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PubMed1991

The natural genomic variability of poliovirus analyzed by a restriction fragment length polymorphism assay.

The genomic variability of poliovirus was examined by analyzing the restriction fragment length polymorphism of a reverse-transcribed genomic fragment amplified by the polymerase chain reaction. The fragment was a 480-nucleotide sequence of the poliovirus genome coding for the N-terminal half of the capsid protein VP1, including antigenic site 1. The identification of a pair of generic primers flanking this fragment allowed its amplification in practically all the poliovirus strains tested so far (more than 150). By using the restriction enzymes HaeIII, DdeI, and HpaII, strain-specific restriction profiles could be generated for the amplified genomic fragment of each of the six reference poliovirus strains tested: one representative wild poliovirus of each of the three serotypes (P1/Mahoney, P2/Lansing, and P3/Finland/23127/84) and the three Sabin vaccine strains. When 21 poliovirus field isolates previously identified as Sabin vaccine-related were tested, they showed restriction profiles identical to those of the originating homotypic Sabin virus, demonstrating the conservation of these profiles during virus replication in humans. These profiles could thus be used as markers for Sabin-derived genotypes. To compare the distribution of poliovirus genotypes in nature before and after the introduction of poliovirus vaccines, the restriction profiles of the amplified genomic fragment of a total of 72 strains of various geographic and temporal origins were determined. Strains isolated before the introduction of polio vaccines displayed a wide diversity of genotypes. In contrast, wild (Sabin unrelated) strains isolated after vaccine introduction, during a single epidemic in a particular geographic area, showed identical or very similar restriction profiles, indicating the circulation of predominant regional genotypes. Our results indicate that the assay we developed for the analysis of the restriction fragment length polymorphism of the poliovirus genome may be used to identify and characterize poliovirus genotypes circulating in nature.

Base Sequence↗
PubMed1992

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.

Amino Acid Sequence↗
PubMed1993

Bacterial expression of a single-chain antibody fragment (SCFV) that neutralizes the biological activity of human interferon-gamma.

Studies with animal models have indicated that neutralizing antibodies against human interferon-gamma (HuIFN-gamma) may be used to treat a number of diseases in man. A major handicap for the implementation of this form of therapy is the immunogenicity of antibodies of non-human origin. Antibody fragments that do not contain parts of the most immunogenic regions may help to circumvent this problem. Therefore, we have constructed several antibody fragments [VH (variable fragment of the heavy chain), Fv (variable fragment) and scFv (single-chain Fv)] derived from a murine hybridoma (D9D10) which produces a neutralizing antibody against HuIFN-gamma. cDNAs encoding the variable domains of the L and H chains of D9D10 were cloned by PCR-based techniques in a suitable E. coli expression system. Bacterial clones are described that produce either VH, Fv or scFv. The Ig fragments were secreted into the periplasm and leaked into the culture supernatant. By SDS-PAGE and immunoblot analysis, the fragments were shown to be of the expected size (15, 14 and 30 kDa for VH, Fv and scFv, respectively). Functionality of the recombinant Ig fragments was tested by an ELISA for HuIFN-gamma binding and by a neutralization assay for the antiviral activity of HuIFN-gamma. Fv as well as scFv, but not VH, were found to bind to HuIFN-gamma and to neutralize its antiviral activity. Since it is found that scFv proteins are more stable at physiological temperatures than the Fv, it may have potential usefulness for the treatment of diseases in which overproduction of IFN-gamma plays a crucial role.

Amino Acid Sequence↗
PubMed1982

Use of substance P fragments to differentiate substance P receptors of different tissues.

The C- and N-terminal fragments of substance P were compared to the parent molecule with respect to their ability to: (a) contract the isolated guinea pig ileum, (b) induce salivation in the rat, (c) excite single cat dorsal horn neurones, and (d) induce scratching by intracranial injections in mice. C-terminal fragments as small as the heptapeptide were potent SP agonists on all assay systems. C-terminal fragments containing five amino acids or less were, at most, only weakly active. The C-terminal hexapeptide was a potent SP receptor stimulant on the isolated guinea pig ileum and, when directly applied by microiontophoresis, on cat dorsal horn neurons. However, the same compound was only 2-5% as potent as substance P in eliciting salivation and scratching in vivo, an indication that this fragment may be especially labile to enzymatic degradation. N-terminal fragments were totally inactive on the isolated guinea pig ileum. On the rat salivation and central nervous system assays, however, N-terminal fragments were capable of weak SP-like activity. It is concluded that SP receptors exist in multiple forms which we have labelled SP1 and SP2 receptors for those insensitive or sensitive to N-terminal fragments, respectively.

Animals↗
PubMed1986

Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth: structure-function correlations.

Heparin-binding fragments derived from the amino- and carboxyl-terminal regions of human plasma fibronectin appear to be at least relatively specific potent inhibitors of the growth of bovine aortic endothelial cells in culture by as yet unknown mechanisms. In order to understand better the sites which subserve this activity, we have compared the relative potency of other major fragments of fibronectin, most of which have dissimilar properties and do not bind heparin. We have also proteolytically digested and chemically modified the most potent of these fragments, the amino-terminal 29-kDa fragment, in order to test whether structural alterations that affect heparin-binding also affect the inhibitory property. Not all chemical modifications that abolished heparin-binding also abolished endothelial cell growth. Neither an amino-terminal 20-kDa nor a carboxyl-terminal 8-kDa subfragment of the 29-kDa fragment bound heparin; however, both were as inhibitory as native 29-kDa fragment. Reduction of the disulfides of the 20-kDa and 8-kDa fragments did not abolish inhibitory activity. We therefore conclude that the activity is not strictly conformation-dependent and that although the inhibitory activity is distributed throughout the 29-kDa segment, it can be expressed by an 8-kDa carboxyl-terminal segment containing residues of the last Type I loop structure.

Cells, Cultured↗
PubMed1992

Spectroscopic studies of fibrinogen and its plasmin-derived fragments.

The secondary structure of human fibrinogen and its plasmin-fragments have been studied by FTIR spectroscopy. The quantitative results for fibrinogen are in good agreement with previous studies using circular dichroism spectroscopy. After treatment of fibrinogen with plasmin in buffer containing Ca2+, two major fragments are produced: fragment E (Mw 45,000) and fragment D (Mw 100,000). Fragment E is shown to contain 50% alpha-helical values, attributed to its coiled-coil portions, and minor beta-strands and turn structures. Its deuteration gives evidence of the presence of solvent-exposed alpha-helical structures. On the other hand, fragment D contains a distribution of secondary structure values of 35% alpha-helix, 29% beta-sheet segments and 17% turn structures. Fragment D itself has two domains: a portion of the original coiled-coil and also a thermally labile globular domain. The coiled-coil portion (Mw 27,000) was isolated and showed a high alpha-helical content (around 70%). The globular domain is estimated to be rich in beta-sheet structures. The spectra of fibrin clots formed in Ca(2+)-containing buffer have a lower amide I/amide II ratio than fibrinogen spectra, which is interpreted as being due to aggregation.

Fibrinogen↗
PubMed1995

Intracellular degradation of apolipoprotein B generates an N-terminal 70 kDa fragment in the endoplasmic reticulum.

Regulated apoB degradation in HepG2 cells occurs in the endoplasmic reticulum (ER), is catalyzed by an N-acetylleucylleucylnorleucinal (ALLN)-sensitive proteinase, and generates a specific 70 kDa fragment (Adeli, K., 1994, J. Biol. Chem. 269, 9166-9175) [corrected]. In the present report, we have characterized the 70 kDa fragment by immunoprecipitation of permeabilized HepG2 cells with a battery of monoclonal antibodies against various sites on the apoB molecule. N-Terminal monoclonal antibodies (1D1 and 2D8) were capable of binding to the 70 kDa fragment suggesting that this polypeptide is an N-terminal fragment of the intact apoB. Subcellular fractionation of permeabilized cells and carbonate extraction resulted in the detection of the 70 kDa fragment in the ER lumen. Endoglycosidase H treatment confirmed that the fragment is N-linked glycosylated. We hypothesize that the ALLN-sensitive proteinase which may be located on the luminal side of the ER membrane, catalyzes an initial cleavage of apoB near the N-terminus generating a 70 kDa fragment, which is then released into the ER lumen.

Animals↗
PubMed1980

Somatomedin-like bioactivities of a growth hormone fragment on embryonic chick cartilage and cultured human fibroblasts.

Somatomedins are growth hormone-dependent peptides which appear to mediate many of the effects of growth hormone in vivo. These peptides are commonly assayed by their enhancement of proteoglycan sulfation in cartilage (or fibroblasts). We now report that fragment A-II (bovine growth hormone, 96--133) stimulates sulfation in chick embryonic cartilage and cultured fibroblasts. Enhancement of fibroblast proteoglycan sulfation by fragment A-II was log-dose dependent with maximal stimulation at 10(-8) M. The 25--100% maximal enhancement by fragment A-II was similar to that reported with a preparation of somatomedin A (Wasteson, A., Uthne, K., Westermark, B. (1973) Biochem. J. 136, 1069--1074). Sulfation of chick cartilage, in the presence of both serum (hypopituitary human) and fragment A-II was greater than the sum of the effects of each substance tested separately. Fragment A-II was tested between 10(-12) and 10(-8) M; maximal stimulation occurred at 5 . 10(-11) M. To our knowledge, no other growth hormone fragment has yet been shown to possess these somatomedin-like bioactivities. Our results suggest that fragment A-II may be very useful as a model peptide to study the actions and mechanism of naturally occurring sometomedins.

Animals↗
PubMed1993

Localization of the approximately 12 kDa M(r) discrepancy in gel migration of the mouse glucocorticoid receptor to the major phosphorylated cyanogen bromide fragment in the transactivating domain.

The intact wild-type mouse glucocorticoid receptor has a theoretical molecular weight of approximately 96 kDa based on amino acid sequence, but on SDS-polyacrylamide gel electrophoresis it migrates as a protein of approximately 98 kDa. It is not known where the unusual primary structure or covalent modification responsible for this anomalous migration is located within the amino acid chain. In the course of examining the pattern of fragmentation of 32P-labeled glucocorticoid receptors from Chinese hamster ovary (CHO) cells containing amplified mouse receptor cDNA, we have found a localized region in the amino-terminal half of the receptor that accounts for this anomalous behavior. Cyanogen bromide treatment of the intact receptor produces a 23.4 kDa (theoretical) fragment consisting of residues 108-324 and containing all of the identified phosphorylated serines within the receptor. We find that the only large resolvable 32P-labeled receptor fragment produced after complete cyanogen bromide cleavage of intact receptors migrates with an apparent molecular weight of approximately 35 kDa. Because the apparent difference between the theoretical and the experimentally observed molecular weights of this cyanogen bromide fragment is essentially the same as the difference between the theoretical and experimental molecular weights of the intact mouse glucocorticoid receptor, we propose that some feature lying within this fragment accounts for slower migration. Although the existence of an additional phosphorylation site lying within the 15 kDa tryptic receptor fragment containing the DNA-binding domain has been contested, we also demonstrate that this fragment of the mouse glucocorticoid receptor is phosphorylated in vivo upon incubation of CHO cells in growth medium containing [32P]orthophosphate.

Animals↗
PubMed1994

The side chains responsible for the dimerization of the estradiol receptor by ionic bonds are lost in a 17 kDa fragment extending downstream from aa 303.

Fragments of 32, 26 and 17 kDa of the porcine estradiol receptor were prepared, all of which contain the ligand-binding site. While dimers of the 32 and 26 kDa fragments like those of intact receptor can be dissociated by protonation, the dimer of the 17 kDa fragment obtained by trypsination of the 26 kDa fragment is resistant to lowering the pH from 7.0 to 6.5 and below. Its dissociation can be achieved by 0.5 M MgCl2 at pH 7.0. All fragments are recognized by the MAB 13H2 in Western blots. The antibody also reacts with native receptor and the three fragments, both in their monomer and dimer states. The combining ratios of antibody with receptor, or its fragments, in the monomer and dimer states and the weakening of the estradiol-receptor bond by antibody attachment support the back to back and head to toe model of receptor dimers.

Amino Acids↗
PubMed2004

Fragmented DNA transport in dendrites of retinal neurons during apoptotic cell death.

Movement of fragmented DNA in dendrites of retinal neurons during the apoptotic cell death was investigated. The time-course of the movement of fragmented DNA in dendrites of retinal neurons undergoing apoptotic cell death induced by intravitreal N-methyl-d-aspartate (NMDA) injection were examined by in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method and fluorescence DNA detection technique by 4',6-diamidino-2-phenylindole (DAPI). The inhibitory effect of axoplasmic transport inhibitor, vincristine was also tested on the NMDA-induced fragmented DNA transport. The movement of fragmented DNA from apoptotic nuclei toward peripheral ends of the dendrites of the retinal neurons was clearly demonstrated. The transport of fragmented DNA, but not fragmentation per se, was completely inhibited by the co-administration of vincristine.

Animals↗
PubMed2006

hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro.

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.

Alkaline Phosphatase↗
PubMed2005

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the method of choice for the analysis of complex peptide mixtures. It combines the separation power of nanoflow LC with highly specific sequence analysis, allowing automated peptide sequencing with high resolution and throughput. For peptide fragmentation, the current experimental setup uses predefined parameters based on the mass-to-charge ratio of the individual precursor. Suitable parameters are typically established by empirical evaluation of fragment spectra of individual peptides used as standards. As a result, nonoptimal fragment spectra are obtained if peptides show fragmentation behavior different from these standards, which often result in the loss of sequence-specific fragment ion information. Here we describe a statistical approach for the systematic evaluation of the quality of individual peptide fragment spectra based on the calculation of their arithmetic mean and standard deviation. The method utilizes the dependence of these parameters on the difference in electric potential across the collision cell to determine the value that results in maximum information content. We show that the method is applicable to fragment spectra generated from a variety of multiply-charged tryptic peptides, over a wide concentration range, and on different types of mass analyzers. We also show how this novel approach can be used to define optimized collision energy settings over a wide mass-to-charge range.

Animals↗
PubMed2006

Evaluation of low energy CID and ECD fragmentation behavior of mono-oxidized thio-ether bonds in peptides.

Thio-ether bonds in the cysteinyl side chain of peptides, formed with the most commonly used cysteine blocking reagent iodoacetamide, after conversion to sulfoxide, releases a neutral fragment mass in a low-energy MS/MS experiment in the gas phase of the mass spectrometer [6]. In this study, we show that the neutral loss fragments produced from the mono-oxidized thio-ether bonds (sulfoxide) in peptides, formed by alkyl halide or double-bond containing cysteine blocking reagents are different under low-energy MS/MS conditions. We have evaluated the low-energy fragmentation patterns of mono-oxidized modified peptides with different cysteine blocking reagents, such as iodoacetamide, 3-maleimidopropionic acid, and 4-vinylpyridine using FTICR-MS. We propose that the mechanisms of gas-phase fragmentation of mono-oxidized thio-ether bonds in the side chain of peptides, formed by iodoacetamide and double-bond containing cysteine blocking reagents, maleimide and vinylpyridine, are different because of the availability of acidic beta-hydrogens in these compounds. Moreover, we investigated the fragmentation characteristics of mono-oxidized thio-ether bonds within the peptide sequence to develop novel mass-spectrometry identifiable chemical cross-linkers. This methionine type of oxidized thio-ether bond within the peptide sequence did not show anticipated low-energy fragmentation. Electron capture dissociation (ECD) of the side chain thio-ether bond containing oxidized peptides was also studied. ECD spectra of the oxidized peptides showed a greater extent of peptide backbone cleavage, compared with CID spectra. This fragmentation information is critical to researchers for accurate data analysis of this undesired modification in proteomics research, as well as other methods that may utilize sulfoxide derivatives.

Electrons↗
PubMed2006

Comparison of random and oriented immobilisation of antibody fragments on mixed self-assembled monolayers.

The sensitivity of immunosensors is strongly dependent on the amount of immobilised antibodies and their remaining antigen binding properties. The use of smaller and well-oriented antibody fragments as bioreceptor molecules influences the final immunosensor signal. The aim of this study was to compare the immunosensor responses of different immobilised antibody fragments, such as F(ab')2 and Fab', with their parental IgG. In addition, we evaluated the oriented versus the random covalent immobilisation method of the Fab' fragments. First, an optimisation of cleavage protocol to generate these F(ab')2 and Fab' fragments was performed. Subsequently, we pursued a study with limited denaturation effects during immobilisation of the bioreceptor molecules and with reduced steric hindrance during antigen binding using mixed self-assembled monolayers (SAM) of thiols as the chemical linking layer. The Surface Plasmon Resonance technique was used to evaluate the degree of immobilisation of the antibody fragments and their parental IgGs on the mixed SAMs and the binding signals of their specific antigens. In this study, we demonstrate that for a particular antibody/antigen system (anti-hIgG/hIgG), the optimised fragmentation protocol in combination with an oriented immobilisation of Fab' fragments on mixed SAMs leads to a >2-fold increase of the antigen binding signals compared to randomly covalent immobilised full-length antibodies.

Animals↗
PubMed2005

Enhanced catalytic efficiency of aminoglycoside phosphotransferase (3')-IIa achieved through protein fragmentation and reassembly.

Many monomeric proteins can be split into two fragments, yet the two fragments can associate to make an active heterodimer. However, for most locations in a protein such a conversion is not feasible, presumably due to inefficient assembly or improper folding of the fragments. For some locations, this can be overcome by fusion of the fragments to dimerization domains that facilitate correct assembly. A variety of heterodimers of aminoglycoside phosphotransferase (3')-IIa (Neo) were created in which the Neo fragments required fusion to a pair of leucine zippers for activity in vivo. However, the ability of these heterodimers to confer kanamycin resistance to Escherichia coli cells was impaired compared to wild-type Neo, primarily due to poor production of soluble protein. The mutations R177S and V198E restored the kanamycin resistance to wild-type levels while maintaining the dependence on leucine zippers for activity. These mutations restored high levels of kanamycin resistance not through an improvement in the production of soluble protein but rather by conferring a large improvement in k(cat)/K(m), surpassing that of Neo. Furthermore, whereas R177S and V198E served to improve k(cat)/K(m) 60-fold in the context of the heterodimer, the same mutations in the context of wild-type Neo had a ninefold negative effect on k(cat)/K(m). This demonstrates the possibility that enzymes with improved catalytic properties can be created through a process involving fragmentation and fusion to domains that facilitate assembly of the fragments.

Amino Acid Sequence↗
PubMed2003

Effects of leptin and leptin fragments on steroid secretion of freshly dispersed rat adrenocortical cells.

The biological actions of leptin on target tissues are mediated via several isoforms of receptors (Ob-Rs), which may differently interact with native leptin and its fragments. Based on the presence in the rat adrenals of at least two Ob-R isoforms and the conflicting findings on the effect of leptin on adrenocortical secretion, we investigated the effects of the native leptin and several leptin fragments (10(-8) and 10(-6)M) on aldosterone and corticosterone secretion from freshly dispersed rat zona glomerulosa (ZG) and zona fasciculata-reticularis (ZF/R) cells. Reverse transcription (RT)-polymerase chain reaction (PCR) showed the expression of Ob-Ra and Ob-Rb mRNAs in both ZG and ZF/R cells. Native murine leptin (1-147) enhanced aldosterone and corticosterone secretion from dispersed ZG and ZF/R cells, and similar effects were elicited by murine leptin fragment 116-130, and human leptin fragments 138-167, 150-167 and [Tyr] 26-39. Human leptin fragment 93-105 was ineffective, while fragment 22-56 decreased corticosterone output without affecting aldosterone secretion. Taken together, our findings indicate that in rat adrenocortical cells leptin and leptin fragments may differently interact with Ob-Rs or interact with different Ob-R isoforms. Moreover, they suggest that (1) the direct adrenocortical secretagogue effect of leptin mainly depends on the C-terminal sequence 116-166; and (2) the N-terminal sequence is not needed for leptin to activate Ob-Rs positively coupled to steroidogenesis, but is possibly responsible for a direct inhibitory effect on glucocorticoid secretion.

Adrenal Cortex↗
PubMed2004

ERK1-2 and p38 MAPK regulate MMP/TIMP balance and function in response to thrombospondin-1 fragments in the microvascular endothelium.

We found that thrombospondin-1 (TSP-1) has opposite functions on angiogenesis depending on the nature of the proteolytic fragment released in vivo by the action of proteases. We studied the effect of the 25 and 140 kDa fragments of TSP-1 generated by its proteolytic cleavage on the cascade of mitogen activated protein kinase (MAPK) activation and matrix-metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) function and expression in microvascular endothelium. Post-capillary endothelial cells (CVEC) isolated from bovine heart were used. The 25 kDa fragment enhanced the upregulation of MMP-2 and -9 and reduced TIMP-2 expression leading to CVEC chemoinvasion. Conversely, the 140 kDa fragment blocked MMP-2 and -9 stimulation and doubled TIMP-2 expression, leading to inhibition of endothelial chemoinvasion induced by fibroblast growth factor-2 (FGF-2). MAPK activity (ERK1-2) was induced by TSP-1 and by the 25 kDa fragment, but not by the 140 kDa fragment which, however, promoted MAPK p38 activation. This evidence indicates that fragments originating from TSP-1 switch the pro- or anti-angiogenic phenotype in endothelium by targeting MAPK cascades with opposite functions on MMP/TIMP balance.

Animals↗