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PubMed1992

Overexpression of the csA cell adhesion molecule under its own cAMP-regulated promoter impairs morphogenesis in Dictyostelium.

The contact site A (csA) glycoprotein is a strictly developmentally regulated plasma membrane component responsible for the EDTA-stable (Ca(2+)-independent) form of intercellular adhesion in Dictyostelium discoideum. Using inverse polymerase chain reaction and a terminator vector we have isolated a 1.6 kb genomic fragment carrying a 1.1 kb upstream region of the csA gene. This fragment had promoter activity in D. discoideum cells, giving rise to a 3'-truncated csA RNA that was regulated like the mRNA of the endogenous gene. Cyclic AMP pulses strongly enhanced transcription from the cloned csA promoter. These findings provide evidence that the cloned region of the csA gene comprises the complete promoter. It contains a G/C-rich octamer motif similar to other cAMP-regulated D. discoideum promoters. When the csA protein was strongly overexpressed under the developmental control of the csA promoter, morphogenesis was substantially altered. Aggregation was delayed, and secondary centres were formed along aggregation streams that led to fragmentation of the aggregates and multiple slug formation. At high cell density a substantial portion of aggregated cells was left behind on the substratum when slugs and fruiting bodies were built. The transformation vector was also employed to rescue a csA-negative mutant, HG1287, from its cell adhesion defect.

Animals↗
PubMed2006

Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates. In this paper, we describe two kinds of method of producing Qbeta replicase. In one kind, both EF-Tu and EF-Ts subunits were expressed with the beta-subunit, and in the other kind, the beta-subunit was genetically fused with EF-Tu and EF-Ts. The fused protein, a single-chain alpha-less Qbeta replicase, was mostly found in the soluble fraction and could be readily purified. These results pave the way for the large-scale production of the highly purified form of this enzyme.

Enzyme Activation↗
PubMed1994

Molecular cloning and the COOH-terminal processing of gp64, a putative cell-cell adhesion protein of the cellular slime mold Polysphondylium pallidum.

The cellular slime mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.

Amino Acid Sequence↗
PubMed2005

Assay and functional analysis of dynamin-like Mx proteins.

Mx proteins are interferon-induced large guanosine triphosphatases (GTPases) that share structural and functional properties with dynamin and dynamin-like proteins, such as self-assembly and association with intracellular membranes. A unique property of some Mx proteins is their antiviral activity against a range of RNA viruses, including influenza viruses and members of the bunyavirus family. These viruses are inhibited at an early stage in their life cycle, soon after host cell entry and before genome amplification. The association of the human MxA GTPase with membranes of the endoplasmic reticulum seems to support its antiviral function by providing an interaction platform that facilitates viral target recognition, MxA oligomerization, and missorting of the resulting multiprotein complex into large intracellular aggregates.

3T3 Cells↗
PubMed1985

Sedimentation equilibrium measurements of the intermediate-size tobacco mosaic virus protein polymers.

Short-column sedimentation equilibrium methods have been applied for the first time to tobacco mosaic virus (TMV) protein (0.1 M ionic strength orthophosphate) at pH 6.5 and at pH 7.0 to estimate molecular weights. Previous sedimentation velocity experiments at pH 6.5, 20 degrees C have led to the conclusion that the major boundary with an S0(20),w value of 24.4 +/- 0.1 S consists of a distribution of polymers which are mainly three-turn, 48-51-subunit helical rod aggregates. The directly measured z-average molecular weights together with sedimentation velocity data are entirely consistent with this assignment of a three-turn aggregate. Molecular weights have also been determined under two conditions where a large mass fraction of the protein sediments with an S0(20),w value of 20.3 +/- 0.2 S. At pH 6.5, 6-8 degrees C, the aggregates in this boundary are metastable and correspond to 50-60% of the preparation. At pH 7.0, 20 degrees C at equilibrium, 65-75% of the protein sediments at 20.3 S. The 20.3S boundary is very similar under both conditions and is interpreted as being composed of a distribution of protein aggregates centered about 39 +/- 2 subunits. This result is important in the interpretation of previous kinetic measurements of TMV self-assembly. The current view is that the 34-subunit structure of TMV protein, in the form of a cylindrical disk which is made up of two 17-subunit layers and has been characterized in single-crystal X-ray diffraction studies, plays a central role in the initial binding steps with RNA. The present results are not consistent with the view that there is a significant concentration of the TMV protein disk structure in solution under the usual conditions of TMV self-assembly.

Hydrogen-Ion Concentration↗
PubMed2006

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid-beta peptide in vivo.

Accumulation of insoluble aggregates of amyloid-beta peptide (Abeta), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimer's disease (AD). Consequently, downregulation of APP, or enhanced clearance of Abeta, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Abeta accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Abeta (HSV-neprilysin), substantially inhibited accumulation of Abeta. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APP(Sw)), resulted in rapid Abeta accumulation. Co-inoculation of LV-APP(Sw) with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Abeta accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Abeta accumulation in the brain, thus providing useful tools to clarify the role of Abeta in AD that may facilitate the development of novel therapies for this important disease.

Alzheimer Disease↗
PubMed2003

Nestin promotes the phosphorylation-dependent disassembly of vimentin intermediate filaments during mitosis.

The expression of the intermediate filament (IF) protein nestin is closely associated with rapidly proliferating progenitor cells during neurogenesis and myogenesis, but little is known about its function. In this study, we examine the effects of nestin expression on the assembly state of vimentin IFs in nestin-free cells. Nestin is introduced by transient transfection and is positively correlated with the disassembly of vimentin IFs into nonfilamentous aggregates or particles in mitotic but not interphase cells. This nestin-mediated disassembly of IFs is dependent on the phosphorylation of vimentin by the maturation/M-phase-promoting factor at ser-55 in the amino-terminal head domain. In addition, the disassembly of vimentin IFs during mitosis appears to be a unique feature of nestin-expressing cell types. Furthermore, when the expression of nestin is downregulated by the nestin-specific small interfering RNA in nestin-expressing cells, vimentin IFs remain assembled throughout all stages of mitosis. Previous studies suggest that nonfilamentous vimentin particles are IF precursors and can be transported rapidly between different cytoplasmic compartments along microtubule tracks. On the basis of these observations, we speculate that nestin may play a role in the trafficking and distribution of IF proteins and potentially other cellular factors to daughter cells during progenitor cell division.

Animals↗
PubMed2006

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Antigens, Surface↗
PubMed1983

DNA-binding proteins of Haemophilus influenzae: purification and characterization of a major intracellular binding protein.

A heat- and acid-stable protein which bound both native and denatured DNA but not RNA was extensively purified from extracts of Haemophilus influenzae Rd strain com-58-A. The active species had an apparent subunit molecular weight of 15,000. The interaction of the protein with denatured DNA appeared to be cooperative, as judged by the sigmoid shapes of binding curves. This cooperativity increased with increasing ionic strength and was more pronounced with sodium ions than with potassium ions. Gel filtration suggested that the native protein formed aggregates in solution. The presence of the binding protein protected single-stranded DNA from the action of S1 endonuclease; approximately 30 nucleotide residues were protected per subunit equivalent of protein. The number of subunit equivalents per cell of this protein has been estimated at 10,000. The protein, which we designate DNA-binding protein II, is most probably a major histone-line protein of H. influenzae.

Chromatography, Affinity↗
PubMed1981

Trypsin enhancement of rotavirus infectivity: mechanism of enhancement.

The infectivity of most rotaviruses is enhanced by treatment with trypsin. We studied the mechanism of enhancement of examining the effect of trypsin on rotavirus infectivity, aggregation, early interactions with host cells, and structure. The results indicated that trypsin does not increase levels of infectious virus by dispersion of aggregates or affect the efficiency or rate of attachment of virus to cells. A fraction of virus that was not infections without trypsin treatment was found to attach to cells, but did not initiate antigen synthesis. When cells were infected with labeled, purified virus, increased levels of uncoated particles were found in cells infected with trypsin-treated virus. Infection of cells with trypsin-treated virus also led to greater levels of RNA synthesis early in the infection. The results suggest that trypsin converts a noninfectious fraction of virus into infectious virus by allowing this fraction to uncoat in the infected cell. Trypsin was found to cleave an 88,000-dalton structural polypeptide of bovine rotavirus generating 67,000- and 20,000-dalton cleavage products.

Animals↗
PubMed1975

LH and its subunits in human pituitary, serum and urine.

Human postmenopausal serum and urine, and saline extracts of human pituitaries were gel-filtered on Sephadex G-100. The eluted fractions were radioimmunoassayed using 3 specific assays for native hLh, hLHalpha and hLHbeta. The elution profile of immunoreactive components in postmenopausal urine exhibited 2 peaks of activity corresponding to those demonstrated for 125I-labeled hLH and hLHalpha. No LHbeta was demonstrated. The immunoreactive components of post-menopausal serum eluted identically to 125I-LH, LHalpha and LHbeta. In addition, a peak of immunoreactive LH material was observed in the pre-LH region of the elution profile, distinctly separate from the void volume. The percent of the total immunoassayable serum LH contained in this peak varied. In elution profiles of all pituitary extracts, LH, LHalpha and LHbeta elution peaks corresponding to the respective labeled preparations were observed. An apparent excess of free alpha subunit was noted. All three antisera demonstrated immunoassayable LH material in the void volume. This large species contained a higher proportion of material reacting in the anti-hLHalpha system than did native hLH. Examination of the void volume material by electron microscopy indicated membrane material, but no ribosomes. Chemical assay failed to detect RNA. When void volume material was subjected to conditions which are known to (a) disrupt membrane continuity and (b) dissociate the noncovalently bonded subunits of native LH, this large species of LH failed to dissociate into smaller molecular species. These studies reveal: (1) an excess of free alpha subunit in the pituitary, serum and urine; (2) a large species of LH in the pituitary which upon analysis does not appear to be simple aggregation of LH, MEMBRANE ENTRAPPED NATIVE LH, OR RIBOSOMAL BOUND LH.

Cell Membrane↗
PubMed2006

Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function.

Metastatic tumor cells that actively migrate and invade surrounding tissues rely on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are membrane protrusions that localize enzymes required for ECM degradation. Little is known about the formation, function, and regulation of invadopodia. Here, we show that invadopodia have two distinct aspects: (a) structural for organizing the cellular actin cytoskeleton to form membrane protrusions and (b) functional for using proteolytic enzyme(s) for ECM degradation. Small interfering RNA (siRNA) inhibition established that organization of invadopodia structure requires cortactin, whereas protease inhibitor studies identified membrane type 1 matrix metalloproteinase (MT1-MMP) as the key invadopodial enzyme responsible for gelatin matrix degradation in the breast carcinoma cell line MDA-MB-231. The inhibition of invadopodial structure assembly by cortactin depletion resulted in a block of matrix degradation due to failure of invadopodia formation. Either protease inhibition or MT1-MMP siRNA depletion moderately decreased the formation of invadopodial structures that were identified as actin-cortactin accumulations at the ventral cell membrane adherent to matrix. The invadopodia that were able to form upon MT1-MMP inhibition or depletion retained actin-cortactin accumulations but were unable to degrade matrix. Examination of cells at different time points as well as live-cell imaging revealed four distinct invadopodial stages: membrane cortactin aggregation at membranes adherent to matrix, MT1-MMP accumulation at the region of cortactin accumulation, matrix degradation at the invadopodia region, and subsequent cortactin dissociation from the area of continued MT1-MMP accumulation associated with foci of degraded matrix. Based on these results, we propose a stepwise model of invadopodia formation and function.

Actins↗
PubMed1998

Advanced glycation endproducts inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in human microvascular endothelial cells.

Several thrombogenic abnormalities are associated with diabetes. To investigate the underlying molecular mechanisms, we examined the effects of advanced glycation endproducts (AGE), non-enzymatically glycated protein derivatives, on the production of prostacyclin (PGI2), an anti-thrombogenic prostanoid, and of plasminogen activator inhibitor-1 (PAI-1), a fast-acting serine protease inhibitor of fibrinolysis, in human microvascular endothelial cells (EC). Firstly, AGE-bovine serum albumin (BSA) but not non-glycated BSA, was found to considerably decrease the production of PGI2 to about two-thirds of the control value. Secondly, quantitative reverse transcription-polymerase chain reaction showed that AGE-BSA increased the EC levels of mRNA coding for PAI-1, this being associated with a concomitant increase in the immunoreactive PAI-1 contents and the anti-fibrinolytic activity. Thirdly, the effects of AGE on PGI2 and PAI-1 syntheses in EC were found to be mediated by a receptor for AGE (RAGE) because antisense DNA against RAGE mRNA could reverse the AGE effects. Further, it was found that AGE decreased the intracellular cyclic AMP concentrations in EC and that cyclic AMP agonists such as dibutyryl cyclic AMP, forskolin and PGI2 analogue reduced the AGE-stimulated PAI-1 production, suggesting the involvement of cyclic AMP in the AGE-signalling pathway. The results thus suggest that AGE have the ability to cause platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis and thereby contributing to the development and progression of diabetic vascular complications.

Cyclic AMP↗
PubMed2000

Detection of abundant sulphate-reducing bacteria in marine oxic sediment layers by a combined cultivation and molecular approach.

The depth distribution and diversity of sulphate-reducing bacteria (SRB) was analysed in the upper intertidal zone of a sandy marine sediment of the Dutch island Schiermonnikoog. The upper centimetre of the sediment included the oxic-anoxic interface and was cut into five slices. With each slice, most probable number (MPN) dilution series were set up in microtitre plates using five different substrates. In the deeper sediment layers, up to 1 x 10(8) cm(-3) lactate-utilizing SRB were counted, corresponding to 23% of the total bacterial count. From the highest positive dilutions of the MPN series, 27 strains of SRB were isolated in pure culture. Sequencing of a 580 bp fragment of the 16S rDNA revealed that 21 isolates had identical sequences, also identical with that of the previously described species Desulfomicrobium apsheronum. However, the diversity of the isolates was higher with respect to their physiological properties: a total of 11 different phenotypes could be distinguished. Genomic fingerprinting by enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) revealed an even higher diversity of 22 different genotypes. A culture-independent analysis by PCR and denaturing-gradient gel electrophoresis (DGGE) revealed that the partial 16S rDNA sequence of the isolated D. apsheronum strains constituted a significant fraction of the Desulfovibrionaceae. The high subspecies diversity suggests that this abundant aggregate-forming species may have evolved adaptations to different ecological niches in the oxic sediment layers.

Colony Count, Microbial↗
PubMed2002

Intracellular deposition, microtubule destabilization, and transport failure: an "early" pathogenic cascade leading to synaptic decline.

Protein deposition is a common event in age-related neurological diseases that are characterized by neuronal dysfunction and eventual cell death. Here, cultured hippocampal slices were infused with the lysosomal disrupter chloroquine to examine the link between abnormal protein processing/deposition and early synaptopathogenesis. Tau species of 55 to 69 kDa increased over several days of treatment with chloroquine, while the protein and message levels of synaptic markers were selectively reduced. Neurons of subfields CA1, CA3, and dentate gyrus accumulated protein deposits recognized by antibodies against paired helical filaments and ubiquitin, and this was accompanied by tubulin fragmentation and deacetylation. The deposition filled the basal pole of pyramidal neurons, encompassing the area of the axon hillock and initial dendritic branching but without causing overt neuronal atrophy. Neurons containing the polar aggregates exhibited severely impaired transport along basal dendrites. Transport capability was also lost along apical dendrites, the opposite direction of deposited material in the basal pole; thus, perpetuating the problem beyond physical blockage must be the associated loss of microtubule integrity. These data indicate that transport failure forms a link between tau deposition and synaptic decline, thus shedding light on how protein aggregation events disrupt synaptic and cognitive functions before the ensuing cellular destruction.

Aging↗
PubMed1990

Characterisation of the Klebsiella pneumoniae nitrogen-fixation regulatory proteins NIFA and NIFL in vitro.

Transcriptional activation by the Klebsiella pneumoniae nitrogen-fixation-specific positive control protein, NIFA, (nifA gene product) has been demonstrated in vitro in S30 extracts from cells which overproduce this protein. The activity of NIFA was dramatically reduced in vitro in the presence of the negative regulatory protein NIFL (nifL gene product) but was not inhibited by the presence of a mutant NIFL protein, NIFL2404. Transcriptional activation from the nifH promoter by NIFA was dependent on the alternative sigma factor, sigma 54, and also required the presence of an upstream activator sequence. NIFA activity was temperature-sensitive in vitro (as it is in vivo) which is due, at least in part, to the intrinsic lability of the protein itself. The majority of overproduced NIFA and NIFL was insoluble after low-speed centrifugation and was inactive in vitro. A low level of less aggregated NIFA protein present in cell extracts was responsible for in vitro activity and this fraction was partially purified.

Bacterial Proteins↗
PubMed2006

Opposing activities protect against age-onset proteotoxicity.

Aberrant protein aggregation is a common feature of late-onset neurodegenerative diseases, including Alzheimer's disease, which is associated with the misassembly of the Abeta(1-42) peptide. Aggregation-mediated Abeta(1-42) toxicity was reduced in Caenorhabditis elegans when aging was slowed by decreased insulin/insulin growth factor-1-like signaling (IIS). The downstream transcription factors, heat shock factor 1, and DAF-16 regulate opposing disaggregation and aggregation activities to promote cellular survival in response to constitutive toxic protein aggregation. Because the IIS pathway is central to the regulation of longevity and youthfulness in worms, flies, and mammals, these results suggest a mechanistic link between the aging process and aggregation-mediated proteotoxicity.

Aging↗
PubMed1972

Effects of treatment with sodium dodecyl sulfate on the ultrastructure of Escherichia coli.

An electron microscopy study has been made of the effects of dissolution of the plasma membrane of Escherichia coli with sodium dodecyl sulfate (SDS) on the organization of the nucleoplasm and the cytoplasm. The alterations observed in time course experiments were related to absorbance changes and to release of macromolecules from the cells. As the cells became plasmolyzed, under the conditions used, the first visible effect of SDS was a collapse of the plasmolysis spaces. This was accompanied by a displacement of the nuclear material which then appeared in broad contact with the redeployed plasma membrane. This initial displacement of nuclear material to the cell border may indicate an association between the nucleoplasm and the plasma membrane. Upon further dissolution of the plasma membrane, the nuclear material receded from the cell margin and contracted into an axial filament. Meanwhile, the cytoplasm dissociated into an amorphous, Pronase-sensitive component and an electron-opaque, granular one sensitive to ribonuclease. The latter represented one continuous area of ribosomal structures surrounding the nucleoplasm, an organization which did not occur when the cells were inhibited with rifamycin before SDS treatment. During prolonged SDS interaction, approximately 65% of the cellular protein, 25% of the ribonucleic acid and 40% of the deoxyribonucleic acid were released from the cells concomitant with the disappearance of the amorphous cytoplasmic part, expansion of the ribosomal aggregate, and rearrangement of the nuclear material at the cell periphery. The observations support the contention that all ribosomal structures bear a direct relationship with the nucleoplasm.

Agar↗

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