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At least 217 records · Page 12Linked to original sources
PubMed1995

Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord.

NF-66, also known as alpha-internexin, has been characterized as a 66 kD mammalian neurofilament (NF) protein whose expression in developing rat brain precedes that of the low molecular weight NF protein (NF-L). NF-66 is thought to assemble into 10 nm diameter intermediate filaments in vitro, although the precise nature of the assembly process remains obscure. Likewise, the ability of NF-66 to polymerize with the low (NF-L), middle (NF-M), and high (NF-H) M(r)NF proteins has not been defined. This investigation describes the reassembly of bovine NF-66 regarding its formation into 10 nm diameter filaments as well as its potential for polymerization with other type IV intermediate filaments. NF-66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Balin et al., Brain Res 556:181-195, 1991), and purified by a combination of high performance liquid chromatography (HPLC) (DEAE anion exchange and hydroxylapatite column chromatography) and gel elution strategies. In vitro reassembly experiments revealed that NF-66 formed approximately 10 nm diameter filaments of varying length; immunoelectron microscopy demonstrated labeling of these filaments by a monoclonal antibody to intermediate filament antigen (IFA), a polyclonal antibody against rat NF-66 and by a monoclonal antibody generated against the core region of NF-M but cross-reactive with NF-66. This report is the first investigation to look at the in vitro interaction between NF-66 and other type IV intermediate filament proteins (NF-H, -M, and -L) and establishes that NF-66 forms heteropolymeric filaments with these other neurofilament proteins, as confirmed by double immunolabeling. These studies suggest that NF-66 could provide a nucleation site for the polymerization of later-expressed proteins during neuronal development.

Animals↗
PubMed1988

Comparison of the dynamics of the membrane-bound form of fd coat protein in micelles and in bilayers by solution and solid-state nitrogen-15 nuclear magnetic resonance spectroscopy.

Solid-state and solution 15N nuclear magnetic resonance experiments on uniformly and specifically 15N labeled coat protein in phospholipid bilayers and in detergent micelles are used to describe the dynamics of the membrane-bound form of the protein. The residues in the N- and C-terminal portions of the coat protein in both phospholipid bilayers and in detergent micelles are mobile, while those in the hydrophobic midsection are immobile. There is evidence for a gradient of mobility in the C-terminal region of the coat protein in micelles; at 25 degrees C only the last two residues are mobile on the 10(9)-Hz timescale, while the last six to eight residues appear to be mobile on slower timescales and highly mobile at higher temperatures. Since all of the C-terminal residues are immobile in the virus particles, the mobility of these residues in the membrane-bound form of the protein may be important for the formation of protein-DNA interactions in the assembly process.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Redistribution of nuclear envelope associated antigen during the mitotic cycle.

Murine hybridomas were generated to DNA/tight binding proteins complex isolated from the residual nuclear structure following a procedure analogous to that yielding "empty" shells of nuclear envelope. A monoclonal antibody designated 2A8 was selected because of its differential immunostaining of mitotic cells of a synchronized mouse fibroblast cell culture L-929. The target antigen was rendered insoluble by a sequence of extractions of isolated nuclei of diverse cell types with detergents, urea, DNase I and alkali thus reproducing some solubility properties of proteins constituting an operationally defined residual nuclear matrix. The cognate polypeptide was localized on a subset of proteins of M(r) 58-65 kDa, 70 kDa in isolated fibroblast nuclear matrices. The functional implication of the antigen in mitosis-related disassembly-assembly process of the nuclear matrix/envelope was detected. At prophase the antibody decorated the nuclear periphery and nuclear envelope fixed inward filaments. A fibrous network of cytoplasmic localization was stained in metaphase. At anaphase the antigen was dispositioned into peripheral fibrogranular clusters of polar orientation predominantly on one side of the nucleus. Proceeding to telophase a spreading fluorescence was manifested over the entire contour of the nuclear periphery to delineate the reforming nucleus. By immunogold electron microscopy of interphase cells the antigen was identified as evenly distributed in chromatin and interchromatin regions. At initiation of chromosome condensation in mitosis the label was detected predominantly in the chromosomal area.

Animals↗
PubMed1994

DNA-protein cooperativity in the assembly and stabilization of mu strand transfer complex. Relevance of DNA phasing and att site cleavage.

The requirements for negatively supercoiled DNA substrates, the cis-acting transposition enhancer and the Escherichia coli HU protein during the phage Mu transposition reaction are relaxed under DMSO-assay conditions. We have used these modified assay conditions to extend studies on the transposition pathway. We show here that linear DNA fragments containing the right end of Mu (attR) and Mu A protein mutually promote the assembly of "high-order" complexes held together by non-covalent protein-DNA and protein-protein interactions. A large subset of these complexes is competent in mediating strand transfer. DNA fragments containing the left end of Mu (attL) as well as non-Mu DNA can be used as targets during strand transfer. The R1 and R2 subsites within attR are required, but R3 is dispensable, in the protein-DNA oligomerization steps as well as in the strand transfer reaction. Proper phasing and spacing between R1 and R2 are central to the reaction. A single base-pair change in the terminal nucleotide that renders attR non-cleavable prevents the assembly of stable high-order complexes, showing that strand cleavage and stabilization of high-order complexes are tightly coupled events. Conversely, pre-cleavage at the attL site allows it to function in the assembly process, albeit at a much lower efficiency than attR. In the presence of HU, the reactivity of pre-cleaved attL is enhanced significantly.

Attachment Sites, Microbiological↗
PubMed1995

The ribosomal DNA loci in Plasmodium falciparum accumulate mutations independently.

Homogeneity of rDNA sequence within a cell is maintained by mechanisms working at the DNA level. The imperative to maintain homogeneity is thought to result from pressure to maintain the sequence of the rRNA transcript. We have investigated the extent of sequence variation within and between members of a species that is unable to utilize some standard mechanisms of rDNA sequence correction. We have compared the sequence of the internal transcribed spacer (ITS1) located between the 18 S rRNA and 5.8 S rRNA genes of five different loci of a single Plasmodium falciparum genotype. The ITS1 sequences are identical at 80 to 91% of the positions among the three asexually expressed genes (A-types) and 75% between the two genes expressed during sporogony (S-types), with only 42 to 57% identity between the types. This is rather startling in that the differences described here for a single genome are greater than those normally seen when comparing rDNA units from distantly related organisms. We observe an apparent conservation of secondary structure within ITS1 sequences from the different transcription units, which would reflect a level of selection at the rRNA but the organism seems to be quite tolerant of primary sequence variation. Investigation of the mature coding region within the 18 S rRNA genes did not reveal sequence variation within A- and S-types from a single genotype. However, comparison of the 18 S rRNA coding region from 17 geographically distinct strains reveals up to 10% sequence variation within a 400 nucleotide region. Hence homogeneity of rRNA units within a species does not seem to be an imperative driven totally by selection at the RNA level. The extraordinary maintenance of homogeneity within rDNA units normally seen within a species appears to have significance beyond those that can be ascribed to the events involved in processing, assembly and function of the ribosome.

Animals↗
PubMed1994

Isoprenylation of large hepatitis delta antigen is necessary but not sufficient for hepatitis delta virus assembly.

Hepatitis delta virus (HDV) encodes two proteins, the small hepatitis delta antigen (SHDAg) and large hepatitis delta antigen (LHDAg). Both proteins are identical except for the presence of additional 19 amino acids at the C terminus of LHDAg. While SHDAg is required for HDV RNA replication, LHDAg inhibits replication and is required together with hepatitis B surface antigen for the assembly of HDV. The C-terminal last 4 amino acids of LHDAg (Cys-Arg-Pro-Gln) is an isoprenylation motif. It has previously been shown that the mutation of the Cys inhibited the assembly of HDV. In order to discern whether this effect is due to change of amino acid residue or abolition of isoprenylation, we constructed several LHDAg mutants of the terminal three amino acid residues and tested their abilities to be packaged with HBsAg by cotransfection experiments. We also made GST-fusion proteins of these mutants and tested their abilities to be isoprenylated in rabbit reticulocyte lysate system. We found that some, but not all, of the substitutions of the amino acid residues other than the Cys also inhibited isoprenylation and that the status of isoprenylation of these mutant proteins correlated well with their abilities to be packaged with HBsAg into virions. This result indicates that isoprenylation, rather than the primary amino acid sequence, is required for LHDAg packaging. Furthermore, we found that the attachment of an isoprenylation motif to SHDAg did not enable it to be packaged with HBsAg and that the deletions of any 5 amino acids in the last 15 amino acids (amino acids 196 to 210) unique to the LHDAg abolished the packaging ability. In contrast, the deletion of 33 amino acids (amino acids 163 to 195) upstream of the last C-terminal 19 amino acids of LHDAg did not interfere with its packaging ability. Therefore, we conclude that the 15 amino acids upstream of the isoprenylation site of LHDAg are also essential for HDV assembly, and a large portion of the alleged C-terminal Pro/Gly-rich region (amino acids 146 to 195) is not required for the assembly process.

Amino Acid Sequence↗
PubMed1994

Characterization of hepatitis B virus core mutants that inhibit viral replication.

We have generated and functionally characterized dominant negative core protein variants of the hepadnaviruses to determine their effects on "wild type" viral replication. Plasmids expressing these constructs were introduced into hepatoma cell lines by transient transfection and effects on wild type woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) replication were evaluated by Southern blot analysis of purified viral core particles. WHV and HBV constructs expressing a truncated core protein fused in frame with the C-terminus of the small surface protein were found to inhibit viral replication by 90-95% due to disruption of the viral nucleocapsid assembly process and preventing encapsidation of pregenomic RNA. The antiviral effects were found to be specific for the targeted virus. These results demonstrate that mutants of hepadnaviral core protein may represent a novel class of antiviral agents.

DNA Replication↗
PubMed1981

Isolation of Drosophila flightless mutants which affect myofibrillar proteins of indirect flight muscle.

A large number of dominant flightless mutants of Drosophila were chemically induced, and their thorax proteins were examined by chemically induced, and their thorax proteins were examined by means of two-dimensional gel electrophoresis (O'Farrell 1975). Among them, 26 lines were found to have deficiency or reduction of some of myofibrillar proteins in indirect flight muscle (IFM). The gel patterns of the mutants could be classified into eleven groups. In general, more than a few polypeptides were either absent or reduced in each mutant line. Although the mutations affect myofibrillar proteins in apparently complex and diverse ways, logical correlations were found among the changes. There are pairs of proteins which always change together when a number of mutants are compared. There are also many pairs in which presence of one protein is necessary, but not sufficient for presence of the other. This suggests that absence of one component leads to disappearance or reduction of others which are either spatially or functionally related to the former. The correlation is possibly due to a hierarchy of the proteins in the myofibrillar assembly processes. Chromosomal loci of eleven typical mutants were examined, and it was found that most of them are located in two small regions of the second and the third chromosomes. IFM myofibrils of these mutants are either abnormal or absent in homozygotes as well as in heterozygotes.

Animals↗
PubMed1991

Outer membrane protein PhoE as a carrier for the exposure of foreign antigenic determinants at the bacterial cell surface.

PhoE protein is an abundant outer membrane protein of the Escherichia coli K-12 outer membrane. This protein can be used as an exposure system to produce foregin antigenic determinants and for their transport to the bacterial cell surface. The system is very flexible, since insertions varying in length and nature could be made in different cell surface-exposed regions of PhoE, without interfering with the assembly process of the mutant proteins into the outer membrane. Two antigenic determinants of the structural VP1 protein of foot-and-mouth disease virus were inserted in different combinations in four cell surface-exposed regions of PhoE. The epitopes were exposed at the bacterial cell surface and they keep their antigenic and immunogenic properties in this PhoE-associated conformation. Immunization of guinea pigs with one hybrid protein, containing a combination of the two epitopes inserted in the fourth exposed region, resulted in complete protection against challenge with the virus. A T-cell epitope of the 65 kDa heat shock protein of Mycobacterium tuberculosis was inserted in the fourth exposed region of PhoE and in vitro proliferation of two T-cell specific clones was demonstrated. Thus, the PhoE exposure system has been shown to be suitable for presentation of both B-cell and T-cell determinants to the immune system. Furthermore, good expression of the hybrid protein in attenuated Salmonella strains, which can be used as live oral vaccines, was shown.

Amino Acid Sequence↗
PubMed1991

The cytochrome c reductase/oxidase respiratory pathway of Paracoccus denitrificans: genetic and functional studies.

Data are presented on three components of the quinol oxidation branch of the Paracoccus respiratory chain: cytochrome c reductase, cytochrome c552, and the a-type terminal oxidase. Deletion mutants in the bc1 and the aa3 complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochrome c552 in electron transport between complex III and IV is presented.

Cytochrome c Group↗
PubMedDate not supplied

Single-stranded DNA binding protein encoded by the filamentous bacteriophage M13: structural and functional characteristics.

The single-stranded DNA binding protein, or gene V protein (gVp), encoded by gene V of the filamentous bacteriophage M13 is a multifunctional protein that not only regulates viral DNA replication but also gene expression at the level of mRNA translation. It furthermore is implicated as a scaffolding and/or chaperone protein during the phage assembly process at the hostcell membrane. The protein is 87 amino acids long and its biological functional entity is a homodimer. In this manuscript a short description of the life cycle of filamentous phages is presented and our current knowledge of the major functional and structural properties and characteristics of gene V protein are reviewed. In addition models of the superhelical complexes gVp forms with ssDNA are described and their (possible) biological meaning in the infection process are discussed. Finally it is described that the 'DNA binding loop' of gVp is a recurring motif in many ssDNA binding proteins and that the fold of gVp is shared by a large family of evolutionarily conserved gene regulatory proteins.

Amino Acid Sequence↗
PubMed1993

Isolation and analysis of vaccinia virus previrions.

Vaccinia virus (VV) virion morphogenesis is a complex sequence of events that occurs late in viral infection that is essential for the production of mature progeny. Electron microscopy studies have identified multiple morphogenic forms of virus particles, apparently assembled in a sequence from immature to mature particles that correlates with distinct physical changes. This assembly process is, however, rather poorly understood at the molecular level. To better characterize the multiple forms of VV previrions, sucrose log gradient fractionation of VV-infected cells was used to separate radiolabeled immature and mature forms of the virus. Depending on time postinfection that the infected cells were harvested, four distinct peaks of acid-precipitable counts could be detected that displayed different rates of sedimentation. Using pulse-chase analysis procedures, the labeled peaks were shown to have precursor-product relationships as slower sedimenting entities chased to faster sedimenting ones with time. These peaks were referred to as A, B, C, and V particles, with A being the initial precursor form found near the top of the gradient and V being the fastest sedimenting product. As the previrions mature, they migrated faster in the gradient and became infectious and resistant to treatment with DNase I. The core protein composition of the A particles was predominantly uncleaved precursors, with only small amounts of the mature core proteins 4a, 4b, 25K, and 23K evident. However, as the sedimentation rate of the particles increased, proteolytic maturation proceeded such that C particles were composed almost exclusively of mature core proteins. Together these results indicate that several distinct and separable forms of VV previrions exist, that VV core protein precursors are associated with the previrions prior to cleavage, and that maturation of the core proteins is coordinately linked to the conversion from noninfectious previrions to infectious viral particles.

Cell Line↗
PubMed1987

A highly efficient procedure for the quantitative formation of intact and viable lysozyme spheroplasts from Escherichia coli.

This paper describes a highly efficient procedure for the quantitative conversion of Escherichia coli cells to spheroplasts utilizing 100- to 1000-fold less lysozyme than in the most efficient procedures used to date. The resulting spheroplasts have intact outer and inner membranes and are fully viable on agar plates. The spheroplasting procedure is a refinement of earlier procedures and enables regulation of the translocation of minute amounts of lysozyme into the periplasmic space of E. coli cells, based on a Ca2+ pretreatment, an EDTA incubation, and a heat shock. About 1000 lysozyme molecules per cell are sufficient for complete spheroplast formation (greater than 98%). Some of the characteristics of these spheroplasts prior to and after recovery are described. It is anticipated that such viable spheroplasts will be useful in the study of fusion of gram-negative cells and other membrane systems, in the introduction of DNA and proteins into refractory gram-negative cells, and in investigating envelope-related synthesis and assembly processes.

Bacteriological Techniques↗
PubMed1994

Self assembly of covalently anchored phospholipid supported membranes by use of DODA-Suc-NHS-lipids.

We present a novel preparation method for the self assembly of covalently anchored phospholipid supported membranes. The surface is gold covered by cysteamine. Vesicles containing DMPC and activated DODA-Suc-NHS-lipids assembled on this surface. The whole self-assembly process is monitored conveniently by Near Infrared Surface Plasmon Resonance (NIR-SPR). Comparing the data to those obtained by Ca(2+)-mediated vesicle fusion, confirmed this interpretation.

Dimyristoylphosphatidylcholine↗
PubMed1986

Larval cuticular morphogenesis in the tobacco hornworm, Manduca sexta, and its hormonal regulation.

The sequential synthesis and deposition of larval cuticular proteins was followed during the final larval molt and the final larval instar of the tobacco hornworm Manduca sexta and correlated with changes in cuticular structure. On the final day of feeding (Day 3) before the onset of metamorphosis many endocuticular proteins were no longer synthesized and new isoelectric variants of 27,000-Da polypeptides were deposited into the cuticle coincident with the formation of lamellae 5- to 10-fold thinner than those previously deposited. Application of a juvenile hormone analog methoprene on Day 1 prevented this change in protein synthesis and in lamellar structure by preventing the observed rise in the intermolt ecdysteroid titer on Day 2. These changes could be induced in vitro by 25-100 ng/ml 20-hydroxyecdysone in the absence of juvenile hormone. Thus, the intermolt change in the lamellar assembly process appears to result from hormone-induced changes in cuticular protein synthesis.

Chitin↗
PubMed1985

Interaction of triethyl lead chloride with microtubules in vitro and in mammalian cells.

The effects of triethyl lead chloride (TriEL) on the in vitro assembly and disassembly of microtubules (MTs) from porcine brain were studied by turbidometry at 350 nm and by electron microscopy. TriEL inhibited MT assembly at 50 microM concentration and caused an almost complete disassembly of preformed MTs. The drug depolymerized MTs more effectively than colchicine. Concentrations higher than 50 microM TriEL caused an aberrant assembly process. Fibers about 10 nm width were formed in addition to aggregates of amorphous material. In vivo TriEL also caused MT depolymerization in interphase and mitotic PtK-1 and Ehrlich ascites tumor (EAT) cells as monitored by indirect immuno-fluorescent staining of tubulin and electron microscopy. The extent of MT depolymerization was concentration- and time-dependent. Recovery occurred as early as 5 min after removal of the drug. The fluorescent actin pattern in PtK-1 cells typical of stress fibers and subcortical filaments seemed not to be altered by the presence of TriEL. The vimentin intermediate filament system was, however, rearranged as a juxtanuclear complex after TriEL treatment. Furthermore, TriEL effected the inhibition of cellular growth (100% inhibition at about 10(-5) M). Cytokinesis is prevented to a great extent, resulting in the formation of binucleate cells which can additionally possess some micronuclei.

Animals↗
PubMed1988

Fibronectin fibril formation involves cell interactions with two fibronectin domains.

Fibronectin fragments and domain-specific antibodies have been used to study the mechanism by which cells reorganize exogenous fibronectin substrata into fibrils. Fibroblasts prevented from protein synthesis, and hence not secreting endogenous fibronectin or other matrix components, reorganized exogenous fibronectin substrata into arrays resembling the matrix of normally cultured cells. Cells also formed fibrils from substrata containing mixtures of cell- and either of two different heparin-binding fibronectin fragments but not from either fragment alone. The gelatin-binding fragment alone or in conjunction with the cell-binding fragment did not promote fibril formation. Antibodies recognizing cell- and either heparin- or the gelatin-binding domains labeled fibrils formed by cells under normal culture conditions or when a substratum of intact fibronectin was used as the sole exogenous source. However, only antibodies recognizing the cell- or either heparin-binding fragment reduced fibrillogenesis from intact fibronectin substrates when added during cell spreading. These data suggest that formation of fibronectin fibrils can occur at the cell surface and that membrane components recognizing the cell- and the heparin-binding domains in fibronectin may cooperate in the assembly process.

Animals↗
PubMed1989

Lateral diffusion of lectin receptors in fibroblast membranes as a function of cell shape.

Anchorage-dependent fibroblasts respond to biochemical growth signals only when attached to and spread on a suitable substrate surface. Attachment of fibroblasts initiates a cytoskeletal assembly process that results in the organization of long actin stress fibers and microtubules which may be required for transmembrane signal transduction. Fibroblasts maintained in suspension, however, remain spherical with no apparent stress fibers or lengthy microtubules. Because of the significant differences in cytoskeletal organisation induced by shape modification, and the resulting possible changes in organization and dynamics of membrane receptors, the technique of fluorescence redistribution after photobleaching (FRAP) was employed to examine the lateral mobility of wheat germ agglutinin (WGA) and succinylated concanavalin A (sCon A) receptors in the plasma membrane of untransformed and Kirsten murine sarcoma virus-transformed Balb/c 3T3 fibroblasts in the spread and spherical state. An examination of FITC-WGA and FITC-sCon A binding to the plasma membrane for both cell lines in a spread or spherical state demonstrated no significant differences in the number of WGA or Con A receptors as a function of shape or transformation. The primary observations from this study are (a) membrane WGA and sCon A receptors in spherical Balb/c 3T3 fibroblasts display mobility 12 times faster than in the spread state, while phospholipid mobility is similar and apparently shape independent, (b) transformed cells in the spread state have WGA and sCon A receptor mobilities similar to those of untransformed cells in the spread state, (c) flat adherent, but not unattached spherical, Balb/c 3T3 fibroblasts are subject to Con A-induced global modulation and (d) transformed cells in the spherical state contain a significant population of cells (approximately 30%) with WGA receptor mobilities faster than those observed in spherical untransformed cells. These observations are discussed in terms of a linked matrix model for membrane protein diffusion.

Actins↗