Biomedical subjects

D G Scraba

Publications and source records attributed to D G Scraba.

At least 37 records · Page 2Linked to original sources

PubMed1990

Studies of the morphology and structure of the plasma lipid transfer particle from the tobacco hornworm, Manduca sexta.

Morphological features of Manduca sexta plasma lipid transfer particle (LTP) have been investigated by electron microscopy. LTP was found to be an asymmetric particle with two major structural features: a roughly spherical head and an elongated, hinged tail. The hinge occurs approximately at the midpoint of the tail section with the two halves forming angles ranging from 30 degrees to 180 degrees. A molecular mass estimate of 1.4 x 10(6) daltons based on the dimensions of LTP suggests that multiple copies (two or three) of each of the three LTP apoproteins exist in the native complex. Limited digestion studies of LTP suggest that apoLTP-III is less susceptible to trypsin cleavage than apoLTP-I or -II, and therefore may be less exposed to the aqueous environment. Digestion for 1 h at a 1:50 trypsin-LTP protein ratio did not alter the flotation properties of LTP or its morphological features; thus, although significant proteolysis occurred, the particle retained its overall structure. Transfer activity, on the other hand, was affected by trypsin digestion with 30 +/- 14% inhibition of LTP activity occurring upon proteolysis at a 1:50 trypsin-LTP protein ratio. Treatment of LTP with phospholipase A2 resulted in the conversion of LTP-associated phosphatidylcholine and phosphatidylethanolamine to their corresponding lyso forms. Phospholipase A2 treatment did not, however, alter the SDS-PAGE profile, transfer activity, flotation pattern, or the microscopic features of LTP. These results suggest that the products of the phospholipase reaction remain associated with the particle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Mengo virus maturation is accompanied by C-terminal modification of capsid protein VP1.

We have demonstrated that the C-terminal ends of the VP1 proteins of Mengo virus undergo a postassembly trimming of three amino acid residues. A variable proportion of the VP1 molecules isolated from purified virions terminate at Glu 277, which corresponds to the initial viral protease 3C cleavage site. The remainder of the VP1 molecules terminates at Leu 274. When chymotrypsin treatment is included in the virus purification procedure, Leu 274 is the exclusive C-terminal amino acid of the VP1 molecules. The trimming process was found to influence the pH-mediated dissociability of virions in vitro: this was demonstrated by sucrose gradient analysis and electron microscopy. Since long-term incubation of purified virions did not alter the C-terminal Glu 277/Leu 274 ratio, it appears that the trimming process is not autocatalytic.

Capsid↗

PubMed1989

Assembly of mutant pilins in Pseudomonas aeruginosa: formation of pili composed of heterologous subunits.

Recently, we reported the degree of N-terminal processing within the cytoplasmic membranes of three mutant pilins from Pseudomonas aeruginosa PAK with respect to leader peptide removal and the methylation of the N-terminal phenylalanine (B. L. Pasloske and W. Paranchych, Mol. Microbiol. 2:489-495, 1988). The results of those experiments showed that the deletion of 4 or 8 amino acids within the highly conserved N terminus greatly inhibited leader peptide removal. On the other hand, the mutation of the glutamate at position 5 to a lysine permitted leader peptide cleavage but inhibited transmethylase activity. In this report, we have examined the effects of these mutant pilins upon pilus assembly in a P. aeruginosa PAO host with or without the chromosomally encoded pilin gene present. Pilins with deletions of 4 or 8 amino acids in the N-terminal region were not incorporated into pili. Interestingly, pilin subunits containing the glutamate-to-lysine mutation were incorporated into compound pili together with PAO wild-type subunits. However, the mutant pilins were unable to polymerize as a homopolymer. When wild-type PAK and PAO pilin subunits were expressed in the same bacterial strain, the pilin subunits assembled into homopolymeric pili containing one or the other type of subunit.

Bacterial Outer Membrane Proteins↗

PubMed1988

Electron microscopy of glycogen degrading enzymes.

Single molecules of glycogen phosphorylase b exhibit images in the electron microscope which are similar in shape and dimension to those derived from X-ray crystallography. Phosphorylase alpha exhibits tetramers but shows dimers in the presence of glucose. Glycogen debranching enzyme appears as a monomer with an unusual crescent or shrimp-like shape, with occasional isologous aggregation to circular dimers. The longest dimension of the monomer is very similar to that of the phosphorylase dimer, 11.5 nm. Strong binding of the debranching enzyme to glycogen is readily visualized in the electron microscope. It is suggested that the distinctive shape of the debranching enzyme may be related to its catalytic function.

Animals↗

PubMed1988

Use of synthetic peptides to map the antigenic determinants of glycoprotein D of herpes simplex virus.

The predictive algorithm Surfaceplot (J.M.R. Parker, D. Guo, and R.S. Hodges, Biochemistry 25:5425-5432, 1986) was used to examine glycoprotein D of herpes simplex virus type 1 (HSV-1) for amino acid residues with a high probability of being exposed on the molecular surface. Based on these data, 11 different peptides corresponding to 10-residue segments in the primary sequence of glycoprotein D and one 20-residue segment were synthesized, conjugated to carrier proteins, and used to generate specific antisera in rabbits. Two synthetic peptides predicted not to be on the surface of glycoprotein D were included as negative controls. The polyclonal antisera against individual synthetic peptide conjugates were in turn evaluated for their ability to recognize both isolated glycoprotein D and intact HSV-1 virions in an enzyme-linked immunosorbent assay. Based on Surfaceplot predictions, eight linear antigenic sites on glycoprotein D were thereby defined from the 12 antipeptide antisera prepared. Four of these sites contained epitopes to which complement-independent neutralizing antibodies could be generated. The latter sites corresponded to sequences 12 to 21, 267 to 276, 288 to 297, and 314 to 323 of the mature protein. An additional peptide sequence, 2 to 21, was found to generate antisera which had potent virus-neutralizing capacity in the presence of complement. Identification of a neutralizing epitope in the sequence 314 to 323 makes it likely that the membrane-spanning region of glycoprotein D is within the subsequent sequence, 323 to 339. Antipeptide antisera prepared in this study from 12 synthetic peptides contained 13 surface sites predicted by Surfaceplot, of which 7 were not predicted by the parameters of Hopp and Woods (Proc. Natl. Acad. Sci. USA 78:3824-3828, 1981). Of these seven sites not predicted by the Hopp and Woods plot, all generated antipeptide antibodies that bound to HSV-1 virions and three of these seven sites generated neutralizing antibodies. In total, 8 of 12 synthetic peptides containing surface regions produced antipeptide antibodies that bound to HSV-1 virions and 5 of these generated neutralizing antibodies. These results suggest the advantages of Surfaceplot in mapping antigenic determinants in proteins.

Algorithms↗

PubMed1987

The atomic structure of Mengo virus at 3.0 A resolution.

The structure of Mengo virus, a representative member of the cardio picornaviruses, is substantially different from the structures of rhino- and polioviruses. The structure of Mengo virus was solved with the use of human rhinovirus 14 as an 8 A resolution structural approximation. Phase information was then extended to 3 A resolution by use of the icosahedral symmetry. This procedure gives promise that many other virus structures also can be determined without the use of the isomorphous replacement technique. Although the organization of the major capsid proteins VP1, VP2, and VP3 of Mengo virus is essentially the same as in rhino- and polioviruses, large insertions and deletions, mostly in VP1, radically alter the surface features. In particular, the putative receptor binding "canyon" of human rhinovirus 14 becomes a deep "pit" in Mengo virus because of polypeptide insertions in VP1 that fill part of the canyon. The minor capsid peptide, VP4, is completely internal in Mengo virus, but its association with the other capsid proteins is substantially different from that in rhino- or poliovirus. However, its carboxyl terminus is located at a position similar to that in human rhinovirus 14 and poliovirus, suggesting the same autocatalytic cleavage of VP0 to VP4 and VP2 takes place during assembly in all these picornaviruses.

Antigens, Viral↗

PubMed1987

The synthesis of a particle-forming cellular protein is enhanced by Mengo virus infection.

We have detected a cellular protein which not only escapes the shutoff of host translation induced by Mengo virus, but is synthesized in increasing amounts during Mengo virus infection. The protein has an apparent molecular weight of 20,000 and is contained in a remarkably stable cytoplasmic particle with a sedimentation coefficient of approximately 16 S in sucrose gradients. In the electron microscope this particle appears as a sphere of approximately 12 nm diameter. The synthesis of the protein is stimulated in mouse L cells and in HeLa cells infected with Mengo virus. Its synthesis cannot be induced, however, by stress (heat) or by infection with reovirus.

Animals↗

PubMed1986

The selection and characterization of human monoclonal antibodies to human cytomegalovirus.

This communication describes the application of Epstein-Barr virus lymphocyte transformation technology to the production of human monoclonal antibodies specific for human cytomegalovirus. A group of such IgG antibodies have been characterized in terms of subclass, light-chain composition, specificity for particular viral proteins and neutralizing capacity. These results have shown that the production of antibodies by transformed lymphocytes is representative of the in vivo human immune response; the antibodies produced may therefore be of therapeutic value. This approach should prove to be useful for the identification of specific virion proteins which are antigenic in humans and for the in vitro evaluation of the immune responses to synthetic peptide vaccines.

Antibodies, Monoclonal↗

PubMed1986

Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli.

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.

Bacterial Proteins↗

PubMed1986

Two monoclonal antibodies specific for different epitopes within the amino-terminal region of F pilin.

Two murine monoclonal antibodies (JEL 92 and 93) specific for adjacent epitopes on F pilin were purified and characterized. JEL 93 immunoglobulin G (IgG) and its Fab fragments were specific for the amino-terminal region and were completely reactive with a synthetic peptide representing the first eight amino acids of F pilin. The acetyl group was demonstrated to be an important part of the epitope, since an unacetylated version of the amino-terminal peptide was 100-fold less reactive with JEL 93 IgG. JEL 92 IgG reacted with the region of F pilin surrounding Met-9, represented by a tryptic peptide derived from the first 17 amino acids. This reactivity was completely abolished by cleavage of the peptide with cyanogen bromide. As shown by electron microscopy, both monoclonal antibodies bound to a vesiclelike structure at one end of purified free pili and did not bind to the sides of the pili, nor did they appear to bind to the tip. When sonication was used to break pili into shorter fragments, the number of binding sites for JEL 92 but not JEL 93 IgG increased as measured by a competitive enzyme-linked immunosorbent assay.

Antibodies, Monoclonal↗

PubMed1986

Toward an in vitro system for picornavirus assembly: purification of mengovirus 14S capsid precursor particles.

Mengovirus 14S subviral protein particles generated in infected L cells and in a cell-free translation system primed with mengovirus RNA were purified by sucrose gradient centrifugation and immunoaffinity chromatography. The preparations from both sources contained essentially pure proteins epsilon, alpha, and gamma, as was demonstrated in terms of virus-specific proteins (by autoradiography) and total protein content (by silver staining of sodium dodecyl sulfate-polyacrylamide electrophoresis gels). These purified proteins sedimented as discrete particles at the 14S position when recentrifuged in sucrose gradients. Although their assembly properties have not yet been studied in detail, preliminary results indicate that during incubation with virion RNA the 14S particles purified from infected cells can form a structure cosedimenting with mature mengovirus.

Animals↗

PubMed1986

The binding of the anthelmintic pyrvinium cation to deoxyribonucleic acid in vitro.

Pyrvinium pamoate, an anthelmintic drug with mutagenic activity, binds to duplex DNA in vitro. Binding markedly enhances the intrinsic fluorescence of the drug, permitting a characterization of the binding reaction to be performed directly through fluorimetric analysis. The monopyrvinium salt, pyrvinium iodide, binds cooperatively to native calf thymus DNA with an intrinsic binding constant of 1.1 X 10(4) M-1 as determined fluorimetrically and 1.4 X 10(4) M-1 as determined by equilibrium dialysis. The respective binding site was estimated to be 2.5 base pairs. A change in the absorption of bound drug as a function of the binding ratio identifies secondary, nonfluorescent binding which is essentially ionic in character. The binding of pyrvinium removes negative supercoils from PM2-ccc-DNA with an experimental efficiency greater than that observed for the intercalating drug chloroquine. Electron microscopy shows that molecules of pBR322 DNA treated with pyrvinium increase in length by as much as 18% over controls, consistent with an increase in DNA base pair separation upon binding. The evidence indicates that the primary interaction of pyrvinium with DNA involves intercalation.

Animals↗

PubMed1984

Picornaviruses of two different genera have similar structures.

Crystals of Mengo virus were used to collect three-dimensional X-ray diffraction data to 7 A resolution. A self-rotation function showed the precise orientation of the Mengo particles in the crystal unit cell. A cross-rotation function against similar data of cubic rhinovirus crystals showed a peak when the orientations of these two icosahedral viruses were superimposed. This demonstrates similarity of capsid construction between two picornaviruses of different taxonomic genera.

Crystallization↗

PubMed1984

Structure of the Mengo virion. VII. Crystallization and preliminary X-ray diffraction analysis.

Crystals of Mengo virions have been grown reproducibly and analyzed by X-ray diffraction. These crystals diffract to a resolution of 7.0 A. The unit cell exhibits cubic symmetry with a = 422 A. The space group is P23, with four virus particles situated on crystallographic threefold axes. Picornavirions from three of the four recognized genera (Study Group on Picornaviridae, Intervirology 10, 165-180, 1978) have now been examined at low resolution by X-ray diffraction: poliovirus type 1 (J. T. Finch and A. Klug, Nature (London) 183, 1709-1714, 1959; J. M. Hogle, J. Mol. Biol. 160, 663-668, 1982); human rhinovirus 14 (J. W. Erickson, E. A. Frankenberger, M. G. Rossmann, G. S. Fout, K. C. Medappa, and R. R. Rueckert, Proc. Natl. Acad. Sci. USA 80, 931-934, 1983); and Mengo virus.

Animals↗

PubMed1984

A model for the structure of fumarate reductase in the cytoplasmic membrane of Escherichia coli.

By a recombinant DNA approach we have prepared Escherichia coli cytoplasmic membranes that are highly enriched in the terminal electron transfer enzyme fumarate reductase. This enzyme is composed of four nonidentical subunits in equal molar ratio. A 69,000-dalton covalent flavin-containing subunit and a 27,000-dalton nonheme iron-containing subunit make up a membrane extrinsic catalytic domain. Two very hydrophobic subunits of 15,000 and 13,000 daltons make up the hydrophobic membrane anchor domain. Electron microscopy of negatively stained membranes shows a characteristic knob-and-stalk-type structure composed of the catalytic domain. The anchor polypeptides have been analyzed for hydrophobic segments and alpha-helical content and a model for their organization within the lipid bilayer is presented. The results reviewed in this paper suggest a model for the fumarate reductase complex in the cytoplasmic membrane.

Bacterial Proteins↗

PubMed1984

The molecular length of measles virus RNA and the structural organization of measles nucleocapsids.

Full-length measles virus RNA molecules isolated from purified virions or nucleocapsids and examined by electron microscopy were 5.12(+/- 0.12) micron in length, corresponding to a molecular weight of 5.2 (+/- 0.1) X 10(6). Purified virions examined by negative staining in the electron microscope exhibited a pleomorphic range of particle sizes varying in diameter between 300 nm and 1000 nm. Purified nucleocapsids had dimensions of 21 nm (diameter) X 1254(+/- 7) nm (length) and a central core of diameter about 5 nm. Full-length nucleocapsids were composed of 204 (+/- 3) protein discs. The pitch of the nucleocapsid helix was calculated to be 6.1 nm and the helix angle, alpha, to be 8 degrees 16'. Approximate volume calculations indicate that each enveloped virus particle contains multiple nucleocapsids.

Animals↗

PubMed1984

Overproduction of fumarate reductase in Escherichia coli induces a novel intracellular lipid-protein organelle.

The expression of fumarate reductase in Escherichia coli has been amplified over 30-fold by utilizing a recombinant plasmid, pFR63 , carrying the fumarate reductase operon. More than 50% of the inner-membrane protein could be accounted for by the enzyme, whereas the total amount of protein associated with the membrane fraction doubled. The membrane accommodated this excess fumarate reductase without reducing the levels of other membrane-associated enzymes. At the same time, the amount of membrane lipid increased such that the lipid/protein ratio remained constant, indicating that the total amount of membrane had doubled. Small alterations in fatty acid composition as well as a large increase in cardiolipin were detected in the fumarate reductase-enriched membranes. The excess membrane was localized in novel tubular structures which were observed in thin-section and negatively stained electron-microscopic preparations. The tubules only appeared after the cytoplasmic membrane became highly enriched in fumarate reductase. They branched from the cytoplasmic membrane and were fumarate reductase. They branched from the cytoplasmic membrane and were composed of an aggregate of fumarate reductase and lipid.

Bacterial Proteins↗

PubMed1984

Interactions of the antitumor agents mitoxantrone and bisantrene with deoxyribonucleic acids studied by electron microscopy.

The interactions of the low cardiotoxic antitumor agents 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9, 10-anthracenedione (mitoxantrone) and 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazoyl-2-yl)hydrazone] (bisantrene) with pBR322 and PM2 DNA have been examined by electron microscopy. Direct evidence was obtained for intercalative binding of both drugs, with mitoxantrone causing a 13% average length increase in pBR322 corresponding to approximately 580 drug molecules per circle at saturation and bisantrene causing an 11% increase in length corresponding to approximately 480 drug molecules bound per circle. Considerations of the known GC preference for non-nearest neighbor binding of the drugs and inspection of the known sequence of pBR322 suggest that the available intercalation sites are occupied and that additional external electrostatic binding of the cationic drugs also occurs. An apparent difference in behavior of mitoxantrone as compared with that of bisantrene in causing no net increase in length of supercoiled pBR322 was shown to be attributable to an offsetting compaction due to extensive supercoiling by mitoxantrone molecules. This conclusion was confirmed by independent experiments with PM2 covalently closed-circular DNA--both native, negatively supercoiled and relaxed--with calf thymus topoisomerase, using ethidium for comparison. Ethidium caused a 21.3 +/- 3.6% length increase in nicked, open-circular PM2-DNA, or 2100 molecules bound per 10,300 base pairs. Mitoxantrone caused a 16.6% length increase in nicked PM2-DNA equivalent to approximately 1700 drug molecules per circle. Electron microscopic measurements on relaxed PM2-DNA with progressively increasing proportions of mitoxantrone (from 1.4:1 to 14:1 drug molecules per base pair) revealed the onset of formation of lacelike networks of DNA circles linked together. This phenomenon, which is not produced by bisantrene, is attributed to inter-DNA links by the charged side arms of mitoxantrone and is in accord with previous reports that mitoxantrone causes severe compaction and distortion of chromatin. Electron microscopic examination of the interaction of six additional mitoxantrone derivatives, two of which produced lacelike DNA networks, revealed strict structural requirements for this phenomenon.

Anthracenes↗