Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.     

ITS1 variation among Stichopathes cf. maldivensis (Hexacorallia: Antipatharia) whip black corals unveils conspecificity and population connectivity at local and global scales across the Indo-Pacific

Coral Reefs - Due to the dearth of molecular markers variable enough to distinguish species of black corals, species delimitation in Antipatharia is still mainly based on morphological traits. One...     

Gluten-free diet impact on leptin levels in asymptomatic coeliac adolescents: one year of follow-up

Coeliac disease, daily more frequently diagnosed in our population, involves many organs also in oligosymptomatic patients and with an adequate nutritional regime. Possible endocrine implications include failure to thrive, pubertal delay and reproduction diseases due to deregulation of GH, FSH and LH secretion. Leptin, an adipose tissue hormone, can be decreased as well and its deficiency could be related to growth and puberty anomalies. We studied 14 asymptomatic coeliac patients in peripubertal age (7.5-13.8 years) and tested their leptin levels in order to correlate them with endocrine and anthropometric data. Before the diet was started leptinaemia (M+/-DS) was: 4.94+/-5.53 ng/ml. In 10/14 patients (71%) leptinaemia was相似文献   

Analysis of F9 embryonal carcinoma cell lactosaminoglycans in relation to their differential expression during induction of differentiation

Teratocarcinoma stem cells can be used to study the events related to early differentiation, and many cell surface changes have been described which correlate with the different stages of early embryogenesis. In this work we analyze the [3H]galactose-labeled glycopeptides derived from the mouse embryonal carcinoma cell line F9. We show that the high-molecular-weight glycopeptides typical of embryonal carcinoma cells are composed of two distinct molecular weight classes, namely H1 and H3, and that retinoic acid-induced differentiation determines a relative increase of the larger peak (H1) which is mainly due to a decrease in the expression of H3 species. We also show that, beside this decrease, there is a greater increase in the expression of lower-molecular-weight species. Furthermore, we present evidence that H1 and H3 species are polylactosaminoglycans N-linked to the peptidic backbone, and that induction of differentiation determines slight modifications in the structure of such species.     

Identification of a novel glycopeptide species that correlates with differentiation of F9 cells

The high-molecular-weight fucosyl glycopeptides of differentiated F9 cells have been analyzed. We found that these high-molecular-weight surface structures contain two components with different molecular weights, the largest of which, peak I, has never before been reported. The material eluting in this peak seems to contain only acidic species. Removal of sialic acid from both the peak I and the peak II species does not eliminate the differences in molecular weight, indicating that the two species have more profound structural differences than can be accounted for by sialic acid. Since peak I glycopeptides were found both in differentiated F9 cells and in two parietal endoderm cell lines, we suggest that its presence is related to parietal endoderm differentiation.     

Requirement of a cell division step for stalk formation in Caulobacter crescentus.

Penicillin G at low concentrations blocked cell division in Caulobacter crescentus without inhibiting cell growth. The long filamentous cells formed after two to three generations under these conditions had a stalk at one pole and usually one or more flagella at the opposite pole. The failure of the filaments to form a second stalk at the flagellated pole indicates that stalk formation was dependent upon completion of a step that was also required for cell division. Two observations support this conclusion. (i) Penicillin did not stop the normal development of synchronous swarmer cells into stalked initiation and stalk elongation. (ii) When the action of penicillin was reversed by the addition of penicillinase to cultures of filaments, stalks were not formed at the nonstalked pole until after cell division had occurred; thus the normal order of development events was maintained: cell division leads to stalk formation. These results are consistent with a model in which the organization of the developmental program for stalk formation occurs before cell division as a consequence of steps that branch from the cell division pathway.     

High-yield and high-degree purification of human alpha-fetoprotein produced by adaptation of the human hepatoma cell line Hep G2 in a serum-free medium

The human hepatoma cell line Hep G2 secretes both albumin and alpha-fetoprotein when grown in the presence of serum. The present report describes how adaptation to growth in serum-free medium results in a progressive switch in the expression of the two proteins; i.e., alpha-fetoprotein becomes the main protein secreted while albumin production is greatly reduced. The culture supernatant obtained, being very enriched in the protein, allows the development of a purification procedure by preparative electrophoresis. By this procedure it is possible to easily obtain large amounts of alpha-fetoprotein from a constant and unlimited source. The availability of these protein preparations should improve the reproducibility and the quality of standardization in clinical immunoassays for alpha-fetoprotein and should permit a more accurate study of the structure and biological functions of the protein.     

Pattern of unequal cell division and development in Caulobacter crescentus

The pattern of asymmetric division has been examined in Caulobacter crescentus (gram-negative aquatic bacteria) by determining the position of the “division site” on cells of different ages. Measurements of cell width and length at this site, which corresponds to the point of eventual cell separation, were made on electron micrographs of cells stained with phosphotungstic acid. The results show that (i) the division site is formed early in the cell cycle and it constitutes the first visible feature on the growing stalked cell to differentiate the incipient swarmer cell, (ii) the division site is located asymmetrically (closer to the swarmer pole than the stalked pole) on the dividing cell, (iii) its position relative to the stalked and swarmer poles does not change during the cell cycle, and (iv) division is consequently unequal, with the swarmer cell always smaller than the stalked cell. The implications of these findings for general models of unequal cell division and stem cell development are discussed.     

Synthesis and characterization of a recombinant fragment of human alpha-fetoprotein with antigenic selectivity versus albumin

A DNA sequence coding for human alpha-fetoprotein amino acid sequence 38-119 was synthesized and cloned in a bacterial expression vector. The alpha-fetoprotein sequence was selected as the least homologous to albumin, since the two proteins have an overall amino acid identity of approximately 38%. A chimeric protein was obtained which was purified by preparative electrophoresis and characterized in its primary structure by fast atom bombardment mass spectometry. About 70% of the alpha-fetoprotein sequence was physically mapped and found to correspond to the amino acids encoded in the synthetic gene. The use of this recombinant protein allowed the selection of monoclonal antibodies recognizing both the recombinant fragment and native alpha-fetoprotein. These antibodies should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.