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PubMed1979

Anion transport across the erythrocyte membrane, in situ proteolysis of band 3 protein, and cross-linking of proteolytic fragments by 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonate.

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents. Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a cross-linking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport, Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the noninhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4'-diisothiocyano dihydrostilbene-2,2'-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control anion transport.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1980

Arrangement of the single-stranded fragments in E. coli bacteriophage BF23 DNA.

Bacteriophage BF23st(0) DNA was denatured with alkali and fractionated by agarose gel electrophoresis. Seven single-stranded fragments (designated Fragments I--VII) were identified as the major constituents of the phage DNA. The presence of several minor fragments which represent minor populations of the phage genome was also observed. The largest fragment (Fragment I) represents the intact strand of phage DNA, whereas the other fragments form the complementary strand. Thus, BF23st(0) DNA carries single-strand interruptions in only one strand. The arrangement of the major fragments in the nicked strand was determined by use of gamma-exonuclease and agarose gel electrophoresis. From the mode of action of this nuclease, and from the kinetics of release or disappearance of the fragments, the polarity of the fragments in BF23st(0) DNA was specified. In addition, the presence of two types of major phage populations differing in their composition of the fragments was demonstrated. One type has an additional nick (yielding Fragment IV and Fragment V) in a specific fragment (Fragment II) of other type.

Chromosome Mapping↗
PubMed2004

Enhancement of scFv fragment reactivity with target antigens in binding assays following mixing with anti-tag monoclonal antibodies.

The phage display Ab library technology has been found to be a useful method to isolate antigen-specific Ab fragments, since the repertoire of antibody specificities is broad and since it bypasses the need of immunization. However, when screening clones isolated from a phage display Ab library, the yield of isolating antigen-specific Ab fragments is low and the rate of false negative results is high. This limitation reflects the low affinity/avidity of Ab fragments and/or the low density of the target antigen. To facilitate the isolation of Ab fragments with a broad range of affinities to antigens of interest from phage display Ab libraries, we have developed a simple method to increase the sensitivity of binding assays to detect the reactivity of single-chain fragments of antibody variable regions (scFv) with target antigens. This method involves the mixing of scFv fragments, expressing a c-myc epitope tag, with anti-tag mAb 9E10 prior to their use in binding assays in order to form stable dimeric Ab fragment-anti-tag mAb complexes. The increase in the reactivity of scFv fragments with the corresponding antigen is observed over a broad range of scFv fragment (6-800 microg/ml) and mAb 9E10 (0.5-30 microg/ml) concentrations, thereby facilitating the testing of scFv fragment preparations with unknown scFv fragment concentrations. Use of this method in binding assays resulted in a twofold increase in the reactivity of low-affinity purified scFv fragments with the corresponding antigen. Moreover, application of this method to screen clones isolated from phage display scFv libraries resulted in a reproducible increase in both the yield of antigen-specific scFv clones and the titer of scFv fragment preparations by a factor of 5 and 2- to 32-fold, respectively. Lastly, this method can be applied in both ELISA and flow cytometry and is independent of the characteristics of the antigen (i.e. whole cells, carbohydrates and purified protein) and/or of the library (synthetic scFv Library (#1), a large semi-synthetic phage display scFv library and the human synthetic VH+VL scFv library (Griffin.1 library)) used. Therefore, the method we have described represents a sensitive, simple and reproducible technique that will facilitate the isolation and use of scFv fragments.

Animals↗
PubMed1998

Anterior chamber metal fragments after phacoemulsification surgery.

PURPOSE: To determine whether metal fragments can be shaken loose from phaco needles during surgery and embed in the iris. SETTING: Private practice, Vernon, British Columbia, Canada, and scanning electron microscope laboratory, Mastel Precision, Rapid City, South Dakota, USA. METHODS: The surfaces and rims of new and used phaco needles and the lumens of halved new needles were examined by scanning electron microscopy (SEM). To determine whether the fragments on the phaco needles were approximately the same size as those seen in the iris, a photograph of an eye with metal fragments imbedded in the iris was projected and the image size of the metal fragments approximated by using their magnification value. The magnification scale of the SEM images was used to determine the size of the metal fragments photographed on the phaco needles. RESULTS: The SEM studies of new phaco needles revealed tiny fragments of metal firmly adherent to the interior, exterior, and rim surfaces. No fragments were detected on the surfaces of the used phaco needles. Two metal fragments in the eye photograph were calculated to be 0.20 x 0.20 mm and 0.15 x 0.20 mm. Those in the SEM photos were calculated to be 0.03 to 0.10 mm. CONCLUSION: Althogh SEM of new titanium phaco needles revealed adherent metal fragments on their lathed surfaces, no fragments were found on used phaco needles. The iris fragments calculated from a projected photograph were slightly larger than those from the SEM micrographs, supporting the conclusion that annealed metal fragments shook loose from the phaco needles. This indicates that ultrasonic activation of a new phaco needle with metal fragments annealed to its surface causes fragments to release and embed in the iris.

Alloys↗
PubMed1983

Immortalization and neoplastic transformation of normal diploid cells by defined cloned DNA fragments of herpes simplex virus type 2.

Diploid Syrian hamster embryo (SHE) cells were passaged after transfection with recombinant plasmids containing herpes simplex virus type 2 (HSV-2) DNA inserts Bgl II focus-forming fragment N, Bgl II transforming fragment C, and EcoRI/HindIII fragment AE. Cultures transfected with salmon DNA or with 0.1-5.0 micrograms of Bgl II fragment N reached crisis and senesced. Those transfected with 0.1-0.5 micrograms of Bgl II fragment C or its left-hand 64% subclone EcoRI/HindIII fragment AE escaped senescence and formed continuous lines. At early passages, these lines as well as isolated clones grew in 2% serum but formed small (less than or equal to 0.1 mm) colonies in 0.3% agarose and were nontumorigenic. Serial passaging of Bgl II fragment C-induced cultures and isolated clones resulted in the appearance of large (greater than 0.25 mm) colonies in agarose followed by tumorigenicity. This behavior was not exhibited by the EcoRI/HindIII fragment AE-induced cultures that remained nontumorigenic after 53 passages. DNA from normal SHE cells exhibited homology to Bgl II fragment C but, under relatively stringent conditions, DNAs from transformed and tumor-derived lines exhibited discrete hybridizing bands comigrating with authentic viral fragments. These results indicate that neoplastic transformation of normal diploid SHE cells by HSV-2 DNA fragments involves at least two distinct steps--i.e., immortalization and conversion to tumorigenicity. EcoRI/HindIII fragment AE representing the left 64% of Bgl II fragment C is sufficient to induce immortalization. However, DNA sequences from both left-hand 64% and right-hand 36% subfragments of Bgl II fragment C are required for tumorigenic transformation.

Animals↗
PubMed1985

pH-induced unfolding of the constant fragment of the immunoglobulin light chain: effect of reduction of the intrachain disulfide bond.

Circular dichroism and proton nuclear magnetic resonance measurements were made to clarify how reduction and reduction plus alkylation of the intrachain disulfide bond affect the conformation and stability of the constant (CL) fragment of the immunoglobulin light chain. The pH titration behavior of the two histidine residues in the intact CL, reduced CL, and reduced and alkylated CL fragments were followed by proton nuclear magnetic resonance spectroscopy. It was shown that reduction of the intrachain disulfide bond does not affect the solution conformation of the immunoglobulin fold, whereas reduction and alkylation results in extensive unfolding of the protein molecule. These results are consistent with the previous results obtained by Goto and Hamaguchi [1979) J. Biochem. 86, 1433-1441) using circular dichroism, fluorescence, and titration of SH groups. The pH-induced unfolding of the reduced CL fragment was compared with that of the intact CL fragment. The stability of the intact CL fragment to acid was much greater than that of the reduced CL fragment. The equilibria of the unfolding by acid could be explained by assuming that the same ionizable groups participate in the unfolding for the reduced CL fragment as for the intact CL fragment, and that the stability of the intact CL fragment is 100 times greater than that of the reduced CL fragment. In the alkaline pH region, the reduced CL fragment was unfolded above pH 8.5, while the intact CL fragment was not unfolded until pH 11.6. The kinetics of the unfolding by alkali of the reduced CL fragment was also studied. The unfolding by alkali of the reduced CL fragment could be explained by assuming that the reduced CL molecule can no longer adopt the folded conformation when either or both SH groups ionize.

Circular Dichroism↗
PubMed2005

Library design for fragment based screening.

According to Hann's model of molecular complexity an increased probability of detection binding to a target protein can be expected when small, low complex molecular fragments are screened with high sensitivity instead of full-sized ligands with lower sensitivity. Analysis of the HTS summary data of Novartis and comparison with NMR screening results obtained on generic fragment libraries indicate this expectation to be true with hitrates of 0.001% - 0.151% observed in the identification of ligands with an IC(50) threshold in the micromolar range in an HTS setup and hitrates above or equal to 3% observed in NMR screening of fragments with an affinity threshold in the millimolar range. It is however necessary to keep in mind that the sets of target studied were not identical for both method and the experience in NMR screening is too limited for a final conclusion. The term hitrate as used here reflects only the success rate in the observation of ligand binding event. It must not be confused with the overall success rate of fragment and high throughput screening in the lead finding process, which can be entirely different, since the steps required to follow-up a ligand binding event to a lead are different for both methods. A survey of fragment-based lead discovery case studies given in the literature shows that in approximately half of the cases the initial hit fragment was discovered by screening a generic library, whereas in the other cases some knowledge about an initial ligands or the protein binding site has been used, whereas systematic virtual screening of fragment databases has been only rarely reported. As comparatively high hitrates were obtained, further consideration to optimize the generic fragment screening library were directed to the chemical tractability of the fragment. As several functional groups preferred by chemists for modification and linking of the fragments are also preferentially involved in interactions between the fragments and the target protein, a set of screening fragments was derived from chemical building blocks by masking its linker group by a chemical transformation which can be later on used in the chemical follow-up of the fragment hit. For example primary amines can be masked as acetamides. If the screening fragment is active the related building block can then be used for synthesis of a follow-up library.

Combinatorial Chemistry Techniques↗
PubMed1995

Bucket-handle tears of the medial and lateral menisci of the knee: value of MR imaging in detecting displaced fragments.

OBJECTIVE: A bucket-handle tear of a meniscus is a longitudinal tear with an attached fragment displaced away from the meniscus. Several MR findings of bucket-handle tears have been reported previously. However, the sensitivity of MR for detecting the displaced fragment is unknown. We determined the sensitivity of MR for detecting a displaced bucket-handle tear and determined how the sensitivity varied with tears of different sizes. We also evaluated whether the three findings of a displaced fragment differed in frequency between medial and lateral tears. MATERIALS AND METHODS: We retrospectively evaluated the MR examinations of 39 patients with arthroscopically proven bucket-handle tears for signs of a displaced meniscal fragment. Thirty-two patients had medial meniscal bucket-handle tears, and seven had lateral tears. Two observers evaluated each examination independently, with discrepancies resolved by consensus of a third reader. Each observer evaluated for three findings of a bucket-handle tear: a double posterior cruciate ligament sign, a flipped meniscus sign, or a fragment in the intercondylar notch. A double posterior cruciate ligament sign is present when the meniscal fragment is displaced anterior to the posterior cruciate ligament, resembling two ligaments. A flipped meniscus sign occurs when the fragment is flipped anteriorly so the anterior horn of the meniscus appears to be enlarged. We tested for differences between menisci in the sensitivity for each sign using Pearson's chi-squared test or Fisher's exact test. We compared the sensitivity of MR for identifying fragments when the tear involved one third, two thirds, or the entire meniscus using Spearman's rank correlation. RESULTS: Overall sensitivity for a displaced fragment was only 0.64. When the bucket-handle tear involved the entire meniscus, the displaced fragment could be seen in 84% of the menisci. The double posterior cruciate ligament sign was seen in 53% of the medial and none of the lateral bucket-handle tears (p = .01). The flipped meniscus sign was noted in 44% of medial and 29% of lateral menisci (p = .68), and a fragment was identified in the intercondylar notch in 66% of medial and 43% of lateral menisci (p = .40). CONCLUSION: MR is sensitive for detecting meniscal fragments in large bucket-handle tears, but is less so in small tears. The flipped meniscus sign and presence of a fragment in the notch were comparably useful findings of a displaced fragment in both medial and lateral menisci. The double posterior cruciate ligament sign was noted only in medial bucket-handle tears.

Adult↗
PubMed2006

Palmar carpal osteochondral fragments in racehorses: 31 cases (1994-2004).

OBJECTIVE: To evaluate records of racehorses with palmar carpal osteochondral fragments and determine whether the fragments were indicators of the severity of pathologic joint changes or prognosis. DESIGN: Retrospective case series. ANIMALS: 31 racehorses. PROCEDURES: Medical records, radiographs, and videos of arthroscopic procedures were reviewed. Information gathered included signalment; location, number, and size of the primary lesion; number and size of palmar carpal fragments; and details pertaining to surgical procedures. Outcome variables were obtained from race records. RESULTS: 31 horses met the selection criteria. Multiple palmar fragments were diagnosed in 58% of horses; small fragments (< 3 mm in diameter) were most common (52% of horses). Fifty-two percent of the horses returned to racing, 48% returned to racing and earned money, and 32% had at least 5 more starts. Horses with multiple fragments had significantly less earnings per start and lower performance index values after surgery than those with 1 fragment. Horses with palmar fragments < 3 mm in diameter were significantly less likely to return to racing and have 5 starts or to win money after surgery than horses with larger fragments. CONCLUSIONS AND CLINICAL RELEVANCE: Palmar carpal osteochondral fragments can be used as an indicator of clinically important joint pathology and as a prognostic indicator in racehorses. Horses with multiple small fragments were less likely to successfully return to racing than horses with only dorsally located carpal fragments or horses with 1 or 2 large palmar fragments. When possible, removal of palmar carpal osteochondral fragments should be considered.

Animals↗
PubMed1992

Low temperature pepsin proteolysis. An effective procedure for mouse IgM F(ab')2 fragment production.

Proteolytic fragments from murine IgM antibodies can be obtained by a variety of enzymatic procedures including pepsin digestion. However, these procedures exhibit considerable variability with respect to the size and yield of fragments. In this report, we describe a single step enzymatic digestion procedure which, when performed at a low temperature, can generate reasonably homogeneous proteolytic fragments with greater than 90% yield. Murine IgM monoclonal antibodies specific for the DNP moiety were used throughout this study. By subjecting intact IgM antibodies to enzymatic digestion with pepsin under mildly acidic conditions at 4 degrees C, covalent and noncovalent F(ab')2 fragments were generated. Under nonreducing conditions, the covalent F(ab')2 fragments migrated with an apparent molecular weight of 134 kDa on SDS-PAGE, while the noncovalent F(ab')2 fragments dissociated into their respective Fab' fragments each exhibiting an apparent molecular weight of 67 kDa. However, when subjected to gel-filtration chromatography in nondenaturing buffers, each of these F(ab')2 fragments eluted in a volume corresponding to 130-140 kDa. Upon extensive reduction, these fragments demonstrated equimolar concentrations of shortened mu chains, termed F'd fragments, and light chains. The binding activity of these F(ab')2 fragments was unaffected by the digestion. The F(ab')2 fragments exhibited the same number of binding sites and binding affinities as their respective homologous reductive subunits when analysed by equilibrium dialysis. To test the efficacy of this procedure, peptic digestions were also performed at 37 degrees C. Considerably lower yields of F(ab')2 fragments were obtained when compared to digestion at 4 degrees C.

Animals↗
PubMed1993

Release of DR molecules from complexes with invariant chain through the formation of a C-terminal 25 kDa invariant chain fragment.

To investigate how class II major histocompatibility complex (MHC) molecules are released from complexes with invariant chain (Ii), we studied a 25 kDa Ii fragment (p25) detected by Western blotting in affinity chromatographed DR preparations. The p25 species corresponds to the non-transmembrane, C-terminal Ii fragment 107-232. It was determined by gel filtration chromatography that the p25 fragment has a relative molecular mass (M(r)) of 46 kDa, indicating that this Ii fragment is present as dimers in B cell lysate. Two independent approaches were followed to demonstrate that generation of the p25 fragment takes place shortly before, or concomitantly to, loading of class II MHC molecules with antigen fragments. First, it was shown that a fraction of the p25 molecules is resistant to endoglycosidase H digestion, indicating that the p25 polypeptide can exit the endoplasmic reticulum (ER) and is transported at least to the cis-Golgi compartment. Second, treatment of class II MHC-positive B cells with leupeptin blocks the formation of p25, further indicating that this Ii fragment is generated in the endosomal compartment. The role of the p25 Ii species in the assembly of complexes between peptides and DR molecules was then investigated. While the p25 fragment was totally unable to prevent binding of a synthetic tetanus toxin peptide to DR molecules, the full-length Ii species (p33/35) effectively inhibited peptide binding, indicating that, by contrast with the p33/35 species, the p25 fragment does not occlude the peptide binding site of DR molecules. We concluded that the p25 fragment, which is produced by proteolytic cleavage at the N-terminal side of Methionine 107, has a decreased affinity for DR molecules as compared with the p33/35 species. Dissociation of the p25 fragment from DR molecules exposes the peptide binding site, which is thus made accessible for antigen fragments. This model of the complexes between DR and antigen fragments proposes that a stretch of Ii prevents peptide binding by occluding the peptide binding site without directly occupying it.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1988

Drug-binding and other physicochemical properties of a large tryptic and a large peptic fragment of human serum albumin.

The diazepam-binding behaviour of a large tryptic and a large peptic fragment of human serum albumin has been studied by circular dichroism and equilibrium dialysis in order to locate the primary diazepam-binding site on the albumin molecule. The analytical set-up of the FPLC was used to find the optimum experimental conditions for isolating the fragments. Conventional columns with a 100-fold higher loading capacity than the analytical FPLC columns were used to isolate large amounts of the fragments. The isolation procedure for the tryptic fragment (45 kDa, domains two and three of the albumin structure) is described in this paper. The description of the isolation procedure for the peptic fragment (46 kDa, domains one and two of the albumin structure) is published elsewhere (Bos, O.J.M., Fischer, M.J.E., Wilting, J. and Janssen, L.H.M. (1988) J. Chromatogr. 424, 13-21). The induced ellipticity of the diazepam-fragment complexes as well as the affinity of diazepam to the fragments turned out to be pH dependent. This pH dependence occurs in the region of the neutral to base transition of the albumin molecule. Difference CD-spectra of the proteins showed that the tryptic fragment and albumin have similar diazepam-binding properties, whereas the peptic fragment has different diazepam-binding properties. This result is in line with our equilibrium dialysis experiments which showed that the affinity of diazepam to the tryptic fragment and to albumin is of the same order of magnitude, whereas the affinity of diazepam to the peptic fragment is several orders of magnitude lower. On the basis of these results, it can be concluded that the tryptic fragment contains the primary diazepam-binding site and the peptic fragment one or more secondary diazepam-binding sites. This means that at least the main part of the primary diazepam-binding site is located in domain three of the albumin structure.

Circular Dichroism↗
PubMed1991

Retrograde, trans-synaptic and transneuronal transport of fragment C of tetanus toxin by sympathetic preganglionic neurons.

The atoxic binding fragment of tetanus toxin, Fragment C, was injected into paravertebral ganglion 14, the avian homologue of the mammalian stellate ganglion. Postinjection survival intervals were varied from 2.5 h to 33 days. Experiments performed at the shortest survival time of 2.5 h showed that Fragment C was retrogradely transported by sympathetic preganglionic axons at a rate greater than or equal to 10 mm/h. At survival times ranging from 5 to 15 h. Fragment C-positive, retrogradely labeled sympathetic preganglionic neurons were observed within the last cervical spinal segment and throughout the first three thoracic spinal cord segments. Sporadic retrograde labeling of sympathetic preganglionic neurons was evident within the fourth and fifth thoracic spinal cord segments. Fragment C-labeled perikarya and dendrites exhibited both diffuse cytoplasmic immunostaining as well as intracellular, perinuclear accumulations of small. Fragment C-positive granules. Retrogradely labeled preganglionic neurons were found within both autonomic subnuclei within avian thoracic spinal cord; the column of Terni and the nucleus intercalatus spinalis. The distribution and numerical density of retrogradely labeled sympathetic preganglionic neurons indicated further that: (a) both myelinated and unmyelinated preganglionic axons appear to be capable of intra-axonally transporting Fragment C; and (b) it is unlikely that there is differential Fragment C labeling of a morphologically distinct population of sympathetic preganglionic neurons within or across subnuclei. Fragment C is transferred out of sympathetic preganglionic somas and dendrites into the surrounding neuropil at an aggregate rate greater than or equal to 5 mm/h. Trans-synaptic transport was evident at postinjection survival times as short as 5 h and continued to increase in density within the sympathetic preganglionic neuropil for 24 h. Fragment C-positive terminal labeling persisted for at least 20 days. At survival times greater than or equal to 1 day. Fragment C-positive puncta and weak intracellular labeling of neurons were evident in areas of the spinal gray outside of the nuclear boundaries of the column of Terni and nucleus intercalatus. The regions showing evidence of trans-synaptic and transneuronal labeling included: (a) a group of small cells dorsal to the column of Terni, (b) lamina V and (c) lamina VII. This expansion of Fragment C-labeled neuronal elements was segmental in organization and co-extensive with the retrograde labeling pattern of sympathetic preganglionic neurons. Spinal interneurons in these regions may provide segmental, monosynaptic input to sympathetic preganglionic neurons. Fragment C leaked into the systemic circulation from the site of injection in paravertebral ganglion 14.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2000

Fibronectin fragments and their role in inflammatory arthritis.

OBJECTIVES: To identify potential immunopathogenic links between fibronectin (Fn) fragmentation and the inflammatory response in chronic joint disease. METHODS: Scientific papers involving studies of Fn fragments and inflammatory processes important in the pathogenesis of arthritis, including chondrolysis, synoviocyte growth and adhesion, polymorphonuclear leukocyte (PMN) and monocyte function, proteolysis, and immune complex activation were reviewed. In addition, reports identifying Fn fragments in synovial fluid (SF) were assessed. RESULTS: A series of Fn fragments have been identified in arthritic SF by several investigators. Fn and fragments ranging from 30 to 200 kd are present in elevated concentrations in inflammatory SF. SF Fn fragments display reduced affinity for fibrin and collagen. The 29- and 50-kd amino terminal fragments mediate release of proteoglycan from articular cartilage by RGD-independent mechanisms. Fn fragments can induce fibroblast gene expression of metalloproteinases or can act as proteinases themselves. A 90-kd plasmin generated fragment possesses homology with streptokinase. Fragments mediate PMN chemotaxis and enhance proliferation of CD4+ lymphocytes as well as binding to the C1q component of complement and influencing the behavior of immune complexes. CONCLUSIONS: Fn fragments can be functionally and biochemically characterized in diseased SF. Modification of fragment formation and inhibition of fragment function may have potential therapeutic value in the interruption of chronic synovial inflammation.

Arthritis↗
PubMed2005

Firefly luciferase enzyme fragment complementation for imaging in cells and living animals.

We identified different fragments of the firefly luciferase gene based on the crystal structure of firefly luciferase. These split reporter genes which encode for protein fragments, unlike the fragments currently used for studying protein-protein interactions, can self-complement and provide luciferase enzyme activity in different cell lines in culture and in living mice. The comparison of the fragment complementation associated recovery of firefly luciferase enzyme activity with intact firefly luciferase was estimated for different fragment combinations and ranged from 0.01 to 4% of the full firefly luciferase activity. Using a cooled optical charge-coupled device camera, the analysis of firefly luciferase fragment complementation in transiently transfected subcutaneous 293T cell implants in living mice showed significant detectable enzyme activity upon injecting d-luciferin, especially from the combinations of fragments identified (Nfluc and Cfluc are the N and C fragments of the firefly luciferase gene, respectively): Nfluc (1-475)/Cfluc (245-550), Nfluc (1-475)/Cfluc (265-550), and Nfluc (1-475)/Cfluc (300-550). The Cfluc (265-550) fragment, upon expression with the nuclear localization signal (NLS) peptide of SV40, shows reduced enzyme activity when the cells are cotransfected with the Nfluc (1-475) fragment expressed without NLS. We also proved in this study that the complementing fragments could be efficiently used for screening macromolecule delivery vehicles by delivering TAT-Cfluc (265-550) to cells stably expressing Nfluc (1-475) and recovering signal. These complementing fragments should be useful for many reporter-based assays including intracellular localization of proteins, studying cellular macromolecule delivery vehicles, studying cell-cell fusions, and also developing intracellular phosphorylation sensors based on fragment complementation.

3T3-L1 Cells↗
PubMed2001

Protein interactions leading to conformational changes monitored by limited proteolysis: apo form and fragments of horse cytochrome c.

Proteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene.

Amino Acid Sequence↗
PubMed1992

Conformational differences between surface-bound and fluid-phase complement-component-C3 fragments. Epitope mapping by cDNA expression.

In previous studies a subset of complement-component-C3 (C3) epitopes, C3(D), expressed in denatured and surface-bound C3 and C3 fragments, has been described. These epitopes were detected by antibodies raised against denatured C3. In the present study we used a cDNA expression strategy to localize epitopes recognized by monoclonal and polyclonal anti-C3(D) antibodies. First, DNAse I digestion of C3 cDNA was used to generate 200-300 bp fragments. These cDNA fragments were expressed as beta-galactosidase-C3 fusion proteins using the lambda gt11 vector. The fusion proteins were tested by Western-blot analysis for reactivity with monoclonal and polyclonal anti-C3 antibodies, and the location of the epitopes were determined by sequencing the cDNA fragments. Affinity-purified polyclonal anti-C3(D) antibodies specific for denatured C3 reacted strongly with the C3 fusion fragments corresponding to segments of the 40 kDa subunit of C3c (residues 1477-1510) and the C3d fragment (residues 1117-1155 and 1234-1294) of C3. Adsorption of the polyclonal antibodies with a mixture of EAC3b and EAC3bi (degradation fragments of C3 bound to sheep erythrocytes) abolished binding to fusion proteins spanning the C3d region, but not the 40 kDa fragment of C3c. No effect was seen with the corresponding soluble C3 fragments. The monoclonal anti-C3(D) antibodies (mAbs) 7D326.1 and 7D331.1, specific for EAC3b and EAC3bi, bound to a fusion protein corresponding to amino acid residues 1312-1404, whereas mAb 7D9.2, specific for EAC3d, reacted with a fusion protein spanning amino acid residues 1082-1118. mAbs 4SD11.1 and 4SD18.1, which did not bind to any physiological C3 fragment, detected a fusion protein covering residues 1477-1510. In summary, the segments of C3 represented by amino acid residues 1082-1118, 1117-1155, 1234-1294 and 1312-1404 accommodate C3(D) epitopes that are expressed by erythrocyte-bound C3 fragments, but not by the corresponding fluid-phase fragment, whereas the segments spanning residues 973-1026 and 1477-1510 contain C3(D) epitopes that are exposed exclusively in denatured C3 and therefore hidden in physiological fragments of the protein.

Animals↗
PubMed2002

Comparison of huntingtin proteolytic fragments in human lymphoblast cell lines and human brain.

Proteolytic fragments of huntingtin (htt) in human lymphoblast cell lines from HD and control cases were compared to those in human HD striatal and cortical brain regions, by western blots with epitope-specific antibodies. HD lymphoblast cell lines were heterozygous and homozygous for the expanded CAG triplet repeat mutations, which represented adult onset and juvenile HD. Lymphoblasts contained NH(2)- and COOH-terminal htt fragments of 20-100 kDa, with many similar htt fragments in HD compared to control lymphoblast cell lines. Detection of htt fragments in a homozygous HD lymphoblast cell line demonstrated proteolysis of mutant htt. It was of interest that adult HD lymphoblasts showed a 63-64 kDa htt fragment detected by the NH(2)-domain antibody, which was not found in controls. In addition, control and HD heterozygous cells showed a common 60-61 kDa band (detected by the NH(2)-domain antibody), which was absent in homozygous HD lymphoblast cells. These results suggest that the 63-64 kDa and 60-61 kDa NH(2)-domain htt fragments may be associated with mutant and normal htt, respectively. In juvenile HD lymphoblasts, the presence of a 66-kDa, instead of the 63-64 kDa N-domain htt fragment, may be consistent with the larger polyglutamine expansion of mutant htt in the juvenile case of HD. Lymphoblasts and striatal or cortical regions from HD brains showed similarities and differences in NH(2)- and COOH-terminal htt fragments. HD striatum showed elevated levels of 50 and 45 kDa NH(2)-terminal htt fragments [detected with anti(1-17) serum] compared to controls. Cortex from HD and control brains showed similar NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa; lymphoblasts also showed NH(2)-terminal htt fragments of 50, 43, 40, and 20 kDa. In addition, a 48-kDa COOH-terminal htt band was elevated in HD striatum, which was also detected in lymphoblasts. Overall, results demonstrate that mutant and normal htt undergo extensive proteolysis in lymphoblast cell lines, with similarities and differences compared to htt fragments observed in HD striatal and cortical brain regions. These data for in vivo proteolysis of htt are consistent with the observed neurotoxicity of recombinant NH(2)-terminal mutant htt fragments expressed in transgenic mice and in transfected cell lines that may be related to the pathogenesis of HD.

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