Human plasma carboxypeptidase N. Isolation and characterization.

Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield. Isolation was accomplished by chromatography on DEAE-cellulose and on a p-aminobenzoyl-L-arginine-Sepharose 6B affinity column. Carbohydrate accounts for 17% of the weight calculated from its amino acid and carbohydrate composition. The enzyme appears to consist of three subunits of Mr = 83,000, 55,000, and 49,000 and contains a significant amount of bound zinc. Purified enzyme preparations are very sensitive to proteolytic degradation but are stable for at least 3 months at 4 degrees.     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Covalent structure of fibrinopeptides from buffaloes breeding in Italy.

The primary structure of fibrinopeptides A and B from buffaloes breeding in Italy has been determined with a view to establishing whether this animal is an autochthonous species or is the result of recent radiation mutation. Some differences exist at the morphologic and physiologic levels between the Indian buffalo and that breeding in Italy, But they do not allow a clear evolutionary line to be traced between these species. The amino acid sequences, if compared with homologous sequences of the Indian buffalo, show one difference in the most variable region of fibrino-peptides A, in particular the substitution of a serine residue by a glycine residue in position 8. This difference supports the hypothesis of the autochthonous origin of the Italian buffalo.     

Soybean hydrophobic protein. Isolation, partial characterization and the complete primary structure

A 9000-Mr protein isolated from a 60% ethanolic extract of soybean (Glycine max) seeds has been characterized and fully sequenced. The protein consists of 80 amino acid residues with four disulfide bonds. It contains a large number of hydrophobic residues and lacks methionine, phenylalanine, tryptophan, lysine and histidine residues. The protein readily crystallizes from water but is quite soluble in aqueous organic solvents like 95% 1-propanol. It aggregates to form large molecules (above 80 kDa) under ordinary denaturing conditions, such as 6 M guanidine X HCl and 8 M urea. Sequence analysis showed that the amino-terminal four-fifths is extremely hydrophobic and most of the acidic residues exist as their amide forms, and only the carboxyl-terminal short segment is rather hydrophilic. A computer search for homology detected an unexpected similarity of this protein to rat prolactin; however, its significance could not be assessed and this protein appears to represent a hitherto unknown protein family. Although no biochemical activity could be detected, the existence in relatively high abundance (approx. 200 mg from 1 kg seeds) of this novel protein may suggest its physiological significance in the plant.     

Human microvessel endothelial cells: Isolation,culture and characterization

Summary Over recent years, interest in endothelial cell biology has increased dramatically with our ability to grow and study endothelial cellsin vitro. While large veins and arteries remain a quick and convenient source of endothelial cells, the great morphological, biochemical and functional heterogeneity that endothelial cells express has necessitated the development of techniques to isolate microvessel endothelial cells from different tissues to create more realisticin vitro models. The majority of isolation procedures employ selective methods to enrich microvessel endothelial cells from tissue homogenates directly, or after a period in culture. These include sieving/filtration, manual weeding, isopycnic centrifugation, selective growth media, and the use of flow cytometry or magnetic beads coupled with specific endothelial cell markers. The establishment of pure endothelial cell populations is important for studying their biochemistry and physiology and there are many morphological, immunological and biochemical criteria which can be used to characterize human endothelial cells. These range from classical markers such as von Willebrand Factor and angiotensin-converting enzyme to novel markers like platelet endothelial cell adhesion molecule-1 (CD31) and the expression of E-selectin on cytokine-activated endothelial cells.     

Human phosphoserine 31 corticotropin1-39. Isolation and characterization

Two distinct forms of corticotropin1-39 (ACTH) were isolated and purified from an extract of three adult human pituitaries by reversed-phase chromatographic techniques. Structural studies indicated that the more polar form of ACTH was phosphorylated at serine residue 31. Approximately 30% of the ACTH was found in the phosphorylated form. A similar proportion of phosphorylated ACTH was observed in extracts of three pituitaries from human fetuses of 15, 17, and 20 weeks gestation. Phosphorylated and nonphosphorylated human ACTH1-39 were found to be steroidogenically equipotent using both an isolated rat adrenal cell bioassay and a cultured human fetal adrenal cell bioassay.     

Human tissue kallikrein. II. Isolation and characterization of human salivary kallikrein

Human salivary kallikrein was isolated from saliva using affinity chromatography on aprotinin-Sepharose and anti-human urinary kallikrein IgG-Sepharose followed by ion exchange chromatography on DEAE-Sepharose. The enzyme preparation had a specific activity of 950 U/mg protein towards the synthetic substrate Ac-Phe-Arg-OEt, a specific biological activity of 2000 KE/mg protein (measured in the dog blood pressure assay) and 0.64 HMW-kininogen-U/mg, corresponding to the liberation of 679 micrograms bradykinin equivalents per mg enzyme per min from HMW-kininogen (using the rat uterus test). In sodium dodecyl sulfate gel electrophoresis one protein band corresponding to a molecular mass of 32 kDa was obtained. The amino-acid composition was determined and isoleucin was found as the only N-terminal residue. The bimolecular velocity constant for the inhibition by diisopropyl fluorophosphate was determined as 8 l x mol-1 x min-1. The dissociation constant Ki of the human salivary kallikrein-aprotinin complex was calculated to be 0.7 x 10(-10)M. The Km and Vmax values for the hydrolysis of the synthetic substrates Ac-Phe-Arg-OEt and D Val-Leu-Arg-Nan were determined. In the enzyme immunoassay for human urinary kallikrein parallel binding curves were obtained.     

Human pituitary thyrotropin. Isolation and characterization of the hormone and its subunits.

Procedures have been described for the isolation of highly purified thyrotropin form frozen or acetone-preserved glands or from side fractions of somatotropin isolation and for the separation of its alpha and beta subunits. The products have been characterized by terminal residue analyses, amino acid composition, carbohydrate content, disc electrophoresis, ultracentrifugation, and biological activity.     

Human gingival fibroblasts: Isolation,characterization, and evaluation of CD146 expression

Gingival fibroblasts (GFs) that exhibit adult stem cell-like characteristics are known as gingival mesenchymal stem cells (GMSCs). Specific mesenchymal stem cell (MSC) markers have not been identified to distinguish GMSCs from GFs. Recently, the cell surface molecule known as cluster of differentiation (CD) 146 has been identified as a potential MSC surface marker. In the present study, we investigated the differentiation potential of GMSCs based on CD146 expression.GFs were isolated by two techniques: tissue explants or enzymatic digestion. GFs were cultured and expanded then magnetically sorted according to CD146 expression. CD146^low and CD146^high cells were collected, expanded, and then tested for stem cell markers by flow cytometry as well as osteogenic and chondrogenic differentiation potential. The differentiation of these cells was analyzed after 21 days using histology, immunofluorescence, real-time quantitative PCR (qPCR), and glycosaminoglycan (GAG) to DNA ratio (GAG/DNA) assays. Positive histological staining indicated osteogenic differentiation of all groups regardless of the isolation techniques utilized. However, none of the groups demonstrated chondrogenic differentiation, confirmed by the lack of collagen type II in the extracellular matrix (ECM) of GF aggregates. Our data suggest that identification of gingival stem cells based solely on CD146 is not sufficient to properly carry out translational research using gingival fibroblasts for novel therapeutic methods of treating oral disease.     

Human placental calreticulin characterization of domain structure and post-translational modifications.

The domain organization and the post-translational modifications of human placenta calreticulin were analysed by MS in combination with proteolytic digestion. Prolonged treatment with trypsin, chymotrypsin, elastase, Staphylococcus aureus V8 protease, or proteinase K all led to a 6- to 7-kDa decrease in the molecular mass of calreticulin. The decrease was found to be due to cleavages in the region around residue 340. In addition, minor fragments resulting from secondary cleavages close to the N-terminus were observed, but no stable fragments of intermediate size were found. These results show that the C-domain of calreticulin is susceptible to proteolytic cleavage and that the N- and P-domains form a proteolytically stable tight association. A disulfide bridge between the first two cysteines was mapped in the N-domain, and the third cysteine was found in the reduced form. No post-translational modifications in the form of glycosylation or phosphorylation were found. A modified form of calreticulin lacking the C-terminal hexapeptide including the KDEL endoplasmic reticulum retention sequon was isolated. Such a truncation may point to a mechanism that allows escape of calreticulin from the endoplasmic reticulum.     

Isolation,characterization, and structure of the folded interphase genome of Drosophila melanogaster

The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% Nonidet P40. The nonviscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranucleosome histones: H2A, H2B, H3, and H4).The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs—that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26°C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres.Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA.The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA-DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360° left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.     

Turkey acrosin. I. Isolation, purification, and partial characterization

Acrosin was extracted from turkey spermatozoa by use of urea together with sonication and freezing, and purified approximately 18-fold by sequential use of chromatofocusing and affinity chromatography. The use of chromatofocusing for the initial purification step proved to be superior to preparative isoelectric focusing. Similar to acrosin from many mammalian species, turkey acrosin was found to be a glycoprotein possessing characteristics of serine proteases. Polyacrylamide gel electrophoresis (PAGE) of the enzyme indicated the presence of two isozymes. Sodium-dodecyl sulfate PAGE under reducing conditions revealed three subunits with approximate molecular weights of 11,700, 13,900, and 15,900.     

Isolation and initial characterization of a lymphocyte cap structure

A method for isolating the cap structure induced by polycationized ferritin on the surface of mouse T-lymphoma cells is described. The procedure, based on the 'density perturbation' approach designed by Wallach and co-workers (Wallach, D.F.H., Kranz, B., Ferber, E. and Fischer, H. (1972) FEBS Lett. 21, 29-33), involves a simple, one-step density gradient centrifugation using metrizamide as the gradient material. The isolated polycationized ferritin cap fraction is approx. 20-fold enriched in plasma membrane relative to the whole cell homogenate and is apparently free of all uncapped membrane. Our initial analysis of the protein composition of the isolated cap structure indicates that there are approx. 30 membrane-bound polypeptides specifically associated with the polycationized ferritin cap fraction. Interestingly, there are at least four phosphorylated membrane-bound polypeptides (mol.wt. approximately 130 000, 100 000, 30 000 and 20 000) which are preferrentially accumulated in the cap fraction. These findings provide further evidence for the selective redistribution of certain surface membrane proteins during lymphocyte capping.     

Human heparanase. Purification, characterization, cloning, and expression.

Heparan sulfate and heparan sulfate proteoglycans are present in the extracellular matrix as well as on the external cell surface. They bind various molecules such as growth factors and cytokines and modulate the biological functions of binding proteins. Heparan sulfate proteoglycans are also important structural components of the basement membrane. Heparanase is an endo-beta-D-glucuronidase capable of cleaving heparan sulfate and has been implicated in inflammation and tumor angiogenesis and metastasis. In this study, we report the purification of a human heparanase from an SV40-transformed embryonic fibroblast cell line WI38/VA13 by four sequential column chromatographies. The activity was measured by high speed gel permeation chromatography of the degradation products of fluorescein isothiocyanate-labeled heparan sulfate. The enzyme was purified to homogeneity, yielding a peptide with an apparent molecular mass of 50 kDa when analyzed by SDS-polyacrylamide gel electrophoresis. Using the amino acid sequences of the N-terminal and internal heparanase peptides, a cDNA coding for human heparanase was cloned. NIH3T3 and COS-7 cells stably transfected with pBK-CMV expression vectors containing the heparanase cDNA showed high heparanase activities. The homology search revealed that no homologous protein had been reported.     

Isolation, characterization, and analysis of Leymus-specific DNA sequences.

Genomic Southern hybridization using labeled total genomic DNA of Leymus mollis as probe showed intense hybridization signals on all restriction enzyme digested DNA from five species of Leymus Hochst., and four species of Psathyrostachys Nevski. Experiments using the same L. mollis probe, but with unlabeled blocking DNA from Psathyrostachys, showed no hybridization at all. These two genera evidently had the same genomic content. Southern hybridization without blocking allowed identification of DNA fragments abundant in Leymus and Psathyrostachys. Fragments potentially specific to Leymus were cloned. Five repetitive DNA clones from L. mollis and L. arenarius were characterized: pLmIs1, pLmIs44, pLmIs51, pLmIs53, and pLaIs56. These clones hybridized to both Leymus and Psathyrostachys on Southern blots - no clone hybridized to only one of these genera. Both Southern blot and fluorescence in situ hybridization (FISH) experiments showed that all the clones contained dispersed repetitive sequences. They painted all and whole chromosomes uniformly except at centromeres, telomeres, and nucleolar organiser regions. Three of these clones, i.e., pLmIs1, pLmIs44, and pLmIs53, were essentially specific to Leymus and Psathyrostachys - little or no hybridization was detected in other genera such as Triticum, Hordeum, Thinopyrum, or Elymus. Sequence analysis further revealed that the clones were part of retroelements. In particular, the clone pLmIs44 produced hybridization profiles suitable for analysis of genetic relatedness among species. The present study shows that Leymus and Psathyrostachys share the same basic genome, Ns, and therefore provides strong evidence for combining these two genera.     

Bovine alpha-fetoprotein. Isolation and characterization.

Bovine AFP was purified by ion exchange chromatograph on C.M. cellulose and DEAE Sephadex A-50, gel filtration and immunosorbent technique. AFP was homogeneous when studied by gel electrophoresis under non denaturing and denaturing conditions, by ultracentrifugation and by immunological methods. The following molecular data were obtained: 1. Sedimentation equilibrium indicated a molecular weight of 66,500 and sedimentation velocity gave s degrees 20, w = 4.71 S. A partial specific volume v = 0.737 ml g-1 was derived from density measurements. 2. From these data, a Stokes radius of 3.26 nm, a diffusion coefficient D20 w = 6.61 10(-7) cm2 sec-1 and a frictional ratio f/fo = 1.21 were calculated. 2. Sodium dodecylsulphate disc electrophoresis suggests a molecular weight of 67,000. 3. Gel filtration pointed to a molecular weight of 75,000. 4. Microheterogeneity of AFP was demonstrated by isoelectric focusing. The isoelectric point of the major component is 4.6. 5. The chemical composition was determined. AFP is a glycoprotein containing 7 per cent carbohydrate including 1.67 per cent hexoses, 2.38 per cent N-acetyl glucosamine and 1.8 per cent N-acetyl neuraminic acid.