone morphogenetic proteins (BMPs) was investigated by in situ hybridization and Northern blot analysis using probes of BMP-2, BMP-4, BMP-6, BMP-7, and growth/differentiation factor (GDF)-5. There was a lag phase for 7 days after femoral osteotomy until gradual distraction was carried out for 21 days at a rate of 0.25 mm/12 h using a small external fixator. The signals of the above BMPs mRNA were not detected in the intact rat bone but they were induced after osteotomy except those for BMP-7. By 4 days after osteotomy, BMP-2 and BMP-4 mRNAs were detected in chondrogenic precursor cells in the subperiosteal immature callus. BMP-6 and GDF-5 mRNA were detected in more differentiated cells in chondroid bone. By 7 days after osteotomy, cartilaginous external callus and bony endosteal callus were formed. Meanwhile, the signals of BMP-2 and BMP-4 mRNAs declined to preoperative levels, whereas the signals of BMP-6 and GDF-5 mRNAs were rather elevated. As distraction was started, the callus elongated and eventually separated into proximal and distal segments forming a fibrous interzone in the middle. Expression of BMP-2 and BMP-4 mRNAs was markedly induced at this stage. Their signals were detected widely among chondrogenic and osteogenic cells and their precursor cells sustaining mechanical tension-stress at the fibrous interzone. BMP-6 and GDF-5 mRNAs were detected exclusively in chondrogenic cells at both ends of the fibrous interzone, where endochondral ossification occurred. But neither mRNA was detected in terminally differentiated hypertrophic chondrocytes. As distraction advanced, the cartilage was progressively resorbed from both ends and new bone was formed directly by intramembranous ossification. There was no new cartilage formation in the advanced stage of distraction. The signals of BMP-6 and GDF-5 mRNA declined by this stage, while those of BMP-2 and BMP-4 were maintained at high level for as long as distraction was continued. After completion of distraction, the fibrous interzone fused and the lengthened segment was consolidated. BMP-2, BMP-4, BMP-6, nor GDF-5 was expressed at this stage. The signals of BMP-7 were not detected throughout the experiment. The present results suggest that excellent and uninterrupted bone formation during distraction osteogenesis owes to enhanced expression of BMP-2 and BMP-4 genes by mechanical tensionstress. Abundant gene products of BMP-2 and BMP-4 could induce in situ bone formation by paracrine and autocrin