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PubMed1986

Production of biologically active fragments of parathyroid hormone by isolated Kupffer cells.

Cleavage of parathyroid hormone (PTH) by isolated Kupffer cells from rat liver was examined. Iodinated PTH labeled at position 43 was converted into two radioactive fragments which were shown by Edman degradation to have residues 35 and 38 as their NH2 termini. Cleavage at these positions is characteristic of cathepsin D. Amino-terminal fragments were detected by bioassay of fractions obtained by high performance liquid chromatography. These fragments eluted in positions characteristic of the 1-34 and 1-37 peptides also previously shown to be produced by purified cathepsin D. The putative 1-37 fragment was rapidly converted to 1-34 upon digestion with cathepsin D, whereas the putative 1-34 fragment was not further digested by this enzyme, behavior previously shown to be characteristic of 1-37 and 1-34 bovine PTH. Fragmentation of PTH as measured by generation of fragments soluble in trichloroacetic acid was inhibited by methylamine, monensin, and ammonium chloride. In addition, monensin significantly inhibited production of both carboxyl- and amino-terminal fragments. Finally, active PTH fragments were also produced by elicited peritoneal macrophages. It is concluded that Kupffer cells, and other macrophages, can produce active fragments of PTH which appear in the medium. These fragments may be generated by cathepsin D within the cells.

Adenylyl Cyclases↗
PubMed1986

Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein.

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.

Amino Acid Sequence↗
PubMed1984

Poly (ADP-Ribose) synthetase. Separation and identification of three proteolytic fragments as the substrate-binding domain, the DNA-binding domain, and the automodification domain.

Poly(ADP-ribose) synthetase of Mr = 120,000 is cleaved by limited proteolysis with alpha-chymotrypsin into two fragments of Mr = 54,000 (54K) and Mr = 66,000 (66K). When the native enzyme is modified with 3-(bromoacetyl)pyridine, both portions of the enzyme are alkylated; however, alkylation of the 54K portions of the enzyme is protected by the addition of the substrate, NAD, or its analog, nicotinamide, suggesting that the substrate-binding site is localized in the 54K fragment. When the enzyme previously automodified with a low concentration of [adenine-U-14C] NAD is digested with alpha-chymotrypsin, the radioactivity is detected exclusively in the 66K fragment. The 66K fragment thus labeled is further cleaved with papain into two fragments of Mr = 46,000 and Mr = 22,000. With these two fragments, the label is detected only in the 22K fragment, but not in the 46K fragment. The 46K fragment binds to a DNA-cellulose column with the same affinity as that of the native enzyme, while the 22K fragment and the 54K fragment have little affinity for the DNA ligand. These results indicate that poly (ADP-ribose) synthetase contains three separable domains, the first possessing the site for binding of the substrate, NAD, the second containing the site for binding of DNA, and the third acting as the site(s) for accepting poly(ADP-ribose).

Animals↗
PubMed1983

Isoelectric focusing of human anti-diphtheria toxoid antibodies: identical spectrotypes of anti-fragment A antibodies with the same IgG subclass and light chain constant regions are expressed in multiple individuals.

The serum antibodies from humans booster-immunized with diphtheria toxoid were analyzed by isoelectric focusing. To restrict the antibody response, we visualized those antibodies that reacted to the Fragment A moiety of diphtheria toxin. Of the 100 normal human donors examined, 20 were estimated to be nonresponders to Fragment A. Sixty-three of the 80 donors who responded to Fragment A had restricted IgG-anti-Fragment A spectrotypes that consisted of three to eight distinct bands. Six series of repeat or shared spectrotypes were delineated among our donor population with repeat frequencies ranging from 0.025 to 0.312. We determined that the repeat spectrotypes were IgG1-kappa anti-Fragment A antibodies. Three percent of our donors had complex IgG-anti Fragment A spectrotypes consisting of greater than 10 bands. In some instances, the complex spectrotypes were composed of simple spectrotypes found among other donors. The complex IgG-anti Fragment A spectrotypes of these donors consisted of both IgG1 and IgG4-anti-Fragment A antibodies. From a statistical analysis, we calculate that only a limited number of IgG-anti-Fragment A spectrotypes are expressed in vivo after booster immunization. Our analysis suggests two groups of IgG-anti-Fragment A spectrotypes exist in our donor population. One IgG-anti-Fragment A spectrotype group is expressed randomly, and a second group is preferentially expressed among individuals in our donor population.

Antibodies, Bacterial↗
PubMed1993

A single chain 19-kDa fragment from bovine thrombospondin binds to type V collagen and heparin.

Location of the type V collagen-binding domain within bovine thrombospondin (TSP) was investigated by using fragments of reduced and alkylated TSP. A fragment of relative molecular mass (19 kDa) was isolated, which inhibited binding of 125I-TSP to type V collagen in a solid-phase binding assay. A direct binding assay using the 125I-labeled fragment confirmed that the fragment actually bound to collagen. The fragment retained specificity for the native structure of type V collagen like the intact TSP molecule. Its binding to the collagen, however, was not inhibited by Ca2+ in contrast to intact TSP. Amino acid sequence analysis of the 19-kDa fragment suggested that this fragment corresponded to Val333-Lys412, a part of the stalklike region in the human TSP primary structure. It was found that the fragment also bound well to heparin in a specific and saturable manner and, furthermore, binding to type V collagen was inhibited by soluble heparin. Removal of the N-linked sugar chain from this fragment resulted in a 14-kDa fragment. The deglycosylated fragment retained the ability to bind to type V collagen as well as heparin. These results suggest that a type V collagen-binding site is present in the 80-residue portion of bovine TSP (Val333-Lys412) and is likely to be identical or lie very close to a heparin-binding site, which exists in the type I repeat structure.

Amino Acid Sequence↗
PubMed1997

Combining beta-core fragment and total oestriol measurements to test for Down syndrome pregnancies.

Recent articles by Cuckle et al., Canick et al., and Isozaki et al. have evaluated urine beta-core fragment as a screening test for Down syndrome in second-trimester pregnancies. They found over four-fold elevation of beta-core fragment levels in Down syndrome pregnancies, and between 62 and 88 per cent detection of this trisomy at a 5 per cent false-positive rate. Urine beta-core fragment may be a superior screening test for Down syndrome pregnancies. In the present study, urinary total oestriol has been evaluated as a marker to use in combination with beta-core fragment in screening for Down syndrome pregnancies. The two markers were evaluated separately in relation to the urine creatinine concentration. To amplify screening performance, we evaluated the ratio of beta-core fragment to total oestriol levels (creatinine-independent). beta-core fragment and total oestriol levels were determined (normalized to creatinine, ng/mg creatinine) in urine samples from 480 unaffected and 12 Down syndrome pregnancies, collected consecutively at a single prenatal diagnosis centre. The median beta-core fragment level in Down syndrome cases was 4.5 MOM. Fifty-eight per cent of Down syndrome cases had beta-core fragment levels exceeding the 95th centile of unaffected pregnancies. The median total oestriol level in Down syndrome cases was 0.33 MOM. Forty-two per cent of Down syndrome cases had total oestriol levels exceeding the 95th centile of unaffected pregnancies. We investigated the ratio of the two determinants (beta-core fragment, ng/ml divided by total oestriol, ng/ml) in our sample set. The median beta-core fragment:total oestriol ratio in Down syndrome cases was 13 MOM. Seventy-five per cent of Down syndrome cases had a ratio exceeding the 95th and the 99.5th centile of unaffected pregnancies. Total oestriol complements beta-core fragment in urine screening for Down syndrome pregnancies. A test measuring the ratio of the two urine determinants may be a significant improvement over current serum methods for detecting Down syndrome.

Adult↗
PubMed1985

Cyanogen bromide cleaves Fc fragments of pooled human IgG at both methionine and tryptophan residues.

An attempt was made to obtain fragments containing the C gamma 2 region by selectively cleaving human Fc fragments prepared from pooled IgG at Met residues using cyanogen bromide. Based on the known locations of Met residues in the Fc regions of human IgG subclasses, fragments between Met 252 and 358, comprising the C gamma 2 domains, were expected from IgGl Gm -1, IgG2, IgG3 and IgG4. Greater fragmentation of the Fc fragments occurred, however, than was predicted. Automated N-terminal sequencing identified five major points (Trp 381, 313 and 277, and Met 397 and 252) and two minor points of cleavage (Met 428 and Trp 417). The majority of cleavage points occurred at Trp rather than Met. Furthermore, cleavage at Met 358, necessary to produce C gamma 2 domains, was not detected. Control experiments verified the integrity of the Fc fragments handled in exactly the same manner without cyanogen bromide exposure and the ability of the same cyanogen bromide preparation to produce the expected cleavages at Met of sperm whale apomyoglobin without fragmentation at Trp. Cleavage at Met 358 did not occur presumably because of the difficulty associated with cyanogen bromide cleavage at Met-Thr peptide bonds. Cleavage at Trp probably occurred by way of halogen promoted oxidation of the indole nucleus with resultant peptide bond fissure. These observations show that cyanogen bromide cleavage of pooled human Fc fragments is not selective for Met, but also cleaves at Trp residues. The resultant fragmentation of the C gamma 2 region coupled with the inability to make the required cleavage at the 358-359 Met-Thr bond resulted in the inability to produce fragments comprising the C gamma 2 domains. The reasons for the selective cleavage at Met in some proteins and the cleavage at both Trp and Met in others are not known.

Chemical Phenomena↗
PubMed1985

Preparation and biologic characterization of fragments containing dimeric and monomeric C gamma 2 domain of rabbit IgG.

A number of fragments derived from acid-treated rabbit IgG by digestion with plasmin have been separated by high-resolution gel filtration. Fragments isolated included a dimer and monomer Facb, named F(acb)2 and Facb, respectively and a heterodimer composed of Facb and Fab subunits, named F(acb)(ab). A C gamma 2 fragment was obtained by papain digestion of Facb. A heterodimer composed of Facb and Fab', named F(acb)(ab'), was also prepared by oxidizing a reduced mixture of these fragments. Fragments thus obtained are classified into two groups--those carrying paired C gamma 2 domains, i.e. F(acb)2, and the disulfide-linked dimeric C gamma 2 fragment; and those having a single C gamma 2 domain, i.e. reduced, alkylated Facb and C gamma 2 fragment, F(acb)(ab) and F(acb)(ab'). These fragments exhibited marked differences in their capacity to activate complement in assay systems of hemolysis and complement consumption by immune complexes or aggregates on polystyrene latex. Fragments of the former group could activate complement but with a definitely reduced efficiency (50%) compared to intact IgG, whereas fragments of the latter group were practically inactive. Although it was not determined whether the C1-binding capacity itself is changed by monomerization of the C gamma 2 domain, the results suggested that the intact paired C gamma 2 module is required at least for the activation process of complement.

Animals↗
PubMed1981

Further characterization of the alternative protein-A interaction of immunoglobulins: demonstration of an Fc-binding fragment of protein A expressing the alternative reactivity.

Intact IgG and fragments F(ab')2 gamma, Fab gamma and Fc gamma from a rabbit anti-protein-A serum (RapA) and corresponding preparations from normal rabbit IgG (NRG) were tested for their inhibitory effect on the binding of protein-A-reactive 125I-IgE and 125I-Fc gamma, respectively, to protein-A-Sepharose. Intact IgG, F(ab')2 gamma and Fab gamma of RapA inhibited the binding of protein-A-reactive 125I-IgE, whereas only intact IgG and Fc gamma fragments from both RapA and NRG inhibited the binding of 125I-Fc gamma to protein A-Sepharose. Further, the functional relationship between RapA and human polyclonal IgG was studied in a nephelometric test system. Intact IgG or fragments of IgG from human polyclonal IgG and rabbit antiprotein-A were found to affect the precipitation between human IgG and protein A in a similar way. Thus F(ab')2 gamma fragments and intact IgG enhanced the precipitation, whereas Fab gamma and Fc gamma fragments inhibited the precipitation. Protein A and an Fc-binding fragment of protein A (fragment B) were tested for their abilities to link different radiolabelled immunoglobulin preparations expressing the alternative and the classical protein-A reactivity to immobilized Fc fragments. All proteins expressing the alternative reactivity were efficiently bound both by fragment B and by protein A, indicating that fragment B, in addition to its classical Fc-binding activity, also expresses the alternative protein-A reactivity.

Animals↗
PubMed1998

Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves.

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by the hydroxyl radical in the lysates of chloroplasts in light (H. Ishida et al. 1997, Plant Cell Physiol 38: 471-479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts, and discuss the mechanisms of the fragmentation. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated under illumination in the presence of KCN or NaN3, which is a potent inhibitor of active oxygenscavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. These results indicate that, as in the lysates, the fragmentation of the LSU in the intact chloroplasts was also caused by the hydroxyl radical generated in light. The fragmentation of the LSU was completely inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU), and only partially inhibited by methyl viologen in the lysates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU.

Antibodies↗
PubMed1989

In vitro packaging of bacteriophage phi 29 DNA restriction fragments and the role of the terminal protein gp3.

Restriction fragments of bacteriophage phi 29 DNA-gp3 (DNA-gene product 3 complex) were packaged in a completely defined in vitro system that included purified proheads, the DNA packaging protein gp16 and ATP. Both left and right end DNA-gp3 fragments were packaged in this system, in contrast to the oriented and selective packaging of left end DNA-gp3 fragments in extracts; left ends could be packaged quantitatively in the defined system, while the packaging efficiency of right ends was generally about threefold lower. In addition, certain internal (non-end) DNA fragments were packaged at efficiencies of about 10% to 15%. Digestion of the gp3 with trypsin or proteinase K reduced the packaging of whole-length DNA by a factor of 2 or 4, respectively, and removal of the gp3 from whole-length DNA or end fragments with piperidine reduced packaging to the level of internal fragments. Though the terminal protein gp3 was non-essential for DNA translocation in the defined system, it stimulated packaging of left and right end fragments, and stabilized packaging of the left end. The packaging of end and internal DNA fragments of the related phage M2Y into phi 29 proheads was similar to that of phi 29 DNA fragments, and certain fragments of lambda DNA were packaged at the efficiency of the internal phi 29 DNA fragments. Selective packaging of DNA-gp3 left ends was restored by the addition of bacterial cell extracts or glycerol to the defined system, and these packaging conditions discriminated between phi 29 and M2Y DNAs that have distinct terminal proteins.

Bacillus subtilis↗
PubMed1990

Enhanced CNS uptake of systemically administered proteins through conjugation with tetanus C-fragment.

No other exogenous protein enters the central nervous system from the circulation as readily as tetanus toxin. We examined the capability of the non-toxic binding fragment of tetanus toxin (C-Fragment) so serve as a vehicle for transport of other proteins into the mouse CNS. Using periodate oxidation of the enzyme horseradish peroxidase (HRP), we synthesized two separate macromolecular complexes, one containing C-fragment and HRP, and the other C-fragment, HRP and a third "test" protein-human IgG. The distribution of C-fragment-HRP was typical of blood borne proteins including native C-fragment, with labeling of all neurons with known projections outside the blood-brain barrier, particularly large spinal motoneurons. C-fragment-HRP conjugates showed superior neuronal labeling to over 100-fold greater quantities of free HRP. Complexes containing C-fragment, HRP and human IgG were internalized by neurons from both intramuscular and intraperitoneal injections. The efficiency of neuronal uptake of IgG in the C-fragment conjugated form was enhanced over 40-fold compared to free IgG. Linkage of a large protein to C-fragment probably leads to enhanced endocytosis of that protein by neuronal terminals projecting outside the blood-brain barrier. C-fragment can serve as a vehicle to allow selected proteins to bypass the barrier and enter the CNS.

Animals↗
PubMed1987

Binding properties of 111In-diethylenetriaminepentaacetic acid (DTPA)-fragment E1,2, a fibrin-specific probe, are dependent on use of protecting complex during attachment of DTPA groups.

Fragments E1 and E2, plasmic degradation products of crosslinked fibrin, bind specifically to polymers of fibrin. A mixture of these fragments, denoted as fragment E1,2, was radiolabeled with 111In after covalently attaching metal chelating groups (diethylenetriaminepentaacetic acid, DTPA) to the fragment, using two approaches. In the first approach, DTPA groups were attached directly to purified fragment E1,2. In the second approach, attachment sites of DTPA groups were directed away from the active region of the molecule by having fragment E1,2 bound in complex, with its active sites protected during the derivatization. Direct attachment of DTPA groups to fragment E1,2 resulted in complete loss of binding to fibrin in vitro. When derivatized in complex, 111In-DTPA-fragment E1,2 retained a higher degree of binding to human fragment DD and human plasma clots in vitro than did radioiodinated fragment E1, even when up to eight DTPA groups were attached per molecule of fragment E1,2.

Fibrin Fibrinogen Degradation Products↗
PubMed2005

Embryo fragmentation as a determinant of blastocyst development in vitro and pregnancy outcomes following embryo transfer.

OBJECTIVE(S): To determine how the type of embryo fragmentation on day 3 affects progression of human embryos to blastocyst and pregnancy rates following embryo transfer. STUDY DESIGN: Retrospective analysis of all in vitro fertilization cycles in patients < or =40 years of age or younger from January 2002 through December 2003, during which time surplus day 3 embryos were transferred to blastocyst medium for extended culture. All embryos (4 cells or more) not suitable for transfer or freezing 72 hours following in vitro fertilization were placed into microdroplets (60 microL) of blastocyst medium and cultured for an additional 48 hours to assess blastocyst formation. Normal blastocyst development required blastulation, a visible inner-cell mass, trophectoderm cells covering 60% of the inner zona surface and thinning of the zona. The rate of blastocyst formation was then analyzed (chi 2 and analysis of variance) against the type of fragmentation 72 hours after insemination. Pregnancy outcomes were analyzed with respect to the pattern of fragmentation in cleaving embryos transferred after 3 days of culture. RESULTS: A total of 1566 embryos were cultured beyond day 3 of development of which 229 (14.6%) reached the blastocyst stage and were frozen. Embryos exhibiting no fragmentation or type I fragmentation had significantly higher blastocyst development rates (27.9% and 19.9%) than embryos with type 2 or 3 fragmentation (13.9 and 8.8, respectively; P < .001). No embryos with type 4 or 5 fragmentation progressed to blastocyst. The average type of fragmentation in transferred embryos correlated with pregnancy outcome and embryo age. CONCLUSION(S): More pervasive embryo fragmentation was associated with a decreasing rate of blastocyst development with day 3 embryos. To the extent that blastocyst development rates of day 3 embryos is an index of embryo viability, our findings establish that careful classification of the type of embryo fragmentation is important in selection of day 3 embryos for transfer. Recent reports of associations among embryo fragmentation, aneuploidy, apoptosis, and patient age support these conclusions.

Adult↗
PubMed1998

Holmium:YAG lithotripsy yields smaller fragments than lithoclast, pulsed dye laser or electrohydraulic lithotripsy.

PURPOSE: The mechanism of lithotripsy differs among electrohydraulic lithotripsy, mechanical lithotripsy, pulsed dye lasers and holmium:YAG lithotripsy. It is postulated that fragment size from each of these lithotrites might also differ. This study tests the hypothesis that holmium:YAG lithotripsy yields the smallest fragments among these lithotrites. MATERIALS AND METHODS: We tested 3F electrohydraulic lithotripsy, 2 mm. mechanical lithotripsy, 320 microns pulsed dye lasers and 365 microns. holmium:YAG fiber on stones composed of calcium hydrogen phosphate dihydrate, calcium oxalate monohydrate, cystine, magnesium ammonium phosphate and uric acid. Fragments were dessicated and sorted by size. Fragment size distribution was compared among lithotrites for each composition. RESULTS: Holmium:YAG fragments were significantly smaller on average than fragments from the other lithotrites for all compositions. There were no holmium:YAG fragments greater than 4 mm., whereas there were for the other lithotrites. Holmium:YAG had significantly greater weight of fragments less than 1 mm. compared to the other lithotrites. CONCLUSIONS: Holmium:YAG yields smaller fragments compared to electrohydraulic lithotripsy, mechanical lithotripsy or pulsed dye lasers. These findings imply that fragments from holmium:YAG lithotripsy are more likely to pass without problem compared to the other lithotrites. Furthermore, the significant difference in fragment size adds evidence that holmium:YAG lithotripsy involves vaporization.

Calculi↗
PubMed2003

Roles of amyloid precursor protein and its fragments in regulating neural activity, plasticity and memory.

Amyloid-beta precursor protein (APP) is a membrane-spanning protein with a large extracellular domain and a much smaller intracellular domain. It is the source of the amyloid-beta (Abeta) peptide found in neuritic plaques of Alzheimer's disease (AD) patients. Because Abeta shows neurotoxic properties, and because familial forms of AD promote Abeta accumulation, a massive international research effort has been aimed at understanding the mechanisms of Abeta generation, catabolism and toxicity. APP, however, is an extremely complex molecule that may be a functionally important molecule in its full-length configuration, as well as being the source of numerous fragments with varying effects on neural function. For example, one fragment derived from the non-amyloidogenic processing pathway, secreted APPalpha (sAPPalpha), is neuroprotective, neurotrophic and regulates cell excitability and synaptic plasticity, while Abeta appears to exert opposing effects. Less is known about the neural functions of other fragments, but there is a growing interest in understanding the basic biology of APP as it has become recognized that alterations in the functional activity of the APP fragments during disease states will have complex effects on cell function. Indeed, it has been proposed that reductions in the level or activity of certain APP fragments, in addition to accumulation of Abeta, may play a critical role in the cognitive dysfunction associated with AD, particularly early in the course of the disease. To test and modify this hypothesis, it is important to understand the roles that full-length APP and its fragments normally play in neuronal structure and function. Here we review evidence addressing these fundamental questions, paying particular attention to the contributions that APP fragments play in synaptic transmission and neural plasticity, as these may be key to understanding their effects on learning and memory. It is clear from this literature that APP fragments, including Abeta, can exert a powerful regulation of key neural functions including cell excitability, synaptic transmission and long-term potentiation, both acutely and over the long-term. Furthermore, there is a small but growing literature confirming that these fragments correspondingly regulate behavioral learning and memory. These data indicate that a full account of cognitive dysfunction in AD will need to incorporate the actions of the full complement of APP fragments. To this end, there is an urgent need for a dedicated research effort aimed at understanding the behavioral consequences of altered levels and activity of the different APP fragments as a result of experience and disease.

Alzheimer Disease↗
PubMed1983

Anomalous electrophoresis of deoxyribonucleic acid restriction fragments on polyacrylamide gels.

A detailed study has been made of the polyacrylamide gel electrophoresis of DNA restriction fragments obtained from two plasmids, pBR322 and p82-6B. Variables studied were molecular weight, gel concentration, temperature, and electric field strength. The retardation coefficients of the larger fragments (greater than 800 base pairs) were independent of molecular weight. The retardation coefficients of the smallest fragments (less than or equal to 300 base pairs) were proportional to Mr1/3, and therefore to the mean geometric radii of the fragments. The logarithm of the relative mobility of all fragments was also proportional to Mr1/3. The anomalous migration of certain fragments on polyacrylamide gels was found to be "transportable" into fragments generated by different restriction enzymes. Anomalous migration was enhanced at lower temperatures and disappeared upon increasing the temperature. A fragment which migrated anomalously slowly migrated even more anomalously when dimerized; dimerizing a normally migrating fragment resulted in the normal migration of the dimerized fragment. Anomalously migrating fragments were found to be localized in distinct regions of the pBR322 circle.

DNA↗
PubMed1984

Circular dichroism and thermal melting of two small DNA restriction fragments of the same molecular weight.

The thermal melting and circular dichroism of two 147 base pair restriction fragments of pBR322 have been studied. The fragment with the higher GC content, 12B, melts at a higher temperature than the other fragment, 12A, as expected. The melting temperatures are proportional to the logarithm of the concentration of NaCl or tris(hydroxymethyl)aminomethane (Tris) buffer, between 1 mM and 0.2 M added salt. In 1 mM Tris buffer, the melting temperatures of the two fragments are nearly equal. The circular dichroism spectra of fragments 12A and 12B in 0.2-10 mM Tris buffer are characteristic of B-form DNA. In 81% ethanol, the circular dichroism spectra of the two fragments are characteristic of A-form DNA. With 1 mM Tris buffer as the supporting electrolyte, fragment 12A exhibits a very sharp B----A transition, with a midpoint at 79% ethanol. However, a biphasic transition is observed for fragment 12B, with midpoints at 73% and 80% ethanol. This biphasic transition may represent the conversion of separate domains of fragment 12B from the B conformation to the A conformation; half of this fragment is much more GC rich than the other half. Methods are also described for preparing polymers of the 12A and 12B fragments.

Base Composition↗