Cloning of the gamma chain of the human IL-2 receptor.

A third subunit, the gamma chain, of the human interleukin-2 receptor (IL-2R) was identified, and a complementary DNA clone encoding this member of the cytokine receptor family was isolated. The gamma chain is necessary for the formation of the high- and intermediate-affinity receptors, which consists of alpha beta gamma heterotrimers and beta gamma heterodimers, respectively. The IL-2R on murine fibroblastoid cells can be internalized after binding IL-2 only if the gamma chain is present; alpha and beta are insufficient for internalization. Thus, the gamma chain is an indispensable component of the functional IL-2R.

[Does aortic cusp repair influence the outcome of valve sparing aortic root replacement?].

BACKGROUND: Outcome of the patients who underwent aortic root replacement with valve sparing procedure concomitant with cusp repair was evaluated. METHODS: Between October 1999 and April 2009, valve sparing aortic root replacement were performed in 62 patients. Isolated valve sparing procedure was performed in 38 patients (control) and concomitant cusp repair was performed in 24 patients (aortic valve plasty: AVP). Cusp prolapse was corrected by plication of Arantius body (n = 13), cusp perforations were closed by pericardial patch (n = 6) or reinforcement of free margin (n = 6). RESULTS: No patient died during the hospital stay and no thromboembolic events occurred in the follow up. Pre-operative grade of aortic insufficiency was 3.0 +/- 0.9 in AVP group vs. 2.5 +/- 1.3 in control (NS). There was no significant difference between both groups regarding age, cardiac function and preoperative aortic root diameter. However, eccentric jet by preoperative transesophageal echocardiography (TEE) was more often in group AVP than in control (p<0.01). Five patients underwent reoperation because of recurrent aortic regurgitation (AR) or hemolysis. Postoperative AR grade were similar in both groups (0.9 +/- 0.5 vs 0.5 +/- 0.6). During follow up, the 5 year freedom from re-operation (aortic valve replacement: AVR) was 85.1+/- 8.2% in AVP and 94.0 +/- 4.1% in control (NS). 5-year-survival was 100% and 95.0 +/- 4.9% (NS), respectively. CONCLUSIONS: Valve sparing aortic root replacement with concomitant cusp repair provided satisfactory midterm result.

Timing for orthotopic liver transplantation in children with biliary atresia: a single-center experience.

INTRODUCTION: Biliary atresia is the most common indication for orthotopic liver transplantation (OLT) in childhood. The purpose of this study was to determine predictive prognostic factors for children with biliary atresia related to the timing for OLT within 15 months after hepatoportoenterostomy (HPE). PATIENTS AND METHODS: We retrospectively analyzed the medical records of 25 children (7 boys and 18 girls) who underwent HPE because of biliary atresia between January 1990 and December 2005 at our center. Data examined included age and pathologic findings at HPE, Pediatric End-Stage Liver Disease score at first admission, whether phototherapy was given, liver function test results and total bilirubin level before and 30 days after HPE, and number of cholangitis events. RESULTS: Twelve children were alive with their native liver, 8 had undergone living donor OLT (all children alive), and 5 had died without OLT. Five- and 10-year survival rates without OLT after HPE were 47.4% and 26.3%, respectively. At univariate analysis, the predictive prognostic factors for children with biliary atresia were total bilirubin level at 30 days after HPE and Pediatric End-Stage Liver Disease score before HPE. At multivariate analysis, the only prognostic factor was total bilirubin level at 30 days after HPE. CONCLUSIONS: In this study, the predictive prognostic factor was total bilirubin level at 30 days after HPE. Orthotopic liver transplantation within 15 months after HPE is needed in children with biliary atresia with a high total bilirubin level at 30 days after HPE.

Living donor liver transplantation for a child with recurrent pediatric adult-type hepatocellular carcinoma.

INTRODUCTION: Pediatric hepatocellular carcinoma (HCC) is an uncommon disease with a poor prognosis. There are few reports about liver transplantation for pediatric adult-type HCC. We experienced a case of living donor liver transplantation (LDLT) for a child with recurrent pediatric adult-type HCC. CASE REPORT: A 12-year-old boy was admitted to the Department of Pediatrics in our institution due to HCC in May 2005. He underwent hepatectomy after 3 courses of chemotherapy in July 2005. After the operation, he had 2 more courses of the same chemotherapy. His posttheraputic course was uneventful for 1 year. However, his alpha-fetoprotein level increased and a computed tomography (CT) scan showed recurrent tumor in his remnant liver in October 2006. He underwent another chemotherapy session immediately. However, CT revealed multiple liver tumors after chemotherapy in December 2006. His mother requested to be an LDLT donor, which was performed on January 23, 2007. The donor operation was a right hepatic lobectomy. The postoperative course of the donor was unremarkable and she has now returned to work. The recipient's posttransplantation course was uneventful and he was discharged at postoperative day 53 and is currently doing well. CONCLUSION: Liver transplantation in conjunction with chemotherapy may have an increasing role in the management of pediatric HCC.

Multiple Re-entry Closures After TEVAR for Ruptured Chronic Post-dissection Thoraco-abdominal Aortic Aneurysm.

INTRODUCTION: Although thoracic endovascular aortic repair (TEVAR) has become a promising treatment for complicated acute type B dissection, its role in treating chronic post-dissection thoraco-abdominal aortic aneurysm (TAA) is still limited owing to persistent retrograde flow into the false lumen (FL) through abdominal or iliac re-entry tears. REPORT: A case of chronic post-dissection TAA treatment, in which a dilated descending FL ruptured into the left thorax, is described. The primary entry tear was closed by emergency TEVAR and multiple abdominal re-entries were closed by EVAR. In addition, major re-entries at the detached right renal artery and iliac bifurcation were closed using covered stents. To close re-entries as far as possible, EVAR was carried out using the chimney technique, and additional aortic extenders were placed above the coeliac artery. A few re-entries remained, but complete FL thrombosis of the rupture site was achieved. Follow-up computed tomography showed significant shrinkage of the FL. DISCUSSION: In treating post-dissection TAA, entry closure by TEVAR is sometimes insufficient, owing to persistent retrograde flow into the FL from abdominal or iliac re-entries. Adjunctive techniques are needed to close these distal re-entries to obtain complete FL exclusion, especially in rupture cases. Recently, encouraging results of complete coverage of the thoraco-abdominal aorta with fenestrated or branched endografts have been reported; however, the widespread employment of such techniques appears to be limited owing to technical difficulties. The present method with multiple re-entry closures using off the shelf and immediately available devices is an alternative for the endovascular treatment of post-dissection TAA, especially in the emergency setting.

The recessive phenotype displayed by a dominant negative microphthalmia-associated transcription factor mutant is a result of impaired nucleation potential.

In the DNA binding domain of microphthalmia-associated transcription factor (MITF), four mutations are reported: mi, Mi wh, mi ew, and mi or. MITFs encoded by the mi, Mi wh, mi ew, and Mi or mutant alleles (mi-MITF, Mi wh-MITF, Mi ew-MITF, and Mi or-MITF, respectively) interfered with the DNA binding of wild-type MITF, TFE3, and another basic helix-loop-helix leucine zipper protein in vitro. Polyclonal antibody against MITF was produced and used for investigating the subcellular localization of mutant MITFs. Immunocytochemistry and immunoblotting revealed that more than 99% of wild-type MITF and Mi wh-MITF located in nuclei of transfected NIH 3T3 and 293T cells. In contrast, mi-MITF predominantly located in the cytoplasm of cells transfected with the corresponding plasmid. When the immunoglobulin G (IgG)-conjugated peptides representing a part of the DNA binding domain containing mi and Mi wh mutations were microinjected into the cytoplasm of NRK49F cells, wild-type peptide and Mi wh-type peptide-IgG conjugate localized in nuclei but mi-type peptide-IgG conjugate was detectable only in the cytoplasm. It was also demonstrated that the nuclear translocation potential of Mi or-MITF was normal but that Mi ew-MITF was impaired as well as mi-MITF. In cotransfection assay, a strong dominant negative effect of Mi wh-MITF against wild-type MITF-dependent transactivation system on tyrosinase promoter was observed, but mi-MITF had a small effect. However, by the conjugation of simian virus 40 large-T-antigen-derived nuclear localization signal to mi-MITF, the dominant negative effect was enhanced. Furthermore, we demonstrated that the interaction between wild-type MITF and mi-MITF occurred in the cytoplasm and that mi-MITF had an inhibitory effect on nuclear localization potential of wild-type MITF.

Isolation and characterization of a sulfated glycoprotein from a transplantable colorectal adenocarcinoma of rats.

A transplantable colorectal adenocarcinoma from rats of the ACI/N strain was extracted with 5 mM EDTA (pH 7.0), and fractionated by gel filtration on Sepharose 4B, followed by preparative poly(vinyl chloride) zone electrophoresis. An acidic glycoprotein (SGP) thus obtained was shown to be Homogeneous by electrophoresis on cellulose acetate membrane and on sodium dodecyl sulfate agarose gel. SGP contained 61.9% carbohydrate, 28.9% total amino acids and 1.4% sulfate. The major monosaccharides in SGP were galactose, glucosamine and galactosamine. Small quantities of sialic acid, L-fucose and mannose were also present. Threonine, serine, proline, glutamic acid and aspartic acid were the major amino acids of the protein moiety.

Further characterization of porcine plasma fibronectin which contains fucosylated carbohydrate chains.

Porcine plasma fibronectin and its functional four fragments produced by cathepsin B digestion were examined for biological, immunochemical and biochemical properties. Native fibronectin, 150-kDa and 130-kDa fragments exhibited similar cell attachment-promoting activity to each other. In an Ouchterlony double immunodiffusion system, these three polypeptides formed a precipitin line with anti-fibronectin antiserum, while the 50-kDa and 30-kDa fragments did not. The 150-kDa and 130-kDa fragments contained free sulfhydryl(s). The glycopeptide fractions were prepared by pronase digestion of porcine and human plasma fibronectin, and radiolabeled with [14C]acetic anhydride. The results of affinity chromatography with concanavalin A and lentil lectin immobilized on agarose indicated that the porcine glycopeptide fraction was different from the human fraction in that a larger part (58%) of the former was bound to lentil lectin. About 90% of this lentil lectin-reactive glycopeptides lost this reactivity upon alpha-L-fucosidase digestion. The glycopeptide fractions were also prepared from three carbohydrate-containing domains. Less than 30% of the radioactivity of the glycopeptide fractions of 150-kDa and 130-kDa fragments was retained on the lentil lectin-agarose, while about 90% of that from the 50-kDa fragment was retained. These results indicate that porcine plasma fibronectin has characteristics very similar to those of human plasma fibronectin and others, but is unique in that it contains fucosylated carbohydrate chains which unevenly distribute through functional domains.

Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen.

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues. Spleen dermatan sulfate exhibited the highest thrombin-inhibitory activity, which may be related to its high content of the disulfated N-acetylgalactosamine residue.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta.

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain. They were also distinguished immunochemically. These data indicate that the intima-media of the aorta contains at least two distinct species of chondroitin sulfate proteoglycan.

Biochemical characterization of a 34 kDa ribonucleoprotein (p34) purified from the spinach chloroplast fraction as an effective phosphate acceptor for casein kinase II.

A 34 kDa ribonucleoprotein (p34) was purified to homogeneity from a 1.0 M KCl extract of spinach chloroplasts and characterized as an effective phosphate acceptor for casein kinase II (CK-II). The N-terminal 21 residues (W-V-A-Q-T-S-E-E-E-Q-E-G-S-T-N-A-V-L-E-G-E) of p34 were 95% identical with the sequence reported for 28RNP (plastid mRNA 3' end processing factor in chloroplast). Moreover, the findings that DNAs as well as RNAs significantly stimulate the CK-II catalyzed phosphorylation of p34 in vitro and induce its conformational change, suggest that the physiological activity of p34-bound RNA or DNA in chloroplast post-transcriptional regulation is controlled by specific p34 phosphorylation by CK-II.

DNA-binding sperm proteins with oligo-arginine clusters function as potent activators for egg CK-II.

The stimulatory effect of DNA-binding sperm proteins (histone and protamine) on the phosphorylation of p98 (ERp99/GRp94, one of the Hsp-90 family of proteins) by egg casein kinase II (CK-II) was investigated in vitro. It was found that (i) phosphorylation of p98 by egg CK-II in vitro is greatly stimulated by poly-Arg, but not by poly-Lys; and (ii) similar stimulation is observed with sperm histones H2B2 and H2B3 (sea urchin) and fish protamines, such as salmine A1 (salmon) and protamine 3a (rainbow trout). These findings suggest that these DNA-binding sperm proteins function as potent activators for CK-II in fertilized eggs. All of these DNA-binding sperm proteins contain at least an oligo-Arg cluster as a common feature, which can interact with an acidic amino acid cluster of the regulatory beta-subunit CK-II.

Structural determination and characterization of a 40 kDa protein isolated from rat 40 S ribosomal subunit.

We have purified a 40 kDa protein from the rat 40 S ribosomal subunit and determined its primary structure by amino acid and cDNA sequencing. The amino acid sequence of the 40 kDa protein shared 29-37% homology with prokaryotic ribosomal protein S2 of eubacteria and chloroplasts, indicating that the protein is a eukaryotic counterpart to prokaryotic S2. Moreover, the amino acid sequence shared 99% identity with those deduced from cDNAs for 68 kDa laminin binding proteins of human, murine and bovine origins. The cDNAs are capable of encoding polypeptides with predicted molecular mass of 33,000 which lacked typical signal sequences, N-linked glycosylation sites and putative transmembrane domains. These results indicate that the cDNAs for 68 kDa laminin binding proteins actually code for the 40 kDa ribosomal protein.

Physiological correlation between glycyrrhizin, glycyrrhizin-binding lipoxygenase and casein kinase II.

By means of glycyrrhizin (GL)-affinity column chromatography, a GL-binding lipoxygenase (gbLOX) was selectively purified from the partially purified soybean LOX-1 fraction. Polypeptide analysis of the purified gbLOX by SDS-PAGE detected two distinct polypeptides (p96 and p94), which were identical to LOX-3 as determined by their partial N-terminal amino acid sequences. Moreover, it was found that (i) phosphorylation of gpLOX by casein kinase II (CK-II) is significantly stimulated by 3 microM GL, but inhibited by 30 microM GL or 10 microM oGA; and (ii) gbLOX activity is enhanced when the enzyme is phosphorylated by CK-II in the presence of 3 microM GL. These results suggest that (i) CK-II is a kinase responsible for the activation of gbLOX through its specific phosphorylation; and (ii) GL is one of the regulatory substances for specific phosphorylation of gbLOX (LOX-3) by CK-II in plant cells.

Isolation and characterization of a sulfated glycoprotein from rabbit small intestine.

Complex saccharides were extracted with 0.9% NaCl at 0 degrees C from rabbit small intestine, then fractionated with cetylpryridinium chloride (CPC), followed by DEAE-Sephadex A-25 column chromatography and by GEON-zone electrophoresis. An acidic glycoprotein (Fr. A-1) thus obtained was shown to be homogeneous by electrophoreses on cellulose acetate membrane and on sodium dodecyl sulfate (SDS)-polyacrylamide gel, as well as by gel filtration on Sepharose 4B. It contained 40.0% protein, 52.7% carbohydrate, and 1.6% sulfate. The principal sugars in the sulfated glycoprotein were galactose, glucosamine, galactosamine, and sialic acid. Small quantities of mannose, L-fucose, and glucose were also present. Glutamic acid, threonine, aspartic acid, alanine, serine, leucine, proline, glycine, valine, and histidine were the major amino acids of the protein moiety. Small amounts of other basic amino acids, sulfur-containing amino acids and aromatic amino acids were also present.

Isolation and characterization of sulfated glycoproteins from the brush border fraction and the soluble fraction of rabbit small intestine.

The brush border fraction (Fr. P2) and the calcium chloride (10 mM)-soluble fraction (Fr. S) were separated from the small intestinal mucosa of rabbit. Sulfated glycoproteins, P-SGP and S-SGP, were then purified from Fr. P2 and Fr. S by zone electrophoresis and gel filtration, respectively. P-SGP and S-SGP were both electrophoretically homogeneous, although their mobilities differed from each other. P-SGP and S-SGP contained 61.8 and 35.% protein, 31.8 and 55.0% carbohdyrate, and 1.1 and 2.7% sulfate, respectively. The major constituent sugars in P-SGP and S-SGP were galactose and glucosamine, while mannose, sialic acid, and L-fucose were minor components. In addition, galactosamine was a major sugar in S-SGP, but a minor one in P-SGP. The major amino acids of the protein moiety of P-SGP were glutamic acid, aspartic acid, glycine, and alanine, while those of S-SGP were threonine, proline, glutamic acid, aspartic acid, serine, and glycine.

Hormonal effects on the sulfation of sulfated glycoprotein in a particulate fraction of the endometrium of rabbit uterus.

A particulate fraction was separated from endometrial scrapings of the uterus of hormone- or sham-treated ovariectomized rabbit. The effects of estrogen and progesterone on the incorporation of [35S]sulfate from 3'-[35S]phosphoadenosine 5'-phosphosulfate (PAPS) into endogenous acceptors in the particulate fraction were investigated. Estrogen increased the incorporation of [35S]sulfate, but progesterone suppressed this effect. The results of DEAE-Sephadex A-25 (Cl-form) column chromatography of the pronase digest of the 35S-labeled substances indicated that the fraction eluted with 0.9 M NaCl (0.9 Fr) was most sensitive to the hormones. The major component in 0.9 Fr was resistant to crude heparinase, whereas the minor component was susceptible to this enzyme. The present observation, together with previous findings, suggested that the former was sulfated glycopeptide and the latter heparan sulfate. The results of the present study indicated that PAPS can serve as the direct "activated" sulfate donor in the enzymatic sulfation of sulfated glycoprotein in the particulate fraction, that the sulfation is greatly stimulated by pre-treatment of the rabbit with estrogen, and that the estrogen effect is suppressed by progesterone.

Hormonal effects on the biosynthesis of sulfated glycoprotein in a microsomal fraction of the endometrium of rabbit uterus.

A crude microsomal fraction (M-Fr) was separated from the endometrial scrapings of uteri of ovariectomized rabbits with or without hormonal treatment. The effects of estrogen and progesterone on the incorporation into M-Fr of L-[U-14C]-fucose and N-acetyl-D-[6-3H]-glucosamine from their nucleotides were investigated. Estrogen increased the incorporation of these sugars, whereas progesterone suppressed this effect. The results of fractionation on a DEAE-Sephadex A-25 (Cl- form) column of the isotope-labelled complex saccharide mixtures, obtained by pronase digestion of the incubation mixtures, indicated that biosynthesis of sulfated glycoprotein was most sensitive to the hormones among the complex saccharides in M-Fr. Thus, a hormonal effects on the biosynthesis of sulfated glycoprotein in the endometrium of ovariectomized rabbit has been unambiguously confirmed at the microsomal level.

Purification and structure of rat erythroid-specific delta-aminolevulinate synthase.

The existence of erythroid form delta-aminolevulinate synthase (ALAS-E) was historically a matter of some controversy. To obtain direct evidence for a unique ALAS-E, we have purified ALAS-E to homogeneity for the first time, from rat reticulocyte lysate. The papain digestion method was used at the initial step of the purification to overcome the difficulty which repeatedly hampered earlier attempts to purify ALAS-E. The size of the purified papain-resistant core catalytic domain of ALAS-E was estimated electrophoretically to be 49,000 Da. The pH optimum (7.6) and apparent Km values for the substrates, glycine (6.5 mM) and succinyl-CoA (2 microM), were similar to those of the non-specific form of delta-aminolevulinate synthase (ALAS-N); but, in contrast to ALAS-N, the substrate inhibition by succinyl-CoA was not evident in ALAS-E. We then isolated cDNA and genomic DNA clones encoding rat ALAS-E. By combining the nucleotide sequence information of the cDNA and genomic clones, the rat ALAS-E precursor is predicted to be composed of 587 amino acids with a calculated molecular mass of 64,841 Da. All the peptide sequences determined directly from the purified protein agreed with those predicted from the nucleotide data, demonstrating the existence of ALAS-E. Analysis of the papain-resistant core domain further revealed that it overlaps with the evolutionally conserved segment that has been noticed by sequence alignment analysis of ALA synthases from various species.