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PubMed1976

Amino-acid sequence of fragment A, an enzymically active fragment from diphtheria toxin.

The amino-acid sequence of Fragment A from diphtheria toxin is reported. Fragment A (molecular weight, Mr, 21,145) is the major enzymically active fragment produced upon activation of the intact toxin (Mr about 60,000) by limited tryptic digestion and reduction. It, or a similar fragment, is believed responsible for the inhibition of protein synthesis in animal cells exposed to the toxin. Fragment A, which corresponds to the amino terminus of the toxin, is shown here to consist of three major forms (190, 192, and 193 residues) resulting from cleavage by trypsin adjacent to any of three closely spaced arginine residues. All three forms are enzymically active.

Amino Acid Sequence↗
PubMed1998

Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45.

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.

Animals↗
PubMed1999

Studies on degradative mechanisms mediating post-translational fragmentation of apolipoprotein B and the generation of the 70-kDa fragment.

It has been well established that the biogenesis of apoB is mediated co-translationally by the cytosolic proteasome. Here, however, we investigated the role of both the cytosolic proteasome as well as non-proteasome-mediated degradation systems in the post-translational degradation of apoB. In pulse-chase labeling experiments, co-translational (0-h chase) apoB degradation in both intact and permeabilized cells was sensitive to proteasome inhibitors. Interestingly, turnover of apoB in intact cells over a 2-h chase was partially inhibitable by lactacystin, thus suggesting a role for the cytosolic proteasome in the post-translational degradation of apoB. In permeabilized cells, however, there was no post-translational protection of apoB by lactacystin. Further investigations of proteasomal activity in HepG2 cells revealed that, following permeabilization, there was a dramatic loss of the 20 S proteasomal subunits, and consequently the cells exhibited no detectable lactacystin-inhibitable activity. Thus, apoB fragmentation and the generation of the 70-kDa apoB degradation fragment, characteristic of permeabilized cells, continued to occur in these cells despite the absence of functional cytosolic proteasome. Similar results were observed when we used a derivative of lactacystin, clastolactacystin beta-lactone, which represents the active species of the inhibitor. Interestingly, however, the abundance of the 70-kDa fragment could be modulated by the microsomal triglyceride transfer protein inhibitor, BMS-197636, as well as by pretreatment of the permeabilized cells with dithiothreitol. These data thus suggest that although the cytosolic proteasome appears to be involved in the post-translational turnover of apoB in intact cells, the specific post-translational fragmentation of apoB generating the 70-kDa fragment observed in permeabilized cells occurs independent of the cytosolic proteasome.

Acetylcysteine↗
PubMed2000

Characterization of the rat DNA fragmentation factor 35/Inhibitor of caspase-activated DNase (Short form). The endogenous inhibitor of caspase-dependent DNA fragmentation in neuronal apoptosis.

Nuclear changes, including internucleosomal DNA fragmentation, are classical manifestations of apoptosis for which the biochemical mechanisms have not been fully elucidated, particularly in neuronal cells. We have cloned the rat DNA fragmentation factor 35/inhibitor of caspase-activated DNase (short form) (DFF35/ICAD(S)) and found it to be the predominant form of ICAD present in rodent brain cells as well as in many other types of cells. DFF35/ICAD(S) forms a functional complex with DFF40/caspase-activated DNase (CAD) in the nucleus, and when its caspase-resistant mutant is over-expressed, it inhibits the nuclease activity, internucleosomal DNA fragmentation, and nuclear fragmentation but not the shrinkage and condensation of the nucleus, in neuron-differentiated PC12 cells in response to apoptosis inducers. DFF40/CAD is found to be localized mainly in the nucleus, and during neuronal apoptosis, there is no evidence of further nuclear translocation of this molecule. It is further suggested that inactivation of DFF40/CAD-bound DFF35 and subsequent activation of DFF40/CAD during apoptosis of neuronal cells may not occur in the cytosol but rather in the nucleus through a novel mechanism that requires nuclear translocation of caspases. These results establish that DFF35/ICAD(S) is the endogenous inhibitor of DFF40/CAD and caspase-dependent apoptotic DNA fragmentation in neurons.

Amino Acid Sequence↗
PubMed1988

Segregation of HLA-DR, -DQ and -DP phenotypes and restriction fragment length polymorphic fragments in two recombinant families.

Members of two families were typed for HLA-DR, -DQ and -DP specificities by means of sera and local PLT bulk reagents. One B:C and one DR:DP cross-over were identified in both families. The restriction fragment length polymorphism (RFLP) was analyzed by southern blotting and the use of DR beta, DR alpha, DQ beta, DQ alpha, DP beta and DP alpha probes. Previous observations concerning typical RFLP patterns for serological DR/DQ- and cellular DP-specificities were confirmed. With respect to recombinant haplotypes, RFLP was informative in both families. All polymorphic fragments-(DRB, DQA and B as well as DPA and B)-segregated together in the HLA-B:DR cross-overs. In the DR:DP recombinant children, the DR and DQ fragments were separated from DP fragments, demonstrating that DP-types as defined by PLT and RFLP map close together. No cross-hybridization between the segregating fragments was detected with the various probes.

Female↗
PubMed1985

Calcium release from intact calmodulin and calmodulin fragment 78-148 measured by stopped-flow fluorescence with 2-p-toluidinylnaphthalene sulfonate. Effect of calmodulin fragments on cardiac sarcoplasmic reticulum.

Calcium release from high and low-affinity calcium-binding sites of intact bovine brain calmodulin (CaM) and from the tryptic fragment 78-148, purified by high-pressure liquid chromatography, containing only the high-affinity calcium-binding sites, was determined by fluorescence stopped-flow with 2-p-toluidinylnaphthalene sulfonate (TNS). The tryptic fragments 1-77 and 78-148 each contain a calcium-dependent TNS-binding site, as shown by the calcium-dependent increase in TNS fluorescence. The rate of the monophasic fluorescence decrease in endogenous tyrosine on calcium dissociation from intact calcium-saturated calmodulin (kobs 10.8 s-1 and 3.2 s-1 at 25 degrees C and 10 degrees C respectively) as well as the rate of equivalent slow phase of the biphasic decrease in TNS fluorescence (kobsslow 10.6 s-1 and 3.0 s-1 at 25 degrees C and 10 degrees C respectively) and the rate of the solely monophasic decrease in TNS fluorescence, obtained with fragment 78-148 (kobs 10.7 s-1 and 3.5 s-1 at 25 degrees C and 10 degrees C respectively), were identical, indicating that the rate of the conformational change associated with calcium release from the high-affinity calcium-binding sites on the C-terminal half of calmodulin is not influenced by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed by the N-terminal half of the molecule. The fast phase of the biphasic decrease of TNS fluorescence, observed with intact calmodulin only (kobsfast 280 s-1 at 10 degrees C) but not with fragment 78-148, is most probably due to the conformational change associated with calcium release from low-affinity sites on the N-terminal half. The calmodulin fragments 1-77 and 78-148 neither activated calcium/calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum nor inhibited calmodulin-dependent activation at a concentration approximately 1000-fold greater (5 microM) than that of the calmodulin required for half-maximum activation (5.9 nM at 0.8 mM Ca2+ and 5 mM Mg2+) of calmodulin-dependent phosphoester formation.

Animals↗
PubMed1988

Refolding of bacteriorhodopsin. Protease V8 fragmentation and chromophore reconstitution from proteolytic V8 fragments.

Staphylococcus aureus protease V8 cleaves bacteriorhodopsin to two main fragments, V-1 and V-2. Proteolytic digestion of the purple membrane integrated protein is carried out in the presence of limited amounts of sodium dodecyl sulfate (0.5 g detergent/g bacteriorhodopsin). The fragment V-1 includes the arylisothiocyanate binding site (Lys41). The V-2 fragment comprises the two C-terminal transmembrane segments of bacteriorhodopsin. Improved renaturation of bacteriorhodopsin and the ternary complex, reformed from its V8 proteolytic fragments, is attained by peptide extraction in chloroform/methanol/0.1 M ammonium acetate and subsequent incorporation into phospholipid/detergent micelles. In the presence of retinal, V8 fragments reform chromophoric ternary complexes. Light-adapted reconstituted chromophores absorb incident light at 560 nm. Protein secondary structures are partially conserved in the course of solvent extraction and are restored in the reconstituted system. Vesicles prepared from the reconstituted complexes show light-dependent proton translocation activity.

Bacteriorhodopsins↗
PubMedDate not supplied

Restriction fragment length polymorphism (RFLP) heterogeneity of HLA-DQ beta genes associated with DNA fragment identical to the DR1-beta DNA structure.

Restriction fragment length polymorphism (RFLP) analyses of DR1 positive peripheral blood leukocytes DNA was carried out. The Taq I digested DNA was hybridized with cDNA probes for HLA-DR and -DQ beta genes. The DR probe detected fragments commonly observed in the DR1 specificity, whereas a new DQ-beta fragment was detected in some DR1 haplotypes when the DQ-beta probe was used. This fragment had an RFLP pattern identical to the DQ-beta fragment typically associated with most DR2 and some DRw6 specificities.

Alleles↗
PubMed1981

Fc fragment activation of T lymphocytes. I. Fc fragments trigger Lyt 1+23- T lymphocytes to release a helper T cell-replacing activity.

Fc fragments of human IgG stimulate both proliferation and polyclonal antibody formation by B lymphocytes. T lymphocytes, although not proliferating in response to Fc fragments, are triggered to produce a T cell-replacing factor, which substitutes for T cells in the Fc fragment-induced polyclonal response. The factor is produced within 24 hr after Fc stimulation. Neither Fab fragments nor whole IgG have the capacity to stimulate the release of this activity. This material is a product of Lyt 1+23-, Ia-T lymphocytes and requires Ia+ adherent accessory cells for production. The role of the accessory cell is to process the Fc fragments to biologically active 14,000-daltons subfragments, which directly trigger T cells to factor production. This material has been called (Fc)TRF to distinguish it from other T cell factors.

Animals↗
PubMed1993

Apoptotic death in epithelial cells: cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation.

To date, apoptosis has been characterized biochemically by the production of 180-200 bp internucleosomal DNA fragments resulting from the activation of an endonuclease(s). The principal morphological feature of apoptosis is the condensation of chromatin and it has been assumed that this may reflect the oligonucleosomal fragmentation pattern. We have re-examined this dogma by comparing the biochemical and morphological features of cell death in several epithelial cell types (HT-29-I1 colon adenocarcinoma, CC164 mink lung, DU-145 human prostatic carcinoma and MCF-7 human breast adenocarcinoma) and one mesenchymal cell line (H11ras-R3 ras-transformed rat fibroblasts). Cell death was induced either by serum deprivation, TGF-beta 1 or etoposide, or by leaving cells to reach confluence. Cell death was assessed with respect to detachment from monolayers, morphological changes and DNA integrity. The DNA-binding fluorophore Hoechst 33258 revealed chromatin condensation patterns consistent with apoptotic cell death in all cell types except MCF-7 cells. Using field inversion gel electrophoresis in conjunction with conventional 2% agarose gel electrophoresis, cleavage of DNA to 50 kbp fragments was observed in all cases except MCF-7 cells. This preceded the appearance of oligonucleosomal fragments in HT-29-I1, CC164 and H11ras-R3 cells. Although the DNA of DU-145 cells fragmented into 50 kbp units, and although the cells exhibited classical apoptotic morphology, no subsequent internucleosomal cleavage was observed. These results suggest that changes in the integrity of DNA indicative of the release of chromatin loop domains occur before cleavage at internucleosomal sites is initiated and that the latter is not an essential step in the apoptotic process.

Animals↗
PubMed2000

Lack of obvious 50 kilobase pair DNA fragments in DNA fragmentation factor 45-deficient thymocytes upon activation of apoptosis.

The DNA fragmentation factor 45 (DFF45/ICAD) is a key subunit of a heterodimeric DNase complex critical for the induction of DNA fragmentation during apoptosis in vivo. To further assess the importance of DFF45 in chromosomal DNA degradation, we induced apoptosis in wild-type control and DFF45 deficient thymocytes and compared the cleavage of chromosomal DNA to 50 kilobase pair size fragments. We found that there is a lack of obvious large chromosomal DNA fragments upon treatments by various apoptotic agents in DFF45 deficient thymocytes. The major organ systems in the DFF45 mutant mice either two months or fifteen months of age appear normal. These results suggest that functional DFF45 is required for cleavage of DNA into both large size and oligonucleosomal size fragments in thymocytes during apoptosis. However, deficiency in DFF45 apparently does not significantly affect normal mouse development and tissue homeostasis.

Age Factors↗
PubMed1997

[Dislocated multiple fragment fractures of the head of the humerus. Does dislocation of the humeral head fragment signify a worse prognosis?].

PROBLEM: The vascularity of the articular fragment is of key importance for the final outcome in three- and four-part fractures of the humeral head. Displacement of the articular segment may compromise the arterial blood supply to the humeral head and result in avascular necrosis. There is still controversy as to whether three-and four-part fracture dislocations (articular fragment outside the glenoid) have an even worse prognosis than displaced three- and four-part fractures. PATIENTS AND METHODS: Between January 1985 and May 1993, 102 patients with three- and four-part fractures of the humeral head were treated by ORIF (mostly tension band wiring) at our institution. In a retrospective study we analysed the functional (Constant 100 point score) and radiological outcome of 67 (66%) of these patients. There were 21 patients with fracture dislocations (FD), n = 5 type B2X, n = 5 type B3X, n = 3 type C2X, n = 8 type C3X, according to the classification of Habermeyer [7]. The "X" represents the dislocation of the articular fragment, whereas the classification to each type is done after reduction of the head. The remaining 46 patients presented with displaced, but not dislocated, three- and four-part fractures (DF), n = 24 type B2, n = 7 type B3, n = 3 type C2, n = 12 type C3. Average follow-up was 25 months (7-72 months). Patients with FD were significantly younger (average age 50 years) than patients with DF (average age 63 years, P < 0.05) and showed a significantly higher incidence of traumatic nerve or plexus lesions (FD 19%, DF 2%, P < 0.05). RESULTS: Concerning the functional results, there was no statistically significant difference between the two groups. The FD patients even showed a slight tendency to better results than patients with DF. This was true for the three-part fractures (average Constant score 78 versus 67 points), as well as for the four-part fractures (average Constant score 62 versus 55 points). The significantly younger age of the FD patients may explain their better results. The entire group of patients with three-part fractures showed a significantly better functional outcome (average Constant score 68 points) than patients with four-part fractures (average Constant score 55 points, P < 0.05). The rate of partial and total avascular necrosis of the humeral head was strongly correlated to the fracture type (number of fragments, fracture of the anatomical or surgical neck, according to the classification of Habermeyer),but again there was no difference between the FD and DF group (B2X: 20%, B3X: 20%, C2X: 33%, C3X: 63%; B2: 25%, B3: 29%, C2: 33%, C3: 67%). Astonishingly, the FD were not associated with an increased rate of avascular necrosis of the humeral head. Three (axillary nerve) out of the five observed primary nerve and plexus lesions had a full neurological recovery after 6-12 months; the two patients with alterations of the brachial plexus showed a slow tendency of improvement at follow-up (12 and 18 months), but still had gross muscular atrophy and impaired sensory function. CONCLUSION: In displaced three-and four-part fractures of the humeral head the dislocation of the articular segment does not seem to increase the risk of avascular necrosis, if treated by timely and careful ORIF with respect to the vascularity. Even with the increased risk of primary nerve and plexus lesions in fracture dislocations, good functional results can be achieved by early operative nerve decompression and fracture stabilization in this middle-aged patient group. However, older patients with displaced or dislocated four-fragment fractures through the anatomical neck (type C3) have a poor chance of a favourable outcome, and therefore primary prosthetic replacement should be considered.

Adult↗
PubMed1998

Similarities in the biodistribution of iodine-labeled anti-Tac single-chain disulfide-stabilized Fv fragment and anti-Tac disulfide-stabilized Fv fragment.

We evaluated the biodistribution and pharmacokinetics of two different iodine-labeled Fv fragments of anti-Tac monoclonal antibody (MAb) in normal and tumor-bearing nude mice. One was a disulfide-stabilized Fv fragment (dsFv), and the other was a single-chain disulfide-stabilized Fv fragment (scdsFv). The scdsFv is a newly developed type of Fv fragment superior to the dsFv in which the VH and VL are linked by covalent bonds through a spacer arm and by an internal disulfide bond. These modifications increase the yield of scdsFv. Both reagents recognize the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The biodistribution of the Fv fragments was evaluated in normal mice co-injected with 50 mg of L-lysine and in a no-lysine control group. Biodistribution was also evaluated in nude mice bearing subcutaneous tumor xenografts derived from IL-2Ralpha-positive ATAC4 cells and receptor-negative A431 cells. These mice were co-injected with 125I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 131I-labeled anti-Tac dsFv (2 microCi/0.7 microg) or with 131I-labeled anti-Tac scdsFv (6 microCi/0.7 microg) and 125I-labeled anti-Tac dsFv (4 microCi/0.7 microg). The biodistribution of 125I-labeled anti-Tac scdsFv and 131I-labeled anti-Tac dsFv was very similar in all organs and the tumors. The renal uptake of both reagents was blocked effectively (<93%) and similarly by lysine. The scdsFv cleared slightly faster from the circulation than did the dsFv because there were more aggregates of dsFv than of scdsFv (3% vs. 1%, respectively). The scdsFv-to-dsFv ratio ranged from 0.79 to 1.20 in all organs at all time points we examined. In conclusion, the first biodistribution study of an scdsFv molecule shows that the scdsFv had a biodistribution very similar to that of the dsFv and seems to be a good alternative to the dsFv because of its higher production yield.

Animals↗
PubMed1986

Cue depreciation: when word fragment completion is undermined by prior exposure to lesser fragments.

The ability to think of a previously studied item has often been shown to be impaired when, in one way or another, the extraitem context is changed from study to test. In a series of five experiments, such impairment is induced in a somewhat different way. A fragment (e.g. r-i--rop) of a just-studied word (raindrop) is shown to be less readily completed if it is presented bit by bit (r------p, r----r-p, r-i--r-p, r-i--rop) rather than all at once (Experiments 1, 3, 4, and 5). No such effect is found if the word has not been studied beforehand (Experiments 2, 3, 4, and 5). This pattern of results occurs even when fragments of studied and nonstudied words occur in the same test and under conditions in which subjects cannot tell whether a given fragment is of a studied or nonstudied word (Experiments 4 and 5). In addition, for words that have been studied beforehand, the impairment is shown to increase systematically with the number of steps involved in the presentation of the word fragment (Experiment 3) and also to persist when the time allowed for completion of the final version of the fragment is increased from 4 s to a full minute (Experiment 5).

Cues↗
PubMed1982

Intracellular stability of diphtheria toxin fragment A in the presence and absence of anti-fragment A antibody.

The erythrocyte ghost function method was used to introduce 125I-labeled diphtheria toxin, its fragments A and B, and two labeled crossreacting material (CRM), mutant proteins CRM176 and CRM197, into cultured mouse cells. Fragment A was relatively stable in mouse cytoplasm at 37 degrees C and at least 80% was recovered from cells after 24 hr of incubation. In contrast, wild-type fragment B and A fragments from CRM176 and CRM197 were unstable and were degraded with half-lives of about 2.5 hr under similar conditions. When a rabbit anti-fragment A IgG fraction was introduced with wild-type A, the rate of degradation of A was accelerated, whereas the rates of degradation of A176 and A197 were retarded by the same antibody. In every instance the degradation rate appeared to be that of the IgG fraction itself with a half-life of about 7.5 hr.

Animals↗
PubMed2002

Characterization of three fragments that constitute the monomers of the human lysyl hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl hydroxylase activity.

Lysyl hydroxylase (LH) catalyzes the formation of hydroxylysine in collagens; three human isoenzymes have been cloned so far. We report here on the purification of all three recombinant isoenzymes to homogeneity from the medium of cultured insect cells, and we demonstrate that they are all homodimers. Limited proteolysis experiments identified two main protease-sensitive regions in the monomers of about 80-85 kDa, corresponding to three fragments A-C (from the N to C terminus), with molecular masses of about 30, 37, and 16 kDa, respectively. Fragment A was found to play no role in LH activity as a recombinant B-C polypeptide constituted a fully active hydroxylase with K(m) values for cosubstrates and the peptide substrate that were identical to those of the full-length enzyme. LH3, but not LH1 and LH2, has also been reported recently (Heikkinen, J., Risteli, M., Wang, C., Latvala, J., Rossi, M., Valtavaara, M., and Myllylä, R. (2000) J. Biol. Chem. 275, 36158-36163) to possess collagen glucosyltransferase activity. We confirm this highly surprising finding here and extend it by demonstrating that LH3 may also possess trace amounts of collagen galactosyltransferase activity. All the glucosyltransferase and galactosyltransferase activity of LH3 was found to reside in fragment A, which played no role in the hydroxylase activity of the polypeptide. This fragment is about 55% identical and 80% similar to the corresponding fragments of LH1 and LH2. However, the levels of the glycosyltransferase activities are so low that they may be of little biological significance. It is thus evident that human tissues must have additional glycosyltransferases that are responsible for most of the collagen glycosylation in vivo.

Amino Acid Sequence↗
PubMed1972

Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. IV. Chemical and cytological characterization and biosynthetic capabilities of fragments obtained by mild procedures.

Membrane-envelope fragments have been isolated from Escherichia coli by comparatively mild techniques. The use of DNAase, RNAase, detergents, sonication, lysozyme, and ethylenediaminetetraacetate were avoided in the belief that rather delicate, but metabolically important, associations may exist between the plasma membrane and various cytoplasmic components. The membrane-envelope fragments have been characterized in terms of their content of major chemical components as well as their electron microscope appearance. Fractions containing membrane-envelope fragments were found to possess appreciable DNA- and protein-synthesizing activities. The fragments were rich in membrane content as determined by reduced nicotinamide adenine dinucleotide (NADH) oxidase activity and deficient in soluble components as measured by NADH dehydrogenase activity. The particulate fraction obtained between 20,000 g and 105,000 g and usually considered a ribosomal fraction was rich in membrane content and had a relatively high capacity for DNA synthesis. Envelope fragments sedimenting at 20,000 g attained very high levels of incorporation of amino acids into protein.

Amino Acids↗
PubMed1983

Segregation and mapping analysis of polymorphic HLA class I restriction fragments: detection of a novel fragment.

An HLA-B7 complementary DNA clone was used as a hybridization probe to analyze the segregation pattern of polymorphic class I restriction fragments in several families whose HLA types had been determined by serological techniques. In one family in which a crossover in the HLA region had occurred, a specific genomic fragment was mapped with respect to the crossover. In another family, a novel genomic fragment present in one child and absent in all other family members was observed. With the exception of this novel fragment, all polymorphic class I fragments observed in this study segregated with a serologically defined parental haplotype, a result consistent with HLA linkage.

Chromosome Mapping↗