Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.     

Multiple expression of keratins, vimentin, and S-100 protein in pleomorphic salivary adenomas.

Immunohistochemical staining for S-100 protein and the intermediate filaments keratin and vimentin, was made in 41 salivary adenomas. In pleomorphic adenomas, great heterogeneity in the staining, as well as multiple and co-expressions of these proteins were found in the outer tumor cells of tubulo-ductal structures and modified myoepithelial cells, but not in the luminal tumor cells. All the outer tumor cells stained for S-100 protein, 97% for K8.12 keratin and 85% for vimentin. Of these cells, 29% showed multiple expression of K8.12 keratin, vimentin, and S-100 protein, and 17% showed co-expression of K8.12 and S-100 protein. Modified and neoplastic myoepithelial cells showed similar expressions of these proteins to those of outer tumor cells; myoepithelioma cells displayed the most complicated pattern, being positive for KL1, PKK1, and K8.12 keratins, vimentin and S-100 protein. In luminal tumor cells there was a heterogeneous expression of KL1 and PKK1 in 82%, and of KL1, PKK1, and K8.12 in only 14.7%. Based on the immunohistochemical findings obtained with different monoclonal antibodies in pleomorphic salivary adenomas, outer tumor cells may be derived from ductal basal cells and luminal tumor cells from intercalated duct cells.     

Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl₃) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 g/ml, whereas a 10-fold excess of iron concentration was required with FeCl₃, Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.     

Denaturation of bacteriorhodopsin by organic solvents

The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.     

Immunohistochemical localization of atrial natriuretic polypeptide (ANP) in human atrial and ventricular myocardiocytes

To date, there have been few immunohistochemical investigations of atrial natriuretic polypeptide (ANP) in human cardiac tissue, especially the ventricles. In this study, myocardial tissue was obtained from two sources: the bilateral atria and ventricles at autopsy; and biopsy tissues from the right auricle and left ventricle of a patient with myocardial infarction undergoing surgery. These tissues were examined by the avidin-biotin immunoperoxidase technique using three kinds of primary ANP-antibodies. ANP-immunoreactivity was observed in the perinuclear region of myocytes of all tissues examined. The intensity of the reaction was stronger in atrial tissue, weaker in ventricular tissue. In the later tissue, the positive-staining myocytes were not part of the pulse-conducting system. Although the tissues we studied were not obtained from normal hearts, our data demonstrates that ANP-reactivity can be detected in ventricular myocytes outside the pulse-conducting system.     

The effects of guanine and cytosine variation on dinucleotide frequency and amino acid composition in the human genome

Summary One hundred twelve human DNA sequences were analyzed with respect to dinucleotide frequency and amino acid composition. The variation in guanine and cytosine (G+C) content revealed: (1) at 2–3 and 3-1 doublet positions CG discrimination is attenuated at high G+C, but TA disfavor is enhanced, and (2) several amino acids are subject to G+C change. These findings have been reported in part for collections of sequences from various species. The present study confirms that in a single organism-the human-the G+C effects do exist. Aspects of the argument that connects G+C with protein thermal stability are also discussed.