gamma -Glutamyl carboxylation: An extracellular posttranslational modification that antedates the divergence of molluscs, arthropods, and chordates

The posttranslational gamma-carboxylation of glutamate residues in secreted proteins to gamma-carboxyglutamate is carried out by the vitamin K-dependent enzyme gamma-glutamyl carboxylase. gamma-Carboxylation has long been thought to be a biochemical specialization of vertebrates, essential for blood clotting. Recently, a gamma-carboxylase was shown to be expressed in Drosophila, although its function remains undefined in this organism. We have characterized both cDNA and genomic clones for the gamma-glutamyl carboxylase from the marine mollusc, Conus, the only nonvertebrate organism for which gamma-carboxyglutamate-containing proteins have been biochemically and physiologically characterized. The predicted amino acid sequence has a high degree of sequence similarity to the Drosophila and vertebrate enzymes. Although gamma-carboxylases are highly conserved, the Conus and mammalian enzymes have divergent substrate specificity. There are striking parallels in the gene organization of Conus and human gamma-carboxylases. Of the 10 Conus introns identified, 8 are in precisely the same position as the corresponding introns in the human enzyme. This remarkable conservation of intron/exon boundaries reveals that an intron-rich gamma-carboxylase was present early in the evolution of the animal phyla; although specialized adaptations in mammals and molluscs that require this extracellular modification have been identified, the ancestral function(s) and wider biological roles of gamma-carboxylation still need to be defined. The data raise the possibility that most introns in the genes of both mammals and molluscs antedate the divergence of these phyla.     

The Usefulness of Transesophageal Echocardiography in the Surgical Management of Older Children with Subaortic Stenosis

Subaortic stenosis is a complex lesion that often presents in older children and adolescents. A clear depiction of the lesion is required for optimization of surgery. Due to the large size of these patients, is not always possible from surface echocardiography. Intraoperative multiplane echocardiography (MTEE) has been performed at our institute in older children for several different congenital heart lesions including many patients with subaortic stenosis. A retrospective analysis of our experience with MTEE in patients with subaortic stenosis was performed to assess its usefulness in the preoperative diagnosis and postoperative assessment of repair. Our results show that intraoperative MTEE was useful preoperatively by correcting or confirming suspected diagnosis, and giving additional details of the lesion in many patients. Postoperatively, MTEE was highly useful in the assessment of repair. We strongly recommend the use of intraoperative MTEE in older children and adolescents with subaortic stenosis.     

Circulating ensembles of tumor-associated cells: A redoubtable new systemic hallmark of cancer

Circulating ensembles of tumor-associated cells (C-ETACs) which comprise tumor emboli, immune cells and fibroblasts pose well-recognized risks of thrombosis and aggressive metastasis. However, the detection, prevalence and characterization of C-ETACs have been impaired due to methodological difficulties. Our findings show extensive pan-cancer prevalence of C-ETACs on a hitherto unreported scale in cancer patients and virtual undetectability in asymptomatic individuals. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of 16,134 subjects including 5,509 patients with epithelial malignancies in various organs and 10,625 asymptomatic individuals with age related higher cancer risk. PBMCs were treated with stabilizing reagents to protect and harvest apoptosis-resistant C-ETACs, which are defined as cell clusters comprising at least three EpCAM⁺ and CK⁺ cells irrespective of leucocyte common antigen (CD45) status. All asymptomatic individuals underwent screening investigations for malignancy including PAP smear, mammography, low-dose computed tomography, evaluation of cancer antigen 125, cancer antigen 19-9, alpha fetoprotein, carcinoembryonic antigen, prostate specific antigen (PSA) levels and clinical examination to identify healthy individuals with no indication of cancer. C-ETACs were detected in 4,944 (89.8%, 95% CI: 89.0–90.7%) out of 5,509 cases of cancer. C-ETACs were detected in 255 (3%, 95% CI: 2.7–3.4%) of the 8,493 individuals with no abnormal findings in screening. C-ETACs were detected in 137 (6.4%, 95% CI: 5.4–7.4%) of the 2,132 asymptomatic individuals with abnormal results in one or more screening tests. Our study shows that heterotypic C-ETACs are ubiquitous in epithelial cancers irrespective of radiological, metastatic or therapy status. C-ETACs thus qualify to be a systemic hallmark of cancer.     

THERMODYNAMIC PARAMETERS OF TRANSFER OF N-ACETYL ETHYL ESTERS OF DIFFERENT AMINO ACIDS FROM ORGANIC SOLVENTS TO WATER

The distribution coefficients of N-acetyl ethyl esters of glycine, diglycine, β-alanine, alanine, valine, norvaline, leucine and norleucine between water and different organic solvents have been measured at different temperatures. Similar distribution coefficients have been measured for simple amides, urea, formamide, acetamide and N-methyl acetamide. From the distribution measurements, ΔG_tr, the free energy of transfer of the solutes from organic solvents to water has been calculated. The temperature dependence of the distribution coefficient values has been utilised to determine the enthalpy of transfer, ΔH_tr, and entropy of transfer ΔS_tr for the above process. From these results similar thermodynamic parameters for the transfer of different nonpolar side chains and peptide groups have been determined and compared with the available data in the literature.     

Metabolism,Uptake, and Transepithelial Transport of the Diastereomers of Val-Val in the Human Intestinal Cell Line,Caco-2

Purpose. The purpose of this study was to determine whether the binding of the diastereomers of Val-Val to the apical oligopeptide transporter(s) could be correlated with their cellular uptake and transepithelial transport. Methods. The Caco-2 cell culture system was used for all experiments. The binding of the diastereomers of Val-Val was evaluated by determining their ability to inhibit [³H]cephalexin uptake. The stability of the diastereomers was determined in a homogenate of Caco-2 cells and in the apical bathing solution over Caco-2 cell monolayers. The cellular uptake and transepithelial transport properties of the individual diastereomers were studied using Caco-2 cell monolayers. Results. 10 mM concentrations of L-Val-L-Val, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val inhibited cellular uptake of [³H]cephalexin (0.1 mM) by 92%, 37%, 70%, and 18%, respectively. When the cellular uptake of Val-Val diastereomers (1 mM) were evaluated, the intracellular concentrations of L-Val-D-Val and D-Val-L-Val were 15 and 50 times higher, respectively, than that of D-Val-D-Val. The cellular uptake of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) (>95%), whereas Gly-Pro had no effect on the cellular uptake of D-Val-D-Val. L-Val-L-Val was not detected in the Caco-2 cells, probably due to its metabolic lability. When the transepithelial transport of the Val-Val diastereomers (1 mM) was determined, L-Val-D-Val, D-Val-L-Val and D-Val-D-Val transport rates were similar. The transepithelial transport of L-Val-D-Val and D-Val-L-Val was inhibited by Gly-Pro (10 mM) 36% and 30%, respectively, while Gly-Pro inhibited carnosine (1 mM) transepithelial transport by 65%. Gly-Pro had no effect on the transepithelial transport of D-Val-D-Val. Conclusions. These results suggest that the major transepithelial transport route of L-Val-D-Val, D-Val-L-Val and D-Val-D-Val is passive diffusion via the paracellular route. The binding of Val-Val diastereomers to the oligopeptide transporter(s) is a good predictor of their cellular uptake, however, the binding is not a good predictor of their transepithelial transport. It appears that the stereochemical requirements for the transporter that mediates efflux of the peptide across the basolateral membrane may be different from the requirements for the apical transporter that mediates cellular uptake.