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PubMed1997

Studies on the Separation, Purification of Beijing Duck Apo A-I CNBr Cleavaged Fragments and the Localization of Their Function Domain.

Apo A-I of Beijing duck was first cleavaged by CNBr into 11 peptide fragments which were further separated and purified by preparative SDS-PAGE and HPLC, then their molecular weights and N-terminal amino acid sequences were also determined. According to the molecular weights and N-terminal amino acid sequences of each fragment the positions of peptide fragments 3-10 in apo A-I molecule were localized at the sites of amino acid sequences 64-240, 74-240, 64-206, 1-163, 1-36, 171-206, 207-240 and 137-170 respectively. The studied results of their main functions are as following: (1) All the fragments could combine with lipids to form lipsomes of various sizes. The fragment Longer, the size will be larger. (2) The ability of the fragments 3-10 to activate LCAT equals to 65%, 52%, 60%, 39%, 8%, 7%, 0% and 2% of that of the whole apo A-I molecule respectively, showing that the activating sequence is localized in amino acid residues 64-136. (3) Only the liposomes formed with fragments 3, 4 and 9 could combine with the HDL receptor of liver cell membrane of Beijing duck; the rest of the liposomes demonstrated essentially no binding ability. As peptide fragments 3 and 4 also contained fragment 9, the result indicated that amino acids 207-240 maybe the fragment that combined with HDL receptor.

Journal Article↗

PubMed2005

[Clinical and prognostic significance of tumor markers cytokeratin 19 fragment, carcinoembryonic antigen, and neuron-specific enolase in lung cancer].

OBJECTIVE: To evaluate the clinical and prognostic significance of the tumor markers cytokeratin 19 fragment, carcinoembryonic antigen and neuron-specific enolase in lung cancer patients. MATERIALS AND METHODS: Serum levels of cytokeratin 19 fragment, carcinoembryonic antigen and neuron-specific enolase were measured using electrochemical luminescence immunoassay in 46 lung cancer patients. Serum levels of cytokeratin 19 fragment, carcinoembryonic antigen, and neuron-specific enolase higher than 3.6 ng/ml, 5.0 ng/ml and 13.0 ng/ml, respectively, were considered as elevated. RESULTS: Cytokeratin 19 fragment, carcinoembryonic antigen, and neuron-specific enolase were elevated in 19.6%, 43.5%, and 63% of patients, respectively. Elevated levels of neuron-specific enolase were detected more frequently in smokers than in ex-smokers (p=0.003). Likewise preoperative levels of carcinoembryonic antigen (p=0.023) and neuron-specific enolase (p=0.007) were statistically higher in smokers than in ex-smokers. A significant correlation was detected between the level of cytokeratin 19 fragments and smoking cumulative exposure (r=0.542, p=0.037). The number of patients with elevated levels of cytokeratin 19 fragment and neuron-specific enolase was higher in more advanced disease than in early lung cancer (p=0.036 and p=0.036, respectively). Preoperative levels of cytokeratin 19 fragment (p=0.017 and p=0.016, respectively) and neuron-specific enolase (p=0.03 and p=0.006, respectively) were significantly associated with more advanced disease and tumor size, as well as tumor histology in non-small cell lung cancer (p=0.03 and p=0.016, respectively). Preoperative levels of cytokeratin 19 fragments were higher in squamous cell carcinoma than in adenocarcinoma (p=0.026). Elevated preoperative serum levels of cytokeratin 19 fragment predict a poor prognosis for lung cancer patients (p=0.007). CONCLUSION: Alteration of serum tumor markers cytokeratin 19 fragment, carcinoembryonic antigen and neuron-specific enolase is associated with particular tumor histology, smoking habit, more advanced disease and poor prognosis.

Adenocarcinoma↗

PubMed1992

Fibronectin fragments cause chondrolysis of bovine articular cartilage slices in culture.

Elevated fibronectin (Fn) and Fn fragment concentrations are found in the synovial fluid of osteoarthritic and rheumatoid arthritic patients. Fn has been shown to affect expression of chondrocytic matrix proteins, and Fn fragments have been shown to elevate gene expression of neutral proteinases in synoviocytes. For these reasons, we tested the effects of Fn fragments on protease release and resultant proteoglycan release from cartilage in serum-free bovine articular cartilage explant cultures. We have found that 1 microM amino-terminal 29- and 50-kDa gelatin-binding Fn fragments caused over a 50-fold enhancement of gelatinolytic and collagenolytic proteinase release with a 23-fold enhancement of proteoglycan (PG) release. Release was significant at fragment concentrations as low as 20 nM. An integrin-binding 140-kDa fragment mixture was the least active fragment, whereas native Fn had little activity. The relative activities of the fragments correlated with their relative abilities to bind to cartilage. The RGDS integrin-recognition peptide also caused release, although sequence mutants did not. PG release was blocked by actinomycin D, cycloheximide, and deoxyglucose. Fn fragment-mediated PG release was decreased in 10% serum by over 10-fold but was still 2-fold greater than in controls. In the presence of insulin-like growth factor-1, PG release was as great as without serum. We suggest that Fn fragments, as found in diseased synovial fluid, may contribute to protease-mediated damage to cartilage.

Animals↗

PubMed1990

Fragmentation of human gallstones using ultrasound and electrohydraulic lithotripsy: experimental and clinical experience.

In vitro fragmentation of gallstones was performed by means of ultrasound (n = 89) and electrohydraulic lithotripsy (n = 83) with success rates of 100% and 93%, respectively. The fragmentation time was 21.9 +/- 52 seconds (mean +/- SEM) for ultrasound and 2.5 +/- 3.4 seconds for electrohydraulic lithotripsy. The energies required were similar to those used safely in the fragmentation of renal and bladder stones. Fragmentation was not related to the composition of the gallstones; there was no statistical difference between the fragmentation times or rates (p greater than 0.05) in cholesterol stones and pigment stones by either method. Fragmentation time was linearly related to gallstone weight for both methods. Comparison of ultrasound and electrohydraulic lithotripsy, using the two-way analysis of variance model, revealed no statistical difference between the two methods in times and rates of fragmentation (p greater than 0.05). Both ultrasound and electrohydraulic lithotripsy offer distinct advantages over the dissolution of gallstones by chemical methods because they are rapid and independent of gallstone composition. In vivo fragmentation of a large pigment common duct stone was also accomplished by means of an electrohydraulic lithotriptor. The stone was discovered in a 79-year-old patient on post-operative T-tube cholangiogram. Chemical analysis of the gallstones removed from the patient during cholecystectomy had revealed a very low cholesterol content. Since the remaining stone could not be dissolved and was too large to be mechanically extracted, it was fragmented through a T-tube, under fluoroscopic guidance and the small fragments were extracted with a dormia basket. The electrohydraulic lithotriptor was selected because it has a flexible probe.

Bile Pigments↗

PubMed1989

Binding properties of monoclonal antibodies against human fragment D-dimer of cross-linked fibrin to human plasma clots in an in vivo model in rabbits.

Two (MA-15C5 and MA-8D3) out of approximately 500 monoclonal antibodies, obtained by fusion of P3X63-Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified fragment D-dimer from human fibrin, demonstrated a more than 1,000-fold higher affinity for fragment D-dimer than for native fibrinogen. MA-15C5 was directed against a neoantigenic determinant only expressed in fragment D-dimer. MA-8D3 reacted equally well with fragment D-dimer of crosslinked fibrin and with fragment D of non-crosslinked fibrin but not with fragment D of fibrinogen. Both monoclonal antibodies did not crossreact with rabbit fibrin and its degradation products. The binding of 125I-labeled Fab fragments to human plasma clots, introduced and aged for 1 hr in the jugular vein of heparinized rabbits was studied. Following injection of an equimolar mixture of Fab fragments derived from MA-15C5 and MA-8D3, the clot to blood ratios of radioactivity increased from 3.2 +/- 1.2 (mean +/- SD) at 4 hr to 7.2 +/- 1.4 at 17 hr. The binding of Fab fragments of MA-15C5 and MA-8D3 was independent of the age (1 to 72 hrs) of the clot and of heparin anticoagulation and was only slightly decreased (by 20%) in the presence of circulating human fibrinogen (90 mg/kg body weight) and of human cross-linked fibrin degradation products at a plasma concentration of 10 micrograms/ml. The binding of Fab fragments of MA-15C5 and MA-8D3 to occlusive human plasma clots in the femoral artery of rabbits was comparable to that of the non-occlusive human plasma clots in the jugular vein.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed1986

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells.

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.

Animals↗

PubMed1983

Quantitative analysis of the association of human hemoglobin with the cytoplasmic fragment of band 3 protein.

The association of the isolated cytoplasmic fragment of band 3 protein with human hemoglobin was studied by rate zonal centrifugation in sucrose density gradients, by quenching of fragment fluorescence by hemoglobin, and by flash photolysis of carbon monoxidebound hemoglobin as a function of fragment concentration. The centrifugation results showed that both proteins interact and that the interaction is abolished upon addition of glyceraldehyde-3-phosphate dehydrogenase. The fractions eluted from the density gradient were analyzed further by spectrophotometric and gel electrophoretic methods. Two types of complexes could be identified, one containing the equivalent of 1 hemoglobin tetramer/dimer of cytoplasmic fragment and another containing 2 tetramers of hemoglobin/dimer of fragment. Flash photolysis and fluorescence-quenching experiments showed that liganded hemoglobin is stabilized as the alpha beta dimer when bound to the fragment, a result almost identical with that seen for membranebound hemoglobin in previous studies. The results further suggest that there are two binding sites for the alpha beta dimer of hemoglobin on one monomer of the cytoplasmic fragment but only one mutually exclusive hemoglobin tetramer binding site, suggesting the possibility of conformational isomerism when the fragment with two dimers bound isomerises to a fragment monomer with one hemoglobin tetramer bound. Finally, despite the stabilization of the dimeric state of liganded hemoglobin when bound to the fragment, estimates of the hemoglobin dimer and tetramer binding constants suggest that the hemoglobin tetramer binds more tightly by about 2 orders of magnitude.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1994

Fragmentation of the Golgi apparatus of motor neurons in amyotrophic lateral sclerosis (ALS). Clinical studies in ALS of Guam and experimental studies in deafferented neurons and in beta,beta'-iminodipropionitrile axonopathy.

Previous morphological immunoenzymatic studies with organelle-specific antibodies have disclosed an apparent fragmentation of the Golgi apparatus in large numbers of motor neurons in 12 cases of sporadic, non-Guamanian amyotrophic lateral sclerosis (ALS) in three cases of other types of motor neuron disease and in one case of a mitochondrial myopathy with cytochrome c oxidase deficiency. Motor neurons with fragmented Golgi apparatus were moderately atrophic; in these cells, discrete immunostained elements of the organelle were twice as many as in normal neurons, and the size of each Golgi element and the percentage of the cytoplasmic area occupied by the Golgi apparatus were reduced (Am J Pathol 1992, 140: 731-737). In this report we have confirmed the fragmentation of the organelle of motor neurons in the spinal cord in six sporadic cases of Guamanian ALS. In four of the six cases the clinical course was 1 to 2 years. The percentages of motor neurons with fragmented Golgi apparatus varied from 38 to 92. Motor neurons from three additional cases of Guamanian ALS of clinical duration from 5 to 7 years did not show fragmentation of the Golgi apparatus. In two cases of Guamanian ALS and in one non-Guamanian ALS, all neurons with ubiquitin-positive skein-like or granular inclusions believed to be pathognomonic for ALS had fragmented Golgi apparatus. To examine whether the fragmentation of the Golgi apparatus results from reactions to either neuronal deafferentation or to lesions of proximal axons, we conducted two experimental studies. In the first study, we examined in cats the Golgi apparatus of deafferented neurons of the dorsal lateral geniculate nucleus. In the second study, we examined the Golgi apparatus of motor neurons in the spinal cord of rats with proximal axonopathy induced by beta,beta'-iminodipropionitrile. In these two experiments, the neuronal Golgi apparatus studied by immunoenzymatic techniques and morphometry, was not fragmented. Taken together, the results of these studies strongly suggest that the fragmentation of the Golgi apparatus of motor neurons in ALS represents an important and perhaps early change of the organelle that may be involved in the pathogenesis of ALS. The fragmentation of the Golgi apparatus of motor neurons is a fairly specific and easily recognizable marker of ALS and may be used together with other criteria for comparisons between the human disease and proposed animal models of the disorder.

Adult↗

PubMed1993

Acceleration of neointima formation in vascular prostheses by transplantation of autologous venous tissue fragments. Application to small-diameter grafts.

We have previously demonstrated rapid and complete endothelialization in synthetic fabric vascular prostheses that have been pretreated with autologous venous tissue fragments. However, significant thrombogenicity has been a major problem when this method has been applied to small-diameter grafts. By masking the positively charged collagen fibrils in the tissue fragments with negatively charged heparin, we were able to overcome this problem. A canine jugular vein was resected, minced into tissue fragments, and suspended. This mixture was sieved through the wall of a highly porous vascular prosthesis with a water porosity value of 4,000 ml/cm2 per minute by pressurized injection, which caused the tissue fragments to be trapped in the graft wall. Tissue-fragmented grafts (7 mm inside diameter, 5.7 cm long) were implanted into the thoracic aorta of 35 dogs. In addition, tissue-fragmented grafts of small diameter (4 mm inside diameter, 3.5 cm long) were pretreated with heparin and implanted into the carotid arteries of 16 dogs (32 grafts). Preclotted grafts without tissue fragmentation were implanted into the thoracic aorta (25 dogs) and carotid arteries (6 dogs, 12 grafts) as controls. Grafts were explanted from 1 to 495 days after implantation. New arterial wall formation was complete throughout the tissue-fragmented grafts within 2 weeks; however, in the control grafts, neointima formation was limited to the anastomotic sites even after 2 months. Twenty small-caliber tissue-fragmented grafts that were pretreated with heparin in the carotid position were patent, but all the control grafts were occluded within 1 week. These results demonstrate that neointima formation can be enhanced in synthetic fabric prostheses; furthermore, long-term patency of vascular grafts of small caliber is possible in dogs with this tissue-fragmentation technique.

Animals↗

PubMed1995

[Experimental study of urinary calculi fragmentation with pulsed laser shockwave, based on their chemical composition].

OBJECTIVES: To attempt to classify the resistance of urinary calculi to shock waves according to the chemical composition of the calculi most frequently treated in clinical practice. METHOD: Seventy four urinary calculi obtained after surgery were submitted to shock waves produced by an experimental neodyme glass phosphate pulsed laser, at a frequency of 532 nanometres, delivering impulses of 4.8 Joules lasting 25 nanoseconds with a recurrence of 1 second. The calculi (25 homogeneous: 90% of one constituent, 49 heterogeneous: > 45% < 90% of one constituent) had to be fragmented down to 2 mm fragments. The total fragmentation energy (TFE) required was the parameter adopted to classify calculi and to compare the fragmentation of various chemical compositions studied by infrared spectrophotometry. The TFE of 25 homogeneous calculi were compared to a microhardness study performed on calculi with the same chemical composition. The fragmentation thresholds energy (TFE) was used to compare the fragmentation thresholds of each type of calculi. Statistical analysis by ascending multiple regression was performed to classify the various factors likely to influence fragmentation. RESULTS: Two groups were able to be significantly distinguished according to whether the TFE required to obtain 2 mm fragments was greater than 200 Joules (cystine, weddellite, brushite, uric acid) or less than 200 Joules (weddellite, carbapatite, struvite). Only cystine and whewellite significantly increased the fragmentation threshold. For the 125 homogeneous calculi, the correlation with microhardness demonstrated an inverse relationship between friability and increased microhardness. A crossover between microhardness and friability to shock waves was only observed for whewellite and cystine. For the 49 heterogeneous calculi, this study showed that when weddellite was the predominant component of a stone, the friability tended to increase. Struvite and whewellite significantly facilitated and decreased fragmentation of heterogenous calculi, respectively. CONCLUSIONS: This study allowed urinary calculi to be classified into three groups: friable (weddellite, carbapatite, struvite), intermediary (brushite, uric acid, whewellite) and resistant (cystine). Although this laser cannot be used clinically, the constants were similar to those of pulsed lasers available in clinical practice and the results of the study corresponded to those already observed empirically by other teams or observed experimentally on a single stone corresponding to each type of chemical composition. The clinical application of this study would be to prospectively compare the results with those observed in clinical lithotripsy.

Humans↗

PubMed1996

Daunorubicin-induced internucleosomal DNA fragmentation in acute myeloid cell lines.

The study was designed to evaluate the implication of apoptosis in myeloid leukemic cell death induced by daunorubicin (DNR) and to identify the possible factors which may influence this process. DNR-induced apoptosis was characterized by morphology and DNA fragmentation in six leukemic myeloid cell lines which expressed different differentiation phenotypes. In phenotypically mature HL-60 and U937 cells, DNR induced typical apoptosis with characteristic morphological changes and intense internucleosomal DNA fragmentation within a narrow concentration range (0.5-2 microM). When these cells were treated with higher doses of DNR, large DNA fragments (100 kbp), but not internucleosomal fragments, were identified. DNR-induced DNA fragmentation in HL-60 and U937 was inhibited by antioxidants such as N-acetylcysteine (N-ac) or pyrrolidine-dithiocarbamate (PDTC). In the phenotypically immature KG1a, KG1, HEL and ML1 cell lines DNR induced no characteristic apoptotic morphological features as well as very low levels of internucleosomal DNA fragmentation, whereas large DNA fragments (200 kbp) were observed in KG1a treated with 7 microM DNR. Since the latter expressed P-glycoprotein (P-gp), the role of P-gp in the lack of apoptotic response to DNR was investigated. One P-gp inhibitor (verapamil) slightly improved DNR-induced DNA fragmentation in KG1a cells whereas the combination of verapamil and buthionine-sulfoximine (BSO), which depletes glutathion store, further increased internucleosomal DNA fragmentation. In conclusion, DNR induced internucleosomal DNA fragmentation in some but not all AML cells; the magnitude of this process being influenced by both intracellular drug concentration and oxidative balance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1976

Characterization of ribosome-protected fragments from reovirus messenger RNA.

The 5'-terminal methylated cap (m7G(5')ppp(5')Gm) in reovirus messenger RNA comprises part of the ribosomes binding site, since attachment of 40 S wheat germ ribosomal subunits to reovirus small (s), medium (m), and large (l) RNA classes conferred almost complete protection of the cap against RNase digestion. After joining of the 60 S ribosomal subunits, however, the cap continued to be protected against T1 RNase within the 80 S initiation complexes formed with only some messenger species; namely the three l-messages, one of the m-messages, and one or two of the s-messages. When protected fragments were recovered from 40 S and 80 S complexes and tested for ability to rebind to ribosomes those fragments which retained the cap were able to rebind most efficiently. The protected fragments recovered from 40 S initiation complexes with several of the s- and m-RNA species were larger than the messenger fragments recovered from 80 S complexes. The medium size class of reovirus RNA, which consists of three messenger species, gave rise to three discrete 5'-terminal fragments after digestion of 40 S complexes with T1 RNase, and to three somewhat smaller fragments after T1 RNase digestion of 80 S complexes. Fingerprints of the T1 oligonucleotides derived from these fragments are consistent with the interpretation that each messenger species within the m-RNA class gives rise to a protected fragment of a unique size and that, with each message, there is extensive overlap between the regions of the message protected by 40 S and 80 S ribosomes. The ratio of the three protected fragments recovered from 40 S complexes with m-RNA was highly reproducible under a given set of binding conditions, but could be shifted by varying the messenger/ribosome ratio in the binding reaction. Thus, one of the fragments, which was preferentially recovered when the ribosome concentration was limiting, could be tentatively identified as the binding site of the most efficiently translated message within the m-RNA class.

Oligoribonucleotides↗

PubMed1998

[Polymorphism of B1-associated DNA fragments on various stages of ontogenesis of mice in tissues of different histogenesis].

Pattern of B1-associated DNA fragments was studied by means of polymerase chain reaction (PCR) in the mouse tissues of different histogenesis of 15 and 20 day old embryos and of adult mice C57B1/6. As many as 20 DNA fragments were revealed on electrophoregrams differing in their molecular masses (m. m.) and amounts of amplified products. DNA fragments varied within a 100-10,000 bp range. The clusters of B1-associated DNA fragments, containing 100-200, 300-400 and 800-1000 bp, were most intensive in all studied electrophoregrams. The B1-associated DNA fragments from muscles of adult mice differed from those of other tissues by the presence of a DNA fragment with 800 bp. A comparative analysis of the spectra of B1-associated DNA fragments from hepatocytes of two inbred strains, C57B1/6 and C3HA, has shown their general similarities in m. m. values. But a significant distinction, that was found, involved the presence of a DNA fragment with m. m. approximately 6000 bp in the spectra of B1-associated DNA fragments from C3HA strain mice, that is absent in the spectra of respective fragments from hepatocytes of C57B1/6 strain mice. The obtained results allow to use the B1-PCR method for studying genome recombination during ontogenesis, intraspecies divergence and also at malignant cell transformation.

Animals↗

PubMed1999

Processing of antibodies to the MHC class II antigen by B-cell lymphomas: release of Fab-like fragments into the medium.

Lym-1, an anti-MHC class II Ab, displayed a unique processing pathway after binding to the surface of Raji B-lymphoma cells, in which Fab-like fragments were gradually released into the medium. The fragments had reduced interchain disulfide bonds. Fragmentation was markedly reduced by inhibitors of intracellular catabolism, namely ammonium chloride, chloroquine and leupeptin. The capacity of the process was high, and fragmentation of approximately 5x10(6) Ab molecules per cell per day was measured directly, in what can be considered to be a minimum estimate. Five other Abs to the MHC class II antigen were tested similarly on Raji and on three other B-cell lymphomas: none showed the same high level of fragmentation seen with Lym-1 binding to Raji, but significant fragmentation did occur with some of the Abs, particularly EDU-1 and L243. The level of fragmentation depended on the cell line as well as on the particular Ab. The other 5 Abs were all catabolized, to low molecular weight material, much more extensively than Lym-1. Part of the difference between Abs can probably be attributed to the fortuitous, preferential labeling of Lym-1 on the light chain, since the data suggest that the Fc fragment is fully degraded while the Fab-like fragment is released into the supernatant. This pathway of Ab processing is likely to be related to the physiology of the MHC class II antigen, which recycles into a mildly proteolytic intracellular compartment.

Animals↗

PubMed1995

Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity.

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.

Amino Acid Sequence↗

PubMed1999

Beta-amyloid peptide fragment 31-35 induces apoptosis in cultured cortical neurons.

A synthetic fragment 31-35 of beta-amyloid peptide was used in cultured cortical neurons to examine whether this smaller sequence could trigger apoptotic degeneration in vitro by using morphological, biochemical and flow-cytometric examinations. The results showed that: (i) neurons treated with fragment 31-35 of beta-amyloid peptide exhibited membrane blebbing, compaction of nuclear chromatin, nuclear shrinkage and nuclear fragmentation; (ii) a typical DNA ladder was revealed by agarose gel electrophoresis following fragment 31-35 of beta-amyloid peptide exposure; (iii) the internucleosome DNA fragmentation was also detected by flow-cytometric examination following fragment 31-35 of beta-amyloid peptide exposure; and (iv) the DNA fragmentation induced by fragment 31-35 of beta-amyloid peptide in the above two examinations could be blocked by co-treatment with aurintricarboxylic acid or actinomycin D. It is suggested that fragment 31-35 of the beta-amyloid peptide may be a shorter sequence of beta-amyloid peptide responsible for triggering an apoptotic process in cultured neurons.

Amyloid beta-Peptides↗

PubMed1976

Fragments of immunoglobulins in human faeces.

Fab-fragments of IgG were easily demonstrated in extracts of faeces from healthy infants and children. Employing immunoelectrophoresis with antisera to whole human serum and to Fab-fragments of IgG, a marked precipitation line, likely to represent such fragments, was evident in the cathodal or gamma-region. Usually no precipitate was formed with antisera specific for gamma-heavy chains or Fc-fragments of IgG. Presumably IgG in the gut is partially destroyed before being excreted with faeces. Results of immunoelectrophoresis, gel filtration as well as polyacrylamide gel electrophoresis, using antisera to alpha-heavy chains, indicated the presence of fragments of IgA in some faecal extracts. Fragments of IgA could not be demonstrated in IgA-deficient patients, and no fragments of either class, or only traces of Fab-fragments of IgG, were found in agammaglobulinaemic patients. No antibody activity against rabbit erythrocytes was found in gel filtrations containing such fragments; any reaction was not observed either in the direct agglutination test or in a modified antiglobulin test.

Agammaglobulinemia↗

PubMed1985

Characterization of the opsonic and monocyte adherence functions of the specific fibronectin fragment that enhances phagocytosis of particulate activators.

The functional opsonic and monocyte adherence domains within the 180,000 m.w. opsonic fibronectin fragment (180K-opFnf) that selectively augments human monocyte phagocytosis of particulate activators of the alternative complement pathway were analyzed with Fab fragments of monoclonal anti-fibronectin antibodies BC7, CE9, BD4, AB3, and CPG1, and with fragments of intact human plasma fibronectin derived by cathepsin cleavage and isolated by affinity chromatography. Monoclonals AB3 and CPG1, which recognize epitopes within 40,000 daltons of the carboxy terminus of intact fibronectin, and the cathepsin D-derived, disulfide-linked fragments that contain these epitopes each inhibited the opsonic function of 180K-opFnf. Monoclonals AB3 and CPG1 inhibited monocyte ingestion of rabbit erythrocytes (Er) by 60 and 50%, respectively, when 180K-opFnf was pretreated with 20 micrograms of these monoclonals, but neither monoclonal affected the enhanced monocyte ingestion of Er pretreated with the fibronectin fragment. The pretreatment of Er with 5 micrograms and 40 micrograms of the disulfide-linked, cathepsin D derivatives isolated from high and low affinity heparin fractions, respectively, inhibited the proportion of ingesting monocytes by 60%, but these types of fragments had little effect when concurrently incubated with the opsonic fragment and Er. Monoclonals CE9 and BD4, which recognize epitopes located adjacent to or within the cell-adhesive domain of intact fibronectin, respectively, inhibited the monocyte adherence function of 180K-opFnf, as evidence by their comparable inhibitory effects when present before or after Er were opsonized with 180K-opFnf. When 20 micrograms of monoclonals CE9 and BD4 were each introduced before and after Er were opsonized with 180K-opFnf, monocyte ingestion was inhibited by 60 and 65% and by 51 and 60%, respectively. At 42 micrograms, cathepsin D-derived, non-gelatin-binding, low affinity heparin fragments that contained both BD4 and CE9 determinants or only the BD4 determinant inhibited monocyte ingestion by 53 and 74%, respectively, when concurrently incubated with 180K-opFnf and target Er, but were without effect when used to pretreat Er before the addition of 180K-opFnf. Thus, the inhibitory effects produced by monoclonals AB3 and CPG1 and by cathepsin D-derived, disulfide-linked fragments containing their corresponding epitopes demonstrated that the opsonic domain within 180K-opFnf is immunologically similar to regions within the carboxy terminus of intact plasma fibronectin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adjuvants, Immunologic↗