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PubMedDate not supplied

Measurement of apoptotic fragments in growing hair follicles following gamma-ray irradiation in mice.

The usefulness of apoptotic fragments assay for investigating radiation response of hair follicles was examined. Frequency was defined as the ratio of the total number of apoptotic fragments to the number of hair follicles per section examined. The curve of dose-effect relationship for the data of apoptotic fragments was obtained by fitting the linear-quadratic model y= a+bD+cD2. When plotting on a linear scale against radiation dose, the line of best fit was y= 0.549 +/- 1.775) + 3.578 +/- 1.236)D + (-0.124 +/- 0.139)D2. The dose-response curves were linear-quadratic and a significant relationship was found between the frequency of apoptotic fragments and dose. The morphological findings of the irradiated groups were typical apoptotic fragments in the matrix region of hair follicles, but the spontaneous occurrence of apoptotic fragments was not observed. Since the apoptotic fragments was not observed. Since the apoptotic fragment assay is simple and reproducible in the whole body irradiation range of 0.5 to 8 Gy, it may be a good tool for evaluating the dose response of low dose radiation in vivo and provide a potentially valuable biological dosimeter for dose distribution determinations following accidental exposure.

Animals↗
PubMed1997

[In vitro study of urinary calculi fragmentation with 4 different systems of lithofragmentation].

The results of shockwave lithofragmentation are conditioned by stone composition. Similarly, the different types of lithofragmenting energy can have different effects on the calculi. The purpose of this experimental study is to establish a relationship between calculi composition and type of energy used for fragmentation, with the results of the lithotriptsy treatment. 60 calculi, 12 for each pure composition selected (calcium oxalate mono and dihydrate, phosphocarbonate, ammonium magnesium phosphate and uric acid) underwent in vitro lithofragmentation with 4 different lithofragmenting sources (electrohydraulic, piezoelectric, ultrasound and pulsed laser). It was carried out a study of fragmentation in particles under 3 mm and pulverization in particles under 0.5 mm, analyzing post-lithotriptsy fragments with scanning electron microscopy associated to X-ray dispersion energy. No relationship was shown between calculi composition and amount of energy supplied for the fragmentation. The lithiasic composition correlates to the fragmentation pattern, so that oxalocalcium calculi undergoes good fragmentation and poor pulverization, infective calculi have poor fragmentation but good pulverization and uric acid calculi show poor fragmentation and pulverization.

Humans↗
PubMed1976

[Transfixion of cervical cord by a glass fragment--report of a case (author's transl)].

Stab wound of the spinal cord caused by accident occurred in the home life is rare absolutely. One case of stab wound of the cervical spinal cord caused by penetrated and retained glass fragment within the spinal canal was reported. A 30-year-old woman was hospitarized on August 16, 1971. with complaint of left hemiparesis, gait disturbance and sensory impairment on the right side. A glass door fell down on the patient's neck on April 14, 1971. and one glass fragment was removed from the patient's left neck by one of the patient's family and no immediate spinal cord symptom appeared and no physician was consulted at that time. One and a half month after the injury the patient rod a motor bicycle and pain and severe stiffness in the shoulder and neck, and headache. These subjective symptom disappeared by rest and same subjective symptom repeated in following five days. Three and a half months after the injury the patient found sensation loss in the right foot and gait distrubance appeared. After the sensory impairment extended to the cervical level which was accompanied by left hemiparesis. On examination, the patient was found to have merked weakness of left limbs, spastic gait and severe impairment of touchpain- and thermosensation below the fifth cervical level but deep sensation was preserved. All tendon reflexes showed marked exaggeration and pathological reflexes were proved. Roentgenograms of the cervical spine revealed a long triangular glass fragment which had been retained in the spinal canal between the first and second cervical vertebrae. The air myelogram suggested the glass fragment had transfixed the cervical spinal cord. Laminectomy was performed and the glass fragment which had a shape of a sharp pointed surgical knife was removed by gentle move in the opposite direction of invation. Following removal of the foreign body, the patient's left hemiparesis recovered to normal state at four months after the operation and right sensory impairment also improved. The cervical spinal cord may be injured in the following way: the right lateral spinothalamic tract may be injured by the skewer injury due to glass fragment but injury of the left same tract may be avoided because of oblique direction of penetration of the glass fragment in the spinal cord. On the other hand, the left pyramidal tract may be compressed by glass fragment and not injured, because left hemiparesis recovered very well postoperatively.

Accidents, Home↗
PubMed1994

Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli.

The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools. This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules. We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector [Ward et al. (1989) Nature 341, 544-546] which originally, when expressed in E. coli, produced an Fab fragment. In an effort to improve the affinity of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P. aeruginosa activity was generated using a 14 amino acid linker [Chaudhary et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1066-1070]. In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments. To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb. The ScAb generated had an antigen binding capacity similar to the parent anti-P. aeruginosa antibody but was superior to the recombinant anti-P. aeruginosa Fab fragment. On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit. Dimeric ScAb had a similar antigen binding profile to the parent antibody.

Antibodies↗
PubMed1997

Preparation and characterization of the F (ab)2 fragments of an aromatase activity-suppressing monoclonal antibody.

The preparation and characterization of the Fab and F(ab')2 fragments of a murine monoclonal antibody specific for aromatase cytochrome P-450 and which is suppressive of estrogen biosynthesis are described. This monoclonal antibody, MAb3-2C2, was purified from murine ascites using protein A affinity chromatography and digested with immobilized papain to produce antibody fragments. The Fab and F(ab')2 fragments were then purified using protein A affinity chromatography and S-200 HR size exclusion chromatography. The Fab fragment was further purified using S-100 HR size exclusion chromatography. Both the Fab and F(ab')2 fragments of the MAb3-2C2 suppressed aromatase activity in a dose-dependent manner. While the F(ab')2 fragment (110 kDa) maintained potent suppressive activity, the Fab fragment (42 kDa) required a higher concentration to suppress aromatase activity as compared to the IgG.

Antibodies, Monoclonal↗
PubMed1992

Analytical control procedures of immunoreactivity for IgG and Fab fragments specific to haptens.

This study investigates immunoreactivity control procedures, i.e., specificity, affinity constant (Ka), and specific active binding sites (SABS), for polyclonal anticolchicine, monoclonal antidigitoxin IgG and Fab fragments, and antidigoxin Fab fragments (Digidot). Preliminary control procedures for IgG and Fab fragment purity indicated that all reagents were immunologically pure. All IgG and Fab fragments exhibited similar cross-reactivity and Ka. No decrease in percentage of Fab fragment SABS was observed after papain cleavage of anticolchicine and antidigitoxin IgG. Nevertheless, only 4.3 +/- 1.2% of nonimmunopurified anticolchicine polyclonal Fab fragments and 76.2 +/- 2.3 to 88.7 +/- 2.5% of different batches of immunopurified anti-digoxin Fab (Digidot) were active, the latter percentage being in the range of the 85% specified by the manufacturer. Only 58 +/- 3% of digitoxin-specific monoclonal IgG was active and 67 +/- 7% of its Fab fragments. Results show the importance of determining the ratio of SABS to presumed total specific binding sites for pharmaceutical monoclonal and polyclonal antibody preparations against haptens.

Antibody Specificity↗
PubMed1976

Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea.

The digestion of human IgG1/K myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic immunoglobulin fragments. Fb'2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the light chains, and the CH1 domains and hinge regions of the heavy chains. The smaller fragment, upFc, has now been characterized. After reduction, three peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the heavy chain. Fragment upFc therefore contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulphide loop. Peptic digestion of IgG1/K proteins in 8M-urea therefore provides a method for isolating from gamma1 heavy chains five homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb'2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two gamma1 heavy chains have been shown to differ in sequence at three positions from the sequence reported for protein Eu.

Amino Acid Sequence↗
PubMed1979

Comparison of the binding of radiolabelled human IgG and Fc fragments to murine spleen cells.

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.

Animals↗
PubMed1975

Immunologically active and structurally similar fragments of protein A from Staphylococcus aureus.

To study the active site(s) in protein A, partial tryptic digestions of the protein and of intact Staphylococcus aureus were performed. Fragments which bind to the Fc-part of human IgG were isolated by affinity chromatography on IgG-Sepharose 4B and purified by ion-exchange chromatography on phosphocellulose. From a partial tryptic digest of pure protein A at 30 degrees C, pH 8.2 for 30 min we have isolated and characterized six active fragments with molecular weights ranging from 6000 to 8000. Two active fragments, obtained in high yields by digestion at pH 7.2 of intact protein-A-containing bacteria, were shown to be similar to two of the six characterized fragments from the digest of pure protein A. All fragments appeared to have similar amino acid sequences, judged by peptide mapping, specific staining and amino acid analysis; some are very possibly overlapping peptides. Each fragment probably contains only one active site region since all are monovalent in the Fc-reaction when studied with a hemagglutination technique. The maximal molar yield of active fragments obtained from the digestion of pure protein A accounts for about 210% of the amount of protein A used. Thus protein A, suggested to consist of repeating units, should exhibit at least three similar if not identical active regions.

Amino Acid Sequence↗
PubMed1979

Immunochemical studies of beef pancreas tryptophanyl-tRNA synthetase and its fragments. Determination of the number of antigenic determinants and a comparison with tryptophanyl- tRNA synthetases from other sources and with reverse transcriptase from avian myeloblastosis virus.

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl-tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl-tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl-tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl-tRNA synthetases. The interaction of antibodies with the fragments of beef tryptophanyl-tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. On third of the antiserum antibodies interacting with the C-terminal fragment of the enzyme (Mr approximately equal to 40000) inhibits its activity whereas the antibodies to the N-terminal fragment (Mr approximately equal to 20000) have no effect on the enzyme activity. The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl-tRNA synthetase. A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl-tRNA synthetase was found to bind 9 (+/- 1) molecules of Fab fragments. Antibodies against tryptophanyl-tRNA snythetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl-tRNA synthetase by radioimmunochemical assays.

Amino Acyl-tRNA Synthetases↗
PubMed1998

Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis.

Apoptosis, or programmed cell death, involves a cascade of regulatory events leading to the activation of specific proteases. However, the key substrates for these proteases remain to be identified. We previously demonstrated that levels of five unidentified polypeptides were specifically increased in neurons from embryonic chicken ciliary ganglia undergoing apoptosis by trophic deprivation. Here we show by microsequencing of two of these polypeptides that they are fragments of actin. One of them represents cleavage of actin at the site of interaction with DNase I. The same actin fragments are also found at early stages of apoptosis in chicken and rat dorsal root ganglion neurons, chicken spinal motoneurons and rat thymocytes. Actin fragmentation may play a role in the apoptotic process, since calpain inhibitors I and II both inhibit neuronal death and suppress actin fragmentation. In contrast, caspase (ICE family) inhibitors, though effective in delaying neuronal death, do not prevent actin cleavage or DNA fragmentation. These results indicate a key role for calpain-like proteases in neuronal programmed cell death and suggest that actin fragmentation in the cell is correlated with subsequent DNA fragmentation.

Actins↗
PubMed1990

Properties of FV and Fab fragments of the antibody McPC603 expressed in E. coli.

The FV and Fab fragments of the phosphorylcholine binding antibody McPC603 were functionally expressed in E. coli. This was achieved by the co-expression and co-secretion of both chains to the periplasm, where correct processing, folding and assembly occurred. Interestingly, the fraction of correctly folded Fab fragment is smaller than that of the Fv fragment in E. coli. The intrinsic hapten binding affinity was shown to be identical for the recombinant FV or Fab fragment, the whole antibody and the Fab fragment obtained by proteolysis from the mouse antibody. Fluorescence and crosslinking analyses showed that the FV fragment dissociates at high dilution, but that it is stabilized by hapten binding. The recombinant FV fragment was shown to have catalytic activity to hydrolyze choline-p-nitrophenyl carbonate and constitutes therefore a promising model system with which the structural requirements of catalytic antibodies can be studied by altering the protein itself.

Animals↗
PubMedDate not supplied

[Two new peptic fragments of human IgA: F(abc)'2Alpha and [F(abc)'2]2alpha (author's transl)].

These experiments were undertaken to study the nature of the fragments produced by pepsin digestion of seric IgA. We describe a new fragment with a MW of 140,000 daltons. This fragment, the first produced during pepsin hydrolysis of monomeric IgA, corresponds to an IgA molecule which has lost its CH3 domain. We propose to call this fragment F(abc)'2alpha. It is gradually digested and transformed into an F(ab)'2alpha fragment. When polymeric IgA are digested, an extra fragment with a MW of 290,000 daltons is produced, which has kept its polymeric structure. It corresponds to a covalent [F(abc)'2]2alpha fragment. These findings suggest that there is an intersubunit disulfide bridge in the Calpha2 domain. These results also indicate that polymeric IgA proteins are more resistant to pepsin digestion than monomeric IgA proteins.

Humans↗
PubMed1979

[Isolation of FAB and Fc fragments from murine immunoglobulin G1].

Two methods of isolating Fab- and Fc-fragments from mouse immunoglobulin G1 are presented. The first method involves fractionation of papain protein hydrolysate on a column with DEAE- (or DE-32)-cellulose adjusted with 0.005 M K-phosphate buffer, pH 8. The Fab-fragment was eluted from the column with the starting buffer. The Fc-fragment was eluted, with the buffer ionic strength being increased to 0.4 M. Another method involves protein fractionation on an ion exchanger adjusted with 0.004 M Tris-H3PO4 buffer, pH 8.5. All the protein was column bound. The Fab-fragment was eluted with 0.04 M Tris-buffer containing a 0.004 M mixture of K-phosphates, pH 8.6. The Fc-fragment was eluted, with ionic strength being raised to 0.4 M with phosphates. As none of the methods assures isolation of absolutely pure Fab- or Fc-fragments, it is requird that cross absorption of antisera with respective immunosorbents may be carried on in order to obtain monospecific antisera to these fragments.

Animals↗
PubMed1994

Improved tumor targeting with chemically cross-linked recombinant antibody fragments.

The construction and use of recombinant chimeric and later fully humanized (CDR-grafted) antibodies to tumor-associated antigens has reduced the immune response generated to these antibodies in clinical studies. However, their long circulating half-life is a disadvantage for tumor imaging and therapy. Fragments such as F(ab')2, Fab', Fv and single chain Fv (scFv) offer faster blood clearance but also lower overall tumor doses. We have examined the tumor targeting of several novel fragments produced by chemical cross-linking of Fab' or scFv to dimeric and trimeric species. To facilitate cross-linking of Fab' fragments, a chimeric B72.3 Fab' fragment has been expressed with a hinge sequence containing a single cysteine residue. B72.3 scFv was also produced with a similar hinge region peptide attached to the COOH terminus to allow cross-linking. These fragments, Fab' delta Cys and scFv' delta Cys were cross-linked with linkers containing two or three maleimide groups to produce dimeric and trimeric molecules with increased avidity for antigen. Cross-linkers were also designed to contain a 12-N-4 macrocycle capable of stable radiolabeling with 90Y. This allowed the production of site-specifically-labeled, fully immunoreactive proteins. Biodistribution studies in the nude mouse LS174T xenograft model with scFv, di-scFv, and tri-scFv demonstrated that these fragments clear extremely rapidly from the circulation and give rise to only low levels of activity accumulated at the tumor. Di-Fab (DFM) and tri-Fab (TFM) however, accumulated relatively high levels of activity at the tumor with high tumor:blood ratios generated, demonstrating improved targeting compared to IgG. cB72.3 90Y-labeled tri-Fab was found not to accumulate in the kidney or the bone, resulting in an attractive antibody fragment for tumor therapy.

Animals↗
PubMed1979

[Structural and immunochemical studies of carp (Cyprinus carpio L.) immunoglobulin. V. Tryptic fragments of carp (Cyprinus carpio L.) immunoglobulin M].

Carp IgM, isolated from normal serum is more sensitive to trypsinization compared to a human myeloma protein IgMGo. Under the same conditions (treatment with trypsin at 56 degrees C for 30 min) carp IgM was degraded to small, mostly dialysable peptides to a larger extent than IgMGo. In both cases the fragmentation resulted in immunoelectrophoretically pure Fab mu and Fc mu fragments. The Fab mu fragments of human IgM (yield: 20% of used IgM material) had a molecular weight of 54,000, the Fc mu fragments (yield: 30%) were a heterogenous mixture as far as molecular sizes concerned with values of about 300,000. For the corresponding fragments of carp IgM we could analyze a molecular weight of about 43,000 for Fab mu (yield: 8%) and for Fc mu (yield 10%) three fractions of 160,000, 130,000 and 90,000. The reductive subunits of Fc mu fragments showed different molecular weights: 39,000 for IgMGo and 45,000 for carp IgM. The anti-fragment antisera prepared in rabbits were monospecific as demonstrated by immunodiffusion.

Animals↗
PubMed1996

Applicability of monoclonal antibody Fab fragments as a carrier of neocarzinostatin in targeting chemotherapy.

Two types of fragments of MAb A7 were produced to improve the efficacy and safety in targeting chemotherapy with neocarzinostatin. In this study, 125I-labeled F(ab')2 and Fab fragments of MAb A7 and 125I-labeled MAb A7 were injected intravenously into mice with pancreatic carcinoma xenografts, and the accumulation of each antibody in the tumors was compared. A greater amount of the 125I-labeled Fab fragments of MAb A7 localized in the tumor 2 h following the injection than was observed with the other probes. Relatively less 125I-labeled MAb A7 localized in the tumor 2 h following the injection than was observed with the other two probes. Moreover, reaction of rabbit antimouse IgC with the Fc portion, which is the most immunopotent region of the Fab and F(ab')2 fragments of MAb A7 and MAb A7, was determined by ELISA; the weakest reaction was observed with the Fab fragments of MAb A7. These results suggest that the Fab fragments of MAb A7 may be more suitable carriers of an anticancer drug that is inactivated rapidly in the blood, such as NCS, in targeting chemotherapy than either intact MAb A7 or the F(ab')2 fragments of MAb A7.

Animals↗
PubMed1997

Levels of urinary beta-core fragment, total oestriol, and the ratio of the two in second-trimester screening for Down syndrome.

Levels of beta-core fragment and total oestriol in second-trimester maternal urine samples were measured in 32 Down syndrome pregnancies and 206 control pregnancies. Beta-core fragment and total oestriol values were corrected for the urinary creatinine level and expressed as multiples of the control medians (MOM). In addition, the ratio of the beta-core fragment level to the total oestriol level, without creatinine correction, was calculated, and expressed as MOM values. The median beta-core fragment, total oestriol, and ratio levels in Down syndrome cases were 5.42, 0.64, and 9.32 MOM, respectively. In the Down syndrome pregnancies, 66 per cent of the beta-core fragment levels were above the 95th centile of control levels, while 22 per cent of the total oestriol levels were below the fifth centile of control levels. In combination with maternal age, measurement of beta-core fragment and total oestriol levels in Down syndrome pregnancy resulted in an 80 per cent detection rate at a 5 per cent false-positive rate. Use of the ratio resulted in a univariate detection rate of 72 per cent. In combination with maternal age, the ratio resulted in a detection rate of 81 per cent at a 5 per cent false-positive rate. Based on this unmatched study, the measurement of a ratio of beta-core fragment to total oestriol levels, without the need for creatinine correction, may be useful in screening for fetal Down syndrome in second-trimester urine.

Adult↗