On the composition of the nucleolus with special reference to its filamentous structure

Summary Different staining procedures, various digestion methods and autoradiographic techniques were employed to study the structure and composition of the nucleolus and of the nucleolonema, after unmasking the latter by adenosine treatment. The presence of DNA, RNA, protein and lipid in these structures has been shown. It has been demonstrated that the filamentous structure within the nucleolus — the nucleolonema— has a core of DNA, around which RNA and protein have accumulated. The structure of the nucleolonema suggests that it is in a highly active state, in synthesizing ribosomal RNA and protein.We take the opportunity to express our gratefulness to the Director, Prof. Dr. Hans Lettré, for providing facilities to work in this Institute. We like to thank our other colleagues, particularly Dr. N. Paweletz, for their valuable help during the course of the investigations.     

Some aspects of the Feulgen reaction in situ

Summary Cytological observations combined with studies on absorption spectra of Feulgen stained normal and lipid — extractet HeLa and ehrlich-Lettré mouse ascites cells were performed after fixation of the cells as well in neutral formaldehyde as in Serra fixative. The effects of formaldehyde treatment of the stained cells to substitute all the free amino groups of DNA bond pararosaniline molecules, were also studied. The results obtained by using DNA samples containing 2% protein and relatively free from protein, led to the conclusion that after acid hydrolysis for a short period purines in DNA become splitted and these released aldehydes react with one or two amino groups of pararosaniline, a triphenylmethane dye (according to the arrangement of purines and pyrimidines in the helices). Some protein molecules also take part in the reaction and substitute some of the free amino groups of DNA bound pararosaniline. Peulgen stained cells fixed in Serra fixative show an absorption maximum at 546–550 m. Under appropriate conditions, as in cells fixed in formaldehyde, other substances e.g. phospholipids and lipoproteins interfere with the reaction by substituting most of the free amino groups of DNA bound pararosaniline molecules. It has been argued that in histochemical reactions monosubstituted pararosaniline molecules should be coloured and further substitution of free amino groups of pararosaniline, bound in DNA helices, does not change the intensity of the colour, but gives a shift in the wavelength of the absorption spectra.It has been suggested that the differential response of the nucleoli to the Feulgen-reaction, depending on whether the cells were fixed in formaldehyde or in Serra fixative, may be due to the formation of a protecting shield around the finely distributed intranucleolar chromatin strands, when formaldehyde is being used. After this fixation lipoproteins and other lipids, present in a relatively high percentage and closely associated with the intranucleolar chromatin strands, are especially well preserved.Evidences have been put foreward in support of the amino alkylsulfonic acid theory of Rumpf (1935) and Hörmann et al. (1958) whereas the amino sulfinic acid theory to explain the Schiffs reaction (Wieland and Scheuing, 1921) was shown not to be in agreement with our results.On leave from the Department of Botany, Calcutta University, 35, Ballygunge Circular Road, Calcutta-19, India; on a fellowship from the German Academic Exchange Service.     

Proteases from human immunodeficiency virus and avian myeloblastosis virus show distinct specificities in hydrolysis of multidomain protein substrates.

The virally encoded proteases from human immunodeficiency virus (HIV) and avian myeloblastosis virus (AMV) have been compared relative to their ability to hydrolyze a variant of the three-domain Pseudomonas exotoxin, PE66. This exotoxin derivative, missing domain I and referred to as LysPE40, is made up of a 13-kilodalton NH2-terminal translocation domain II connected by a segment of 40 amino acids to enzyme domain III of the toxin, a 23-kilodalton ADP-ribosyltransferase. HIV protease hydrolyzes two peptide bonds in LysPE40, a Leu-Leu bond in the interdomain region and a Leu-Ala bond in a nonstructured region three residues in from the NH2-terminus. Neither of these sites is cleaved by the AMV enzyme; hydrolysis occurs, instead, at an Asp-Val bond in another part of the interdomain segment and at a Leu-Thr bond in the NH2-terminal region of domain II. Synthetic peptides corresponding to these cleavage sites are hydrolyzed by the individual proteases with the same specificity displayed toward the protein substrate. Peptide substrates for one protease are neither substrates nor competitive inhibitors for the other. A potent inhibitor of HIV type 1 protease was more than 3 orders of magnitude less active toward the AMV enzyme. These results suggest that although the crystallographic models of Rous sarcoma virus protease (an enzyme nearly identical to the AMV enzyme) and HIV type 1 protease show a high degree of similarity, there exist structural differences between these retroviral proteases that are clearly reflected by their kinetic properties.     

Deletion and insertion mutants of the multidrug transporter

The multidrug transporter is a 170,000-dalton membrane glycoprotein which confers multidrug resistance through its activity as an ATP-dependent efflux pump for hydrophobic, cytotoxic drugs. To determine the essential structural components of this complex membrane transporter we have altered an MDR1 cDNA in an expression vector by deletion and insertion mutations. The structure of the transporter deduced from its amino acid sequence suggests that it consists of two homologous, perhaps functionally autonomous, halves each with six transmembrane segments and a cytoplasmic ATP-binding domain. However, several carboxyl-terminal deletions, one involving 53 amino acids, the second removing 253 amino acids, and an internal deletion within the carboxyl-terminal half of the molecule, totally eliminate the ability of the mutant transporter to confer drug resistance. An internal deletion of the amino-terminal half, which removed residues 140-229, is also nonfunctional. Small carboxylterminal deletions of up to 23 amino acids leave a functional transporter, although the removal of 23 COOH-terminal amino acids reduces its ability to confer colchicine resistance. Insertions of 4 amino acids in a transmembrane domain, and in one of the two ATP-binding regions, have no effect on activity. These studies define some of the limits of allowable deletions and insertions in the MDR1 gene, and demonstrate the requirement for two intact halves of the molecule for a functional multidrug transporter.     

Expression of a multidrug resistance-adenosine deaminase fusion gene

A novel fusion gene has been created in which the expression of a dominant selectable marker, the human multidrug resistance gene, is directly linked to the expression of human adenosine deaminase cDNA. The chimeric gene was inserted between the long terminal repeats of a Harvey murine sarcoma virus expression vector and used to transfect drug-sensitive human KB carcinoma cells. Transfectants were selected in increasing concentrations of colchicine and found to contain multiple copies of the intact fusion gene, which is stably and efficiently expressed. A membrane-associated 210-kDa human P-glycoprotein-adenosine deaminase fusion protein is synthesized which retains function of the multidrug transporter and also exhibits adenosine deaminase activity. The data indicate that the human multidrug resistance gene may be used as a dominant selectable marker to introduce other genes in the form of gene fusions into cultured cells.     

Domain II mutants of Pseudomonas exotoxin deficient in translocation

Pseudomonas exotoxin (PE) kills mammalian cells in a complex process that involves cell surface binding, internalization by endocytosis, translocation to the cytosol, and ADP-ribosylation of elongation factor 2. PE is a three-domain protein in which domain I binds to the cell surface, domain II promotes translocation into the cytosol, and domain III carries out ADP-ribosylation. To determine how translocation occurs, we have mutated all the arginine residues in domain II and found that mutations at positions 276 and 279 greatly diminished the cytotoxicity of PE and mutations 330 and 337 substantially reduced cytotoxicity. Biochemical studies indicate that after internalization into an endocytic compartment, the PE molecule undergoes a specific and saturable intracellular interaction, and this interaction is deficient in an Arg276----Gly mutant. Our data suggest that the translocation process of PE involves a specific interaction of Arg276 (and possibly Arg279, Arg330, and Arg337) with components of an intracellular compartment.     

Pseudomonas exotoxin: chimeric toxins

Pseudomonas exotoxin binds to and enters cells by receptor-mediated endocytosis. Within the cell it requires exposure to low pH to enable it to translocate to the cell cytoplasm where it inhibits protein synthesis by ADP-ribosylating elongation factor 2. The toxin has three main structural domains whose functions are: Ia, cell binding; II, translocation; and III, ADP-ribosylation. Key amino acids have been identified within each domain that are required for the function of the toxin. Chimeric toxins were made originally by using chemical cross-linking reagents to couple Pseudomonas exotoxin (or other toxins) to cell-binding proteins. More recently, a variety of Pseudomonas exotoxin-related chimeric toxins have been made by gene fusion technology. These chimeric toxins may be useful clinically for treating various diseases and experimentally for understanding receptor function.     

Lipid production of revertants of Ufa mutants from the oleaginous yeast Apiotrichum curvatum

Summary From six unsaturated fatty acid auxotrophs (Ufa mutants) of the oleaginous yeast Apiotrichum curvatum blocked in the conversion of stearic to oleic acid, were isolated revertants able to grow in the absence of unsaturated fatty acids, in a search for strains that can produce cocoa butter equivalents. A broad range in the percentage of saturated fatty acids (%SFA) was observed in the lipids of individual revertants (varying from 27%–86% SFA), compared with the wild-type (44% SFA). Further analysis of fatty acid composition indicated that: (i) not all six Ufa mutants had the same genetic background and (ii) one specific Ufa mutation could be reverted in more than one way. Revertants that produced lipids with a %SFA>56%, were examined further. These strains were cultivated for 50 generations and half of them produced lipids with high %SFA after that time and were defined as stable. The viability of revertant strains with extremely high %SFA (>80%) may be explained by our finding that polar lipids, which are part of yeast membranes, contained much more polyunsaturated fatty acids and a significantly lower %SFA than neutral (storage) lipids. One revertant (R25.75) was selected that was able to produce lipids in whey permeate at a rate comparable with wild-type A. curvatum and with a fatty acid composition and congelation curve comparable with cocoa butter. Offprint requests to: A. Ykema     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.