Bidirectional Glenn anastomosis in staged treatment of patients with single ventricle: comparison between on-pump and off-pump technique

Introduction of the bidirectional Glenn procedure in patients with functional single ventricle [BDG] in Fontan candidates would improve clinical outcomes for all candidates underwent BDG and staged Fontan operation [total cavopulmonary connection TCPC]. We discuss indications for the bidirectional cavopuimonary connection. The Bi-directional Cavo-pulmonary [Glenn,] shunt is Commonly Performed Procedure for a variety of Cyanotic Congenital heart diseases that lead eventually to single ventricle repair. We report and Compare the results of Bi-directional Glenn operation done in University of Leipzig, heart Centre. Germany and Assiut University Paediatric Heart Surgery Centre, Egypt. Between November 2008 and July 2010 in Leipzig heart Centre and between March 2010 and March 2012 in Assiut University Paediatric Heart Surgery Centre, 26 patients [16 in Leipzig and 10 in Assiut] aged 3 months to 5 years received a bidirectional Glenn Shunt with the use of a temporary shunt [in Assiut = Group I] or cardiopulmonary bypass [CPB] [in Leipzig = Group II]. Arterial oxygen saturation rose from 78% +/- 8.5% preoperatively to 90% +/- 4.3% postoperatively. Hemodynamic studies showed a mean postoperative superior vena cava pressure of 13 +/- 2 mm H[2]O. No patient had desaturation and the shunt was taken down, 1 required reexploration for bleeding [Leipzig Group], and 1 needed prolonged drainage of 9 to 19 days [Assiut group], 1 of whom had chylothorax [Assiut group]. Hospital stay, was 9.3 +/- 3.5 days. There were one postoperative deaths [1.8%.] from cerebral haemorrhage [Assiut Group]. The bidirectional Glenn shunt remains an excellent palliative procedure as a preliminary step to a Fontan operation, or as an integral part of a Fontan or modified Fontan operation when the procedure is deferred because of age, weight, or cardiac malformations characterized by a hypoplastic right or left ventricle

Modulation of Telomerase Activity and Human Telomerase Reverse Transcriptase Expression by Caspases and Bcl-2 Family Proteins in Cisplatin-Induced Cell Death / 대한진단검사의학회지

BACKGROUND: Human telomerase is a ribonucleoprotein polymerase, which synthesizes telomeric repeat sequences, and human telomerase reverse transcriptase (hTERT) has been identified as the catalytic subunit, as well as the rate-limiting component, of telomerase. In this study, we attempted to identify the modulators of telomerase, and to determine the molecular mechanisms underlying cisplatin-induced apoptosis. METHODS: To determine the role of telomerase in cisplatin-induced apoptosis, we measured telomerase activity and analyzed apoptosis using PI and trypan blue staining. Also, we inhibited the caspase activations using Z-VAD-fmk to analyze the effects on expression of hTERT protein. Finally, we induced the transient co-expression of the Bcl-2 and Bak genes in HEK293 cells, and then, the telomerase activity and expression of hTERT were evaluated. RESULTS: In the Bcl-2-overexpressing HeLa cells, telomerase activity was more enhanced, and cell death was reduced to 40-50% that of the mock controls. This finding suggests that Bcl-2-induced telomerase activity exerts an antiapoptotic effect in cisplatin-induced death. As caspase activation was inhibited via Z-VAD-fmk, the hTERT protein was recovered in the mock controls, but not in the Bcl-2-overexpressing cells. This suggests that the expression of hTERT can be regulated by caspases, but Bcl-2 was located within the upstream pathway. Moreover, when the Bcl-2 and Bak genes were co-transfected into the HEK293, both telomerase activity and hTERT protein were prominently reduced. CONCLUSIONS: Bcl-2-induced telomerase activity inhibits cisplatin-induced apoptosis in HeLa cells, and can be regulated via both caspases and the interaction of Bcl-2 and Bak.

C-Terminal Serine Rich Region of IKK-beta Interacts with Bfl-1, a Homologue of Bcl-2 / 대한진단검사의학회지

BACKGROUND: Bcl-2 family proteins play a central role in regulating apoptosis. In human, over 20 members of this family have been identified to date. Bfl-1, a member of the Bcl-2 family, has been known to retard apoptosis in various cell lines. However, the function of Bfl-1 remains unclear. METHODS: In order to investigate the Bfl-1 function, we employed yeast two-hybrid system to identify the proteins which are capable of interacting with Bfl-1. The interaction of inhibitor kappaB kinase-beta (IKK-beta) and Bfl-1 was confirmed using glutathione S-transferase pull down assays. To determine which regions of IKK-beta were required for interaction with Bfl-1, we constructed 12 deletion mutants of IKK-beta and 5 deletion mutants of Bfl-1. RESULTS: Bfl-1 interacted with the C-terminal region of IKK-beta which is a subunit of IKK complex, and IKK-beta activity is very important in the NF-kappaB related pathway. In addition, the amino acids 673-745 of IKK-beta were important for Bfl-1 interactions, and amino acids 1-484 of Bfl-1, including Bcl-2 homology domains (BH1, BH2, BH3, BH4), were crucial for IKK-beta interactions. CONCLUSIONS: IKK beta C-terminus contains many serine residues as binding partner of Bfl-1. Our results suggested that Bfl-1 is involved in the NF-kappaB activation through interaction of IKK-beta and Bfl-1. Further studies need to be performed to understand functions of the IKK-beta and Bfl-1 associated with the regulation of the NF-kappaB activation pathway.

Anti-Cancer Efficacy of Anti-CEA IgG3 in a Syngeneic Carcinoembryonix Antigen Tumor Model / 대한면역학회지

Development of antibody-based cancer therapies will be greatly facilitated if antibodies are better standardized in two fundamental issues that are specificity analysis of antibody reactivity and the detailed biodistribution and pharmacokinetic profile of antibodies. In the current endeavor we attempted to use an antibody binding specificity to target the tumor in a syngeneic carcinoembryonic antigen (CEA) tumor model. CEA, a 180 kDa glycoprotein, expressed at high levels on the surface of nearly all tumors of the gastrointestinal tract was used a potential target for antibody immunotherapy of gastrointestinal carcinomas. Using the CEA model antibody-based cancer therapy directed against CEA has been evaluated in a syngeneic animal model of disseminated disease. We constructed mouse/human chimeric anti-CEA IgG3, which has been evaluated for the specificity for CEA and the detailed biodistribution and pharmacokinetic profiles. Anti-CEA IgG3 heavy chain was expressed with the expected 180kDa molecular weight, assembled as H2L2 forms with a co-expressed mouse/human chimeric anti-CEA light chain, and were secreted. On FACS the purified anti-CEA IgG3 specifically recognized the mouse colon adenocarcinoma cell line MC-38 transduced with CEA (MCA32a), but not MC 38 without expressing CEA. After subcutaneous injection in C57BL/6 mice the half- lives of anti-CEA IgG3 and an irrelevant anti-dansyl IgG3 showed the bi-phasic kinetic patterns, and their pharmacokinetics of the distribution and the elimination were similar in mice. However, the biodistribution patterns of anti-CEA IgG3 were very different from those of anti-dansyl IgG3. Anti-dansyl IgG3 was mainly distributed into kidney until 72 hours, but anti-CEA IgG3 was slowly rernoved from blood and distributed into liver, kidney, spleen, and tumor. It is note worthy that anti- CEA IgG3 increased in targeting MCA32a tumor expressing human CEA by time, but the targeting to MC38 tumor was negligible. Thus, the increased targeting of anti- CEA IgG3 made MCA32a tumor grow slowly

Affinity Improvement of Antibody-Avidin Fusion Proteins for Biotin / 대한면역학회지

To generate drug delivery vector to locales in the body, genetic engineering and expression techniques have been used to produce antibody avidin fusion proteins. Chicken avidin has been fused to mouse-human chimeric IgG3 immediately after the hinge with a flexible linker (H-Flex-Av) and at the end of CH2 (CH2-Av). Fusion heavy chains were expressed with the expected molecular weight, assembled as H2L2 forms with a co-expressed light chain, and were secreted. The expression level of H- Flex-Av was 1~10 ug/ml/10(8)/24 hrs, but that of C2-Av was a very little (0.08~0.9 ug/ ml/10(8)/24 hrs). The resulting H-Flex-Av and CH2-Av fusion proteins continued to bind antigen dansyl and also bound biotinylated bovine serum albumin; both H-Flex-Av and CH2-Av had shown to retain 3-4 times higher relative affinity than that of CH3-Av in ELISA. Importantly the fact that both avidin fusion proteins had a higher relative affinity suggests that these avidin fusion proteins can be effectively used to deliver biotinylated ligands such as drugs and peptides to a certain locale, such as the brain.

Purification and cloning of glyoxalase II from rat liver

Glyoxalase (GLO) II, which is a component of GLO system and catalyze the conversion of S-lactoyl-glutathione to D-lactate, was purified 1488 fold from rat liver by two steps of Affigel blue and carbobenzoxyglutathione-Sepharose 4B affinity chromatography. The molecular weight of the enzyme was estimated to be 29 kDa which is similar to those from other species. The sequence of N-terminal 9 amino acid residues was determined to be MGIRLLPAT. This was then used to synthesize degenerative primers. cDNA clone was isolated by first synthesizing cDNA from RNA and then PCR amplification. The sequence of cDNA clone was determined by serial sequencing analysis.