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At least 1,621 records · Page 90Linked to original sources
PubMed1996

DNA sequence analysis of Prinker-modified restriction fragments after collection from capillary electrophoresis with replaceable matrices.

This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.

Automation↗
PubMed1991

An improved method for the detection of DNA fragmentation.

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.

Animals↗
PubMed1989

Cleaving the prohead RNA of bacteriophage phi 29 alters the in vitro packaging of restriction fragments of DNA-gp3.

In vitro packaging of restriction fragments of the bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) in the defined system was dependent on prohead RNA. Truncated prohead RNAs were obtained by in situ RNase A digestion, isolated and sequenced. Proheads having the intact 174 base RNA were compared to proheads having RNAs of 120, 95, 71, 69 or 54 bases for the capacity to package the DNA-gp3 left and right ends and internal (non-end) fragments generated by the restriction enzymes EcoRI, HpaI and BstNI. Proheads with the 174 or 120 base RNAs packaged both left and right ends; internal fragments were packaged more efficiently by proheads with the 120 base RNA. Proheads with the 95 base RNA packaged DNA-gp3 left ends and internal fragments efficiently, but lost the capacity to package right ends. Only internal fragments were packaged by proheads with the 71 base RNA, and proheads having 69 or 54 base RNAs were inactive. RNA-free proheads were effectively reconstituted with purified 174 and 120 base RNAs to produce particles similar in biological activity to the proheads from which the RNAs were isolated. The 95 base RNA was the smallest RNA of the group that could reconstitute the prohead and direct fragment packaging, although packaging was inefficient. Alteration of the specificity of DNA fragment packaging with truncated prohead RNAs has delineated RNA domains that function in DNA-gp3 recognition and prohead binding.

Bacteriophage lambda↗
PubMed1991

Facilitation of sexual receptivity in the female rat by C-terminal fragments of LHRH.

Ovariectomized female rats implanted with cannulae directed bilaterally towards the ventromedial nucleus of the hypothalamus (VMH) were primed with estrogen (2 micrograms) and tested for sexual receptivity before and after infusion of the C-terminal LHRH fragment, Ac-LHRH5-10. Animals responsive to the Ac-LHRH5-10 fragment were tested at two-week intervals for responsiveness to other LHRH fragments modified in the C-terminal or saline in the following order: LHRH5-10, des-Gly6LHRH, LHRH5-9OH, saline, and LHRH7-9OH. The lordosis-to-mount (L/M) ratio was used as an index of sexual receptivity. The quality of lordotic posturing and the number of proceptive and resistive behaviors were also recorded for each test. A one-way ANOVA performed on the difference in L/M between pre- and postinfusion tests revealed a significant overall treatment effect. The nonacetylated LHRH5-10 fragment and the LHRH5-9OH fragment were effective in enhancing the L/M ratio, whereas des-Gly6LHRH and LHRH7-9OH were not. The enhancement of sexual behavior by C-terminal fragments of LHRH was associated with a lack of proceptive behavior and moderate levels of resistive behavior. The results indicate that several C-terminal fragments of LHRH are capable of elevating the L/M ratio and suggest that amino acids in positions 6 through 9 may be important for this effect.

Amino Acid Sequence↗
PubMed1992

Acetaminophen-induced cytotoxicity in cultured mouse hepatocytes: effects of Ca(2+)-endonuclease, DNA repair, and glutathione depletion inhibitors on DNA fragmentation and cell death.

Hepatotoxic alkylation of mouse liver cells by acetaminophen is characterized by an early loss of ion regulation, accumulation of Ca2+ in the nucleus, and fragmentation of DNA in vitro and in vivo. Acetaminophen-induced DNA cleavage is accompanied by the formation of a "ladder" of DNA fragments characteristic of Ca(2+)-mediated endonuclease activation. These events unfold well in advance of cytotoxicity and the development of necrosis. The present study utilized cultured mouse hepatocytes and mechanistic probes to test whether DNA fragmentation and cell death might be related in a "cause-and-effect" manner. Cells were isolated by collagenase perfusion, cultured in Williams' E medium for 22-26 hr, and exposed to acetaminophen. Aurintricarboxylic acid, a general Ca(2+)-endonuclease inhibitor, and EGTA, a chelator of Ca2+ required for endonuclease activation, significantly decreased DNA fragmentation at 6 and 12 hr and virtually abolished cytotoxicity. N-Acetylcysteine also eliminated DNA fragmentation and cytotoxicity. 3-Aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase-stimulated DNA repair, failed to alter the amount of DNA fragmentation at 6 hr but substantially increased acetaminophen cytotoxicity in hepatocytes at 12 hr. With the exception of when DNA repair was inhibited by 3-aminobenzamide, Ca2+ accumulation in the nucleus, DNA fragmentation, and hepatocyte death varied consistently and predictably with one another. Collectively, these findings suggest that unrepaired damage to DNA contributes to acetaminophen-induced cell death in vivo and may play a role in necrosis in vivo.

Acetaminophen↗
PubMed1983

Mapping thymidine kinase-deficient mutants of vaccinia virus by marker rescue with hybrid plasmid DNAs containing portions of the HindIII-J fragment of virus DNA.

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK- vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.

DNA Restriction Enzymes↗
PubMed1982

The influence of calcium ions on the conformation of fibrinogen fragment D: the use of chemical cross-linking agents.

Fibrinogen fragment D prepared in the presence of calcium ions (fragment D[Ca++]) shows qualitatively similar cross-linking patterns with dimethyl suberimidate, dimethyl adipimidate and tetranitromethane. Fragment D prepared in the presence of EDTA (fragment D[EDTA]) gives a consistently different pattern with these reagents. In the case of fragment D[EDTA] there is much more intermolecular cross-linking suggesting that the loss of the C-terminus of the gamma-chain remnant results in fragment D adopting a more open conformation. Neither the addition of 2M urea nor EDTA to fragment D[Ca++] alters its cross-linking pattern suggesting that the proposed conformational change follows cleavage of a plasmin susceptible bond which is normally protected by the presence of calcium ions.

Calcium↗
PubMed1983

Influence of fibrinogen fragments on human lymphocyte thymidine uptake.

Fragments of human fibrinogen were tested for ability to suppress activation of human blood lymphocytes by the lectin phytohaemagglutinin (PHA). Measurement of cellular 14C-thymidine uptake was used as an indicator of DNA synthesis and cell proliferation. The tested fragments were obtained by plasmin digestion or by CNBr fragmentation of fibrinogen. Inhibition of uptake, with good dose response relationship, was demonstrated for the plasmic fragment PL-3 and for the CNBr fragment Hol-DSK. Digests of the latter with trypsin and plasmin gave the same effects as the intact fragment. However, reproducibility was poor with PL-3. Hol-DSK gave better reproducibility, but with pronounced interbatch variations. Other fragments of fibrinogen were tested, but gave no effect in the studied concentrations. The reason for the poor reproducibility is discussed.

Chromatography, Gel↗
PubMed1985

Structural studies and "in vivo" and "in vitro" pharmacological activities of heparin fractions and fragments prepared by chemical and enzymic depolimerization.

Two heparin families (SM- and FM- heparins) have been fractionated with barium by selective precipitation at different temperatures. Fragments were obtained from these two fractions by enzymic and chemical degradation. Some structural features and pharmacological activities of FM- and SM-heparins as well as the fragments are now reported. The fragments, prepared by ascorbate/H2O2 oxidation (M.W. 4,500) or heparitinase II (M.W. 5,500), are enriched with trisulfated disaccharide units whereas heparin (M.W.12,500) and FM-heparin (M.W. 10,200) contain also N-sulfated and N-acetylated disaccharides. The chemical and enzymic fragments show 1/3 to 1/5 of the anticoagulant activity of heparin by the USP and APTT methods. The LPL releasing activity is also low in the fragments as well as the AXa activity measured by the chromogenic method. On the other hand the AXa activity measured by the Yin and Wessler procedure is two times higher in the fragments when compared to heparin. The fragments resulting from heparin after heparitinase II degradation and ascorbate/H2O2 oxidation were rich in trisulfated disaccharide units whereas heparin and FM-heparin contained also measurable amounts of monosulfated and N-acetylated disaccharide units. The mechanism of ascorbate oxidation and the structural requirements for the pharmacological activities of heparin is discussed in view of these and other findings.

Animals↗
PubMed1986

Binding of 125I-labelled fibrin(ogen) fragments to platelets and to immunoprecipitated glycoprotein IIb-IIIa complex.

To further investigate which parts of the fibrinogen molecule that are responsible for its binding to the fibrinogen receptor on human platelets, the following approaches were made: The glycoprotein IIb-IIIa complex (the putative fibrinogen receptor) was immunoprecipitated in crossed immunoelectrophoresis of Triton X-100-extracts of platelets against antibodies to whole platelet proteins. Subsequently, the immunoplates were incubated with 125I-labelled, plasmin- or CNBr-cleaved fibrinogen fragments (pre-X,X,Y,D,Degta,Efg,N-DSK) or fibrin fragments (E1,N-dsk), characterized by partial sequenation. The immunoplates were exposed to X-ray films, and binding of the fragments to the glycoprotein IIb-IIIa complex was examined. The findings were compared to the results obtained from studies on binding of the same fragments to intact gel-filtered platelets after ADP-stimulation. The following conclusions were made: All fragments except Efg and Degta bound to the immunoprecipitated GPIIb-IIIa complex as well as to ADP-stimulated platelets suggesting that at least two sequences in the E domain and one in each of the D domains of fibrinogen are involved in binding to the platelet receptor. The GPIIb-IIIa complex is the only surface-located platelet antigen that binds fibrinogen and the aforementioned fragments. The binding of the fragments to the receptor is dependent on divalent cations.

Antibodies↗
PubMed1990

Polymerization of fibrinogen-derived fragment X and subsequent rearrangement of fibers.

Polymerization of purified fragment X preparations, obtained from a late stage 2 plasmin digest of fibrinogen, was studied by turbidimetry and electron microscopy. The thrombin-induced polymerization of fragment X caused a rapid increase in turbidity followed by a slow decrease. The initial turbidity increase of fragment X polymers was considerably greater than that of normal fibrin. Electron microscopy carried out at different time points in the reaction revealed that initially there was a great deal of lateral aggregation of fragment X fibers. At later times, electron microscopy showed that there was more dispersal or disruption of the fibers, corresponding to the decrease in turbidity. The slow decrease in turbidity was apparently caused by the rearrangement and/or splitting apart of fragment X fibers, starting from a state similar to a very coarse clot, with much aggregation of fibers, to a delicate meshwork, similar to a fine clot. These changes in clot and fiber structure may result from cleavages partly in the carboxy-terminal region of the gamma chains and the amino-terminal region of the B beta chain of fragment X or the influence of other non-clottable fragments present in these preparations.

Biopolymers↗
PubMed1979

Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis.

When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60 degrees C in a concentration gradient of a urea-formamide mixture, each Eco RI fragment of lambda or E. coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment has been retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a two-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the Eco RI digest of E. coli DNA. Corresponding fragments from an isogenic lambda lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.

Coliphages↗
PubMed1994

Improvement of attachment rate of bovine embryos after transfer with trophoblastic fragments.

Fragments of trophoblastic vesicles have been shown to improve embryo development when used in co-culture. We wished to investigate the effect of trophoblastic fragments on establishment of pregnancy after embryo transfer. Embryos (n=129) frozen by direct immersion in liquid nitrogen (Group 1), 82 embryos frozen by the slow freezing method (Group 2) and 55 fresh embryos stored at 20-22 degrees C for 21-22 h (Group 3) were transferred into recipient heifers. The embryos of each group were divided in 2 batches. Batch 1 embryos (control) were transferred without trophoblastic fragments, Batch 2 embryos of each group (experimental) were transferred in combination with frozen-thawed trophoblastic fragments at a rate of 5 to 7 fragments per embryo. The fragments were produced from 16-17 day bovine embryos frozen by vitrification. Two months following the embryo transfers it was established that the attachament rate for Batch 1 embryos in Groups 1, 2, 3 was 33%, 39% and 48%, respectively; for Batch 2 embryos in the same Groups the rate was 55%, 56% and 74%, respectively. We conclude that trophoblastic fragments promote embryo attachment, but their effect does not continue beyond the time of attachment.

Journal Article↗
PubMed1994

Polymerization of actin induced by actin-binding fragments of caldesmon.

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Gałazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.

Actins↗
PubMed1992

Sequence and analysis of the BamHI "D" fragment of Shope fibroma virus: comparison with similar regions of related poxviruses.

Differences observed in the virulence of two related leporipoxviruses are closely tied to a particular region of their genomes. For the virulent poxvirus of this pair, malignant rabbit fibroma virus (MV), this region is the BamHI "C" fragment, which is 10.7 kb. For the avirulent poxvirus, Shope fibroma virus, SFV, this region is the corresponding BamHI "D" fragment, which is 13.1 kb. As part of our attempt to understand the virulence of these two viruses, we sequenced these two DNA fragments. The sequence for the BamHI "C" fragment of MV is reported elsewhere (Strayer et al., 1991). We report here the sequence for SFV's BamHI "D" fragment and resultant open reading frames, and compare both DNA and open reading frame structures to those of MV and other known poxviruses. The BamHI "D" fragment of SFV contains 12 open reading frames of 100 amino acids or more, arranged similarly to orf's in MV and vaccinia. Striking similarities between SFV and MV are seen in certain parts of this restriction fragment, including substantial stretches of DNA in which the two viruses are identical. Clear homologies exist between these leporipox virus genomes and those of other related poxviruses. To understand the pathogenesis of virus infection, one must appreciate the structure of those viral genes that play important roles in infection.

Base Sequence↗
PubMed1986

Comparative studies of heparin and heparin fragments: distribution and toxicity in the rat.

To control blood levels of heparin during extracorporeal therapy, the use of a blood filter containing heparinase, a heparin-specific enzyme that cleaves heparin to small fragments with less anticoagulant activity, has been proposed. These fragments have anti-factor Xa activity but no anti-thrombin activity. The potential toxicity of heparin fragments as compared to heparin was examined in rats by identifying presumptive sites of drug-related toxicity by whole-body autoradiography and by histological examination of major organs. Radioautograms of rats sacrificed 5 hr after dosing with [35S]heparin fragments indicated no potential targets different from what was observed in rats dosed with [35S]heparin. In addition, the faster urinary clearance of heparin fragments resulted in a lower concentration of these fragments than of heparin in all common target organs. No hemorrhages or other lesions were found in rats injected intravenously with heparin fragments (100 mg/kg) and sacrificed after 5 hr. In addition, no mortality or delayed toxic effects were observed in a similar group of animals sacrificed 2 weeks after dosing. In contrast, 80% of rats injected with heparin (100 mg/kg) showed hemorrhages of the lungs at the time of necropsy.

Animals↗
PubMed1996

Isolation of chromatin fragments released by apoptotic cells.

A two-step procedure for isolation of chromatin fragments released from apoptotic cells is described. The first step includes extraction of the chromatin fragments by a hypotonic treatment of isolated nuclei. The second step provides stabilization of the extracted chromatin fragments and their collection by a high-speed centrifugation. The procedure ensures not only a complete extraction and collection of the chromatin fragments but also intactness of their components. A number of control experiments confirmed this statement: they showed that the percentage of recovered fragmented DNA does not exceed 10% from the total DNA of apoptotic cells and that it is specific in both nature and chromosomal localization; the experiments reveal, moreover, that the protein components of the fragments including the well known histone and non-histone species possess also ability to support in in vitro conditions their specific phosphorylation. Developed for recovery of chromatin fragments from mouse erythroleukemia cells, spontaneously entering apoptosis, the procedure is practically applicable for all eukaryotic cell systems sharing programmable death.

Acetylation↗
PubMed1990

Retrograde transneuronal transport properties of fragment C of tetanus toxin.

The atoxic fragment C of tetanus toxin reliably undergoes retrograde direct and transneuronal transport, but the full extent of its transport capabilities has not been examined. The primary visual pathways provide an excellent system for investigating, for the first time, the possibility of anterograde direct and transneuronal transport of fragment C. Following injection into the eye of the rat and rabbit, fragment C, localized with a monoclonal antibody, underwent anterograde direct transport to all retinorecipient areas. From these areas, fragment C exhibited retrograde transneuronal transport, strongly and reliably labeling regions that project to retinorecipient areas, including layers V and VI of visual cortex, the parabigeminal nucleus, the suprageniculate nucleus, and the reticular thalamus. In contrast, the absence of fragment C in regions receiving only input from, but not projecting to, retinorecipient areas, most notably layer IV of visual cortex, provides strong evidence for the lack of anterograde transneuronal transport. Thus, while fragment C can be induced to undergo anterograde direct transport by injection into the eye, it exhibits only retrograde transneuronal transport. These characteristics suggest that fragment C of tetanus toxin is a consistent and reliable retrograde transneuronal marker for the elucidation of central nervous pathways.

Animals↗