Structure of cDNA of cytosolic aspartate aminotransferase of chicken and its expression in E. coli.

The structure of the mRNA of chicken cytosolic aspartate aminotransferase has been determined by analysis of cDNA and genomic clones. Two transcripts of different length were found that appear to arise from the alternate use of 2 polyadenylation signals in the 3' untranslated region. The expression product of the full-length construct in E. coli proved to be catalytically active and possessed the expected molecular weight.

Evolutionary and biosynthetic aspects of aspartate aminotransferase isoenzymes and other aminotransferases.

The mitochondrial and cytosolic isoenzymes of aspartate aminotransferase are homologous proteins. Both are encoded by nuclear DNA and synthesized on free polysomes. The organization of their genes is very similar, five out of a total of eight introns are located at the same nucleotide position. A variant consensus sequence was observed at the 3' splice site of introns of genes of imported mitochondrial proteins which may reflect the existence of splicing factors specific for the genes of this particular group of nuclear-encoded proteins. To date the amino acid sequences of 22 aminotransferases are known. A rigorous analysis yielded clear evidence that aspartate, tyrosine, and histidinol-phosphate aminotransferases are homologous proteins despite their low degree of sequence identity. The evolutionary relationship among the vitamin B6-dependent enzymes in general appears less clear. Conceivably, their common structural and mechanistic features are dictated by the chemical properties of pyridoxal 5'-phosphate rather than being due to a common ancestor of their protein moieties. In agreement with this notion, the ubiquitous active-site lysine residue that forms a Schiff base with the coenzyme can be replaced in the case of aspartate aminotransferase by a histidine residue without complete loss of catalytic competence.

[Diagnosis of colorectal carcinoma]. / Diagnostika kolorektálneho karcinómu.

The authors analyzed a group of 207 patients operated on for colorectal carcinoma at the 1st Department of Surgery, Medical Faculty of Comenius University, in the years 1994-1998. They detected a high number of patients in stages C and D according to Dukes' classification--53.1% and a high percentage of patients with liver metastases (42%). The number of urgent operations in this five year follow-up increases gradually, which signalizes no improvement in early diagnostics of colorectal carcinoma. The first symptom of this disease was in 37% of cases bowel obstruction (ileus) or another complication of the underlying disease. It is clear from this analysis, that the early diagnostics of colorectal carcinoma is not improving. The authors also analyse the possibilities of improving this situation and the new possibilities of early diagnostics of colorectal carcinoma. (Tab. 2, Ref. 13.)

The abdominal compartment syndrome.

The abdominal compartment syndrome has received considerable attention only recently. It may be defined as adverse physiologic consequences that occur as a result of an acute increase in the intraabdominal pressure. The most common causes of ACS are haemorrhage, visceral oedema, pancreatitis, bowel distension, venous mesenterial obstruction, abdominal packs, tense ascites, peritonitis, tumor. The mostly affected organ systems include cardiovascular, pulmonary, renal, central nervous and splanchnic. The diagnosis depends on the recognition of the clinical syndrome followed by an objective measurement of intraabdominal pressure, preferably that of the urinary bladder. The treatment consists of adequate fluid resuscitation and surgical decompression when necessary. (Tab. 1, Ref. 29.).

[Reconstruction of the gastrointestinal tract after esophagogastrectomy]. / Rekonstrukcia gastrointestinálneho traktu po ezofágogastrektómii.

The authors deal with the problems of reconstruction of gastrointestinal continuity after esophagectomy. They present the advantages of the stomach which is an especially good mediastinal as well as retrosternal substituent. One case of restoration of gastrointestinal continuity after both esophagectomy and gastrectomy by a jejunal loop with anastomosis on the neck is also presented.

[Laparoscopic vagotomy in the treatment of recurrent duodenal ulcer]. / Laparoskopická vagotómia v liecbe recidivujúceho ulkusu duodéna.

The paper represents our results of laparoscopic vagotomies. In 1993 was the first successful laparoscopic vagotomy in Slovakia performed, at the 1st Department of Surgery, Faculty Hospital, Bratislava. From this time 10 operations with front superselective and dorsal truncal vagotomy were performed. Effectiveness of vagotomy was controlled after 12 months by examination of the gastric acidity. Decrease of gastric acidity in average above 61% was reached. Laparoscopic vagotomy, despite dominant conservative treatment of peptic ulcer, is the method of choice, if the conservative treatment is unsuccessful. (Fig. 3, Ref. 6.)

[Inflammatory complications in reconstructive surgery after esophagectomy]. / Zápalové komplikácie rekonstrukcných operáctí po ezofágektómii.

The authors evaluated in their retrospective study the portion of inflammatory complications on morbidity and mortality at restorative operations after oesophagectomies, performed at Ist Department of Surgery of the Teaching Hospital of Commenius University in Bratislava in the year of 1975-1998. The inflammatory complications still remain a serious problem after oesophagectomy with a great portion on postoperative mortality.

[Alternative use of the EEA stapler in subtotal esophagectomy]. / Alternatívna aplikácia EEA staplera po subtotálnej ezofágektómii.

Authors describe the successful usage of EEA stapling by reconstruction after subtotal oesophagectomy of carcinoma in the middle third of oesophagus. EEA stapling was pushed in transorally and oesophagogastric anastomosis was made. Neither pre- or post-operative complications occurred by this procedure. Patient left the hospital on 14th day afer the operation. Authors consider transoral application of EEA stapling as effective alternative in creation of oesophagogastric anastomosis after subtotal oesophagectomy.

[Gastroduodenal hemorrhage]. / Súcasná problematika krvácania z gastroduodena.

Haemorrhage is a serious complication of peptic ulcer disease. It is an indication for urgent diagnostic and therapeutic endoscopy, which is at present the method of first choice. All patients with gastroduodenal peptic ulcer bleeding, who underwent endoscopy in Ist Department of Surgery in Bratislava between January 1995 and December 1999, were considered for retrospective study. A total of 291 patients (195 male and 96 female) underwent urgent endoscopy with a finding in 34.7% of patients with gastric ulcer and in 65.3% patients duodenal ulcer. The finding was Forrest I in 23%, Forrest II in 25.7% and Forrest III in 51.3% patients. Endoscopic hemostasis was used in 12.37% of patients. A first haemorrhage was found in 82.9% patients, a recurrent one in 17.1% patients. 41.5% of patients had positive peptic ulcer history. Surgical treatment was indicated in cases, when bleeding was not controlled by endoscopic means, or in cases of recurrent bleeding within 48 hours in 19 patients (6.52%).

Triple arginines in the cytoplasmic tail of endomannosidase are not essential for type II membrane topology and Golgi localization.

Endomannosidase is a Golgi-localized endoglycosidase, which provides an alternate glucosidase-independent pathway of glucose trimming. Using a protease protection assay we demonstrated that Golgi-endomannosidase is a type II membrane protein. The first 25 amino acids of this protein, containing the cytoplasmic tail and the transmembrane domain, were sufficient for Golgi retention of fused reporter proteins alpha1-antitrypsin or green fluorescent protein. However, shortening or deletion of the transmembrane domain prevented Golgi localization, while lengthening it partially reduced Golgi retention of the enzyme. Substitution of the highly conserved positively charged amino acids within the cytoplasmic tail had neither an effect on type II topology nor on the inherent Golgi localization of the enzyme. In contrast, cytoplasmic tail-deleted rat endomannosidase possessed an inverted topology resulting in endoplasmic reticulum mislocalization. Thus, proper topology rather than the presence of positively charged amino acids in the cytoplasmic tail is critical for Golgi localization of rat endomannosidase.

A single tryptophan residue of endomannosidase is crucial for Golgi localization and in vivo activity.

Golgi-endomannosidase provides an alternate glucosidase-independent pathway of glucose trimming. Activity for endomannosidase is detectable in various tissues and cell lines but not in CHO cells. Cloning of CHO cell endomannosidase revealed that the highly conserved Trp188 and Arg177 of vertebrate endomannosidase were both substituted by Cys. The Trp188Cys substitution was functionally important since it alone resulted in endoplasmic reticulum (ER) mislocalization of endomannosidase and caused the greatly reduced in vivo activity. These effects could be reversed in cells with a back-engineered Cys188Trp CHO cell endomannosidase, in particular N-glycans of alpha1-antitrypsin became fully processed. The intramolecular disulfide bridge of CHO cell endomannosidase formed with the additional Cys188 was not solely responsible for the reduced enzyme activity since endomannosidase with engineered Cys188Ala or Ser substitutions did not restore enzyme activity and was ER mislocalized. Thus, the conserved Trp188 residue in endomannosidase is of critical importance for correct subcellular localization and in vivo activity of the enzyme.

Endomannosidase processes oligosaccharides of alpha1-antitrypsin and its naturally occurring genetic variants in the Golgi apparatus.

Endomannosidase provides an alternate glucose-trimming pathway in the Golgi apparatus. However, it is unknown if the action of endomannosidase is dependent on the conformation of the substrate. We have investigated the processing by endomannosidase of the alpha1-antitrypsin oligosaccharides and its disease-causing misfolded Z and Hong Kong variants. Oligosaccharides of wild-type and misfolded alpha1-antitrypsin expressed in castanospermine-treated hepatocytes or glucosidase II-deficient Phar 2.7 cells were selectively processed by endomannosidase and subsequently converted to complex type oligosaccharides as indicated by Endo H resistance and PNGase F sensitivity. Overexpression of endomannosidase in castanospermine-treated hepatocytes resulted in processing of all oligosaccharides of wild-type and variants of alpha1-antitrypsin. Thus, endomannosidase does not discriminate the folding state of the substrate and provides a back-up mechanism for completion of N-glycosylation of endoplasmic reticulum-escaped glucosylated glycoproteins. For exported misfolded glycoproteins, this would provide a pathway for the formation of mature oligosaccharides important for their proper trafficking and correct functioning.

Expression of oligo/polyalpha2,8-linked deaminoneuraminic acid and megalin during kidney development and maturation: mutually exclusive distribution with polyalpha2,8-linked N-acetylneuraminic acid of N-CAM.

The expression of homopolymers of alpha2,8-linked deaminoneuraminic acid (oligo/polyalpha2,8-KDN) and of megalin during rat kidney development was investigated using immunocytochemistry and immunoblotting, and compared to homopolymers of alpha2,8-linked N-acetylneuraminic acid (polyalpha2,8-Neu5Ac) of the neural cell adhesion molecule (N-CAM). Both, oligo/polyalpha2,8-KDN and megalin were found in early proximal tubules of embryonic day 18 kidneys. In addition, megalin, but not oligo/polyalpha2,8-KDN, was detectable in late S-shaped bodies and early capillary loop stages. Until postnatal day 7, oligo/polyalpha2,8-KDN and megalin immunoreactivity was present, not only in convoluted but also in straight proximal tubules, and then restricted to the convoluted part as in adult kidney. Immunoblotting revealed increasing megalin expression until postnatal week 3 of kidney development, when the level corresponded to adult kidney. Combined immunoprecipitation/immunoblot analyses showed a steady level of oligo/polyalpha2,8-KDN on megalin throughout development. This was in striking contrast to the expression of polyalpha2,8-Neu5Ac and N-CAM, which was highest in early embryonic kidney, undetectable in kidneys of 3-week-old rats, and mutually exclusive with oligo/polyalpha2,8-KDN in its distribution. These findings demonstrated the coincidence of oligo/polyalpha2,8-KDN and megalin expression and the first appearance of proximal tubules, and revealed the high degree of specialization of the biosynthetic machinery for protein polysialylation in kidney.

Unconventional antigen retrieval for carbohydrate and protein antigens.

Aldehyde fixation of tissues often adversely affects the reactivity of cellular proteins with antibodies. A most commonly used retrieval technique in immunohistochemistry is high-temperature microwave heating of sections from formaldehyde-fixed and paraffin-embedded tissues. Here we report that pretreatment of paraffin and ultrathin cryosections with N-glycanase F to remove N-glycosidically linked oligosaccharides can result in a dramatic increase in specificity and intensity of immunogold labeling for sugar moieties present on O-glycosidically linked oligosaccharides. This is demonstrated in the immunolocalization of poly alpha2,8 KDN (KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) of megalin in rat kidney. The mechanism of this retrieval procedure is most probably based on the elimination of sterical hindrance by large N-glycosidically linked oligosaccharides. Furthermore, we demonstrate that exposure of ultrathin cryosections to acidic conditions (pH 5.5) at ambient temperature prior to immunogold labeling can result in an increased labeling intensity. This effect was observed for megalin immunoreactive sites in proximal tubular epithelia of rat kidney. It is proposed that the mechanism of this retrieval procedure is based on the depolymerization of methylen and polymethylen bridges introduced by formaldehyde in the acidic milieu.

Megalin in normal tissues and carcinoma cells carries oligo/poly alpha2,8 deaminoneuraminic acid as a unique posttranslational modification.

In rat kidney, megalin, a member of the low density lipoprotein receptor gene family, is the sole glycoprotein which carries oligo/poly alpha2,8 deaminoneuraminic acid (KDN) as a posttranslational modification. We have investigated immunoprecipitated megalin from rat brain, lung and placenta, mouse yolk sac carcinoma and megalin synthesizing carcinoma cell lines, for presence of this unique glycan structure. Our immunoblot analysis revealed the presence of oligo/poly alpha2,8 KDN on megalin in all the studied normal tissues and carcinoma cells. Furthermore, it is demonstrated to be part of oligosaccharides O-glycosidically linked to megalin.

Expression of a cDNA encoding the glucose trimming enzyme glucosidase II in CHO cells and molecular characterization of the enzyme deficiency in a mutant mouse lymphoma cell line.

Glucosidase II is an ER resident glycoprotein involved in the processing of N-linked glycans and probably a component of the ER quality control of glycoproteins. For cloning of glucosidase II cDNA, degenerate oligonucleotides based on amino acid sequences derived from proteolytic fragments of purified pig liver glucosidase II were used. An unamplified cDNA library from pig liver was screened with a 760 bp glucosidase II specific cDNA fragment obtained by RT-PCR. A 3.9 kb glucosidase II cDNA with an open reading frame of about 2.9 kb was obtained. The glucosidase II sequence did not contain known ER retention signals nor hydrophobic regions which could represent a transmembrane domain; however, it contained a single N-glycosylation site close to the amino terminus. All studied pig and rat tissues exhibited an mRNA of approximately 4.4 kb with varying tissue expression levels. The authenticity of the identified cDNA with that coding for glucosidase II was proven by overexpression in CHO cells. Mouse lymphoma PHAR 2.7 cells, deficient in glucosidase II activity, were shown to be devoid of transcripts.

Poly (alpha 2,8-deaminoneuraminic acid) is expressed in lung on a single 150-kDa glycoprotein and is an oncodevelopmental antigen.

Homopolymers of alpha 2,8-linked N-acetylneuraminic acid [poly(alpha 2,8-Neu5Ac)] of the neural cell adhesion molecule NCAM have been shown to be temporally expressed during lung development and represent a marker for small cell lung carcinoma. We report the presence of a further polysialic acid in lung that consists of oligo/polymers of alpha 2,8-linked deaminoneuraminic acid residues [poly (alpha 2,8-KDN)], as detected with a monoclonal antibody in conjunction with a specific sialidase. Although the various cell types forming the bronchi, alveolar septs, and blood vessels were positive for poly (alpha 2,8-KDN) by immunohistochemistry, this polysialic acid was found on a single 150-kDa glycoprotein by immunoblot analysis. The poly(alpha 2,8-KDN)-bearing glycoprotein was not related to an NCAM protein based on immunochemical criteria. The expression of the poly (alpha 2,8-KDN) was developmentally regulated as evidenced by its gradual disappearance in the rat lung parenchyma commencing 1 week after birth. In adult lung the blood vessel endothelia and the smooth muscle fibers of both blood vessels and bronchi were positive but not the bronchial and alveolar epithelium. The poly (alpha 2,8-KDN)-bearing 150-kDa glycoprotein became reexpressed in various histological types of lung carcinomas and cell lines derived from them and represents a new oncodevelopmental antigen in lung.