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PubMed1976

Small fragments from the A subunit of cholera toxin capable of activating adenylate cyclase.

Exposure of cholera toxin to membrane particles prepared from sarcoma 180 cells gives rise to a variety of fragments which are capable of activating adenylate cyclase [ATP:pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. A major component of these fragments has an apparent molecular weight in the 8,000-10,000 range. The smallest stimulatory fragment has a molecular weight of approximately 1400. The small size of the fragments is confirmed by Sephadex gel filtration, in the presence of either sodium dodecyl sulfate or formic acid. These fragments are produced from holotoxin or its A subunit by protease(s) found in sarcoma membrane particles. Production of fragments appears optimal in 40-60 min at 30 degrees and pH 7, and is prevented by protease inhibitors. The ability of the small fragments to activate adenylate cyclase is reversed by anti-holotoxin, but not anticholeragenoid, antibodies. These fragments require NAD for the activation of adenylate cyclase and are fully active after heating at 90 degrees for 5 min (pH 7).

Adenylyl Cyclases↗
PubMed1978

Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders.

Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [(32)P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained beta-like gene sequences assayed with beta globin cDNA probe. One beta-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three beta gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing beta-like genes were absent from deltabeta thalassemic DNA and one new fragment containing beta-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of beta-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with deltabeta thalassemia.

Base Sequence↗
PubMed1980

Transformation of rat embryo fibroblasts by cloned polyoma virus DNA fragments containing only part of the early region.

Recombinant plasmids containing either the entire polyoma viral genome or one or the other of the two HindIII fragments of polyoma virus DNA were constructed and cloned in Escherichia coli X1776, and their DNAs were individually tested for the capacity to transform an established line of rat cells. The recombinant plasmids containing the entire polyoma genome and those containing the HindIII-1 fragment of polyoma DNA (45-1.4 map units) efficiently transform rat cells, whereas the plasmids containing the HindIII-2 fragment (1.4-45.0 map units) do not. The properties of many independent transformed cell lines established by infection with the cloned HindIII-1 fragment were determined. In contrast to the parent cell line, rat cells transformed with the cloned HindIII-1 fragment grow to high saturation densities, form colonies with high efficiency in dilute agar suspension, produce high levels of plasminogen activator, and display a disorganized arrangement of actin cables. By all criteria examined, these cells transformed by fragments are indistinguishable from cells transformed by whole polyoma viral DNA. Cellular DNA prepared from many HindIII-1 fragment-transformed cell lines was analyzed for the presence and arrangement of polyoma viral sequences by Southern blot-hybridization. In all cases examined, only those viral sequences contained within the HindIII-1 fragment of polyoma DNA were detected. These data establish a strong correlation between polyoma DNA sequences mapping within a restricted portion of the early region and the induction and maintenance of the transformed phenotype.

Animals↗
PubMed1980

Cloning of reiterated and nonreiterated herpes simplex virus 1 sequences as BamHI fragments.

Over 95% of the herpes simplex virus type 1 (strain F) DNA sequences have been cloned as BamHI fragments in the pBR322 plasmid. With one exception, all of the cloned fragments have the same electrophoretic mobilities and restriction enzyme cleavage sites as do the authentic fragments derived from the BamHI digests of the viral genome. The exception is the BamHI B fragment mapping at the right end of L component in the prototype arrangement of the DNA. Thus, a small deletion mapping near the left end of the fragment was present in two independently derived plasmids. Included in the collection of plasmids are several clones containing DNA sequences that span the junction between the L and S components of the virus DNA. Several plasmids containing the junction fragment were found to be sufficiently stable to permit the preparation of large amounts of the DNA fragment for fine-structure mapping of the restriction enzyme cleavage sites. Preliminary studies on one cloned fragment (BamHI G) have shown that it is biologically active in marker rescue of a temperature-sensitive mutation and in transfer of a plaque morphology marker.

Cloning, Molecular↗
PubMed2003

Ectodomain cleavage of ErbB-4: characterization of the cleavage site and m80 fragment.

Ectodomain cleavage of the ErbB-4 receptor tyrosine kinase generates a membrane-associated fragment of 80 kDa (m80) that has been subjected to N-terminal sequencing. The sequence obtained shows that the N terminus of this fragment begins with Ser-652 of ErbB-4. When a 12-residue peptide corresponding to ErbB-4 residues 646-657 was incubated with recombinant tumor necrosis factor-alpha-converting enzyme, fragments representing residues 646-651 and 652-657 were obtained. These data indicate that ectodomain cleavage of ErbB-4 occurs between His-651 and Ser-652, placing the cleavage site within the ectodomain stalk region approximately 8 residues prior to the transmembrane domain. Several experiments have characterized other aspects of the m80 ErbB-4 fragment. Inhibition of ErbB-4 tyrosine kinase activity with pan-ErbB tyrosine kinase inhibitors indicates that kinase activity is stringently required for heregulin-dependent, but not 12-O-tetradecanoylphorbol-13-acetate-induced, ErbB-4 ectodomain cleavage and formation of the m80 fragment. When the m80 ErbB-4 fragment is generated by cell treatment with heregulin or 12-O-tetradecanoylphorbol-13-acetate, the fragment associates with intact ErbB-2. However, this fragment does not associate with the intact ErbB-4 molecule.

ADAM Proteins↗
PubMed2004

The role of extra fragment at the C-terminal of cytochrome b (Residues 421-445) in the cytochrome bc1 complex from Rhodobacter sphaeroides.

Sequence alignment of cytochrome b of the cytochrome bc1 complex from various sources reveals that bacterial cytochrome b contain an extra fragment at the C terminus. To study the role of this fragment in bacterial cytochrome bc1 complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc1 complexes with progressive deletion from this fragment (residues 421-445) were generated and characterized. The cytbDelta-(433-445) bc1 complex, in which 13 residues from the C-terminal end of this fragment are deleted, has electron transfer activity, subunit composition, and physical properties similar to those of the complement complex, indicating that this region of the extra fragment is not essential. In contrast, the electron transfer activity, binding of cytochrome b, ISP, and subunit IV to cytochrome c1, redox potentials of cytochromes b and c1 in the cytbDelta-(427-445), cytbDelta-(425-445), and cytbDelta-(421-445) mutant complexes, in which 19, 21, or all residues of this fragment are deleted, decrease progressively. EPR spectra of the [2Fe-2S] cluster and the cytochromes b in these three deletion mutant bc1 complexes are also altered; the extent of spectral alteration increases as this extra fragment is shortened. These results indicate that the first 12 residues (residues 421-432) from the N-terminal end of the C-terminal extra fragment of cytochrome b are essential for maintaining structural integrity of the bc1 complex.

Amino Acid Sequence↗
PubMed2005

Expression of an uncleavable N-terminal RasGAP fragment in insulin-secreting cells increases their resistance toward apoptotic stimuli without affecting their glucose-induced insulin secretion.

Apoptosis of pancreatic beta cells is implicated in the onset of type 1 and type 2 diabetes. Consequently, strategies aimed at increasing the resistance of beta cells toward apoptosis could be beneficial in the treatment of diabetes. RasGAP, a regulator of Ras and Rho GTPases, is an atypical caspase substrate, since it inhibits, rather than favors, apoptosis when it is partially cleaved by caspase-3 at position 455. The antiapoptotic signal generated by the partial processing of RasGAP is mediated by the N-terminal fragment (fragment N) in a Ras-phosphatidylinositol 3-kinase-Akt-dependent, but NF-kappaB-independent, manner. Further cleavage of fragment N at position 157 abrogates its antiapoptotic properties. Here we demonstrate that an uncleavable form of fragment N activates Akt, represses NF-kappaB activity, and protects the conditionally immortalized pancreatic insulinoma betaTC-tet cell line against various insults, including exposure to genotoxins, trophic support withdrawal, and incubation with inflammatory cytokines. Fragment N also induced Akt activity and protection against cytokine-induced apoptosis in primary pancreatic islet cells. Fragment N did not alter insulin cell content and insulin secretion in response to glucose. These data indicate that fragment N protects beta cells without affecting their function. The pathways regulated by fragment N are therefore promising targets for antidiabetogenic therapy.

Animals↗
PubMed2002

Radiation-induced peroxidation and fragmentation of lipids in a model membrane.

PURPOSE: To ascertain the possibility of fragmentation processes in lipid membranes when acted upon with gamma-radiation under various conditions, and to evaluate fragmentation processes quantitatively in comparison with the lipid peroxidation processes. MATERIALS AND METHODS: Phospholipids as components of multilamellar liposomes exposed to 137Cs gamma-rays at dose-rates of 0.06 and 0.33 Gy s-1 in the dose range 0-18 kGy. Peroxidation products were determined spectrophotometrically and fragmentation products were analysed using thin-layer chromatography and GLC methods. RESULTS: Phosphatidylglycerol containing unsaturated fatty acid residues and free hydroxyl groups underwent both peroxidation and fragmentation when its aqueous dispersions were treated with ionizing radiation. gamma-Radiolysis of multilamellar liposomes led to the formation of peroxidation products, as well as phosphatidic acid and hydroxyacetone, fragmentation products of the initial lipid. Comparable radiation-chemical yields of conjugated-diene products and phosphatidic acid indicate that the probabilities of oxidation and fragmentation processes to occur are approximately equal in this case. In contrast to lipid peroxidation, lipid fragmentation is not accelerated but suppressed by oxygen and appears to be a prevailing process in de-aerated solutions. The phosphatidic acid formed as a result of phosphatidylglycerol fragmentation can intensify the lipid peroxidation. CONCLUSIONS: Treatment of lipid membranes with ionizing radiation leads to the destructive processes within both hydrophobic and hydrophilic moiety of lipids.

Dose-Response Relationship, Radiation↗
PubMed1995

Separation of DNA fragments induced by ionizing irradiation using a graded-field gel electrophoresis.

A method is described that allows a separation of X-ray induced DNA fragments by graded-field gel electrophoresis. Synchronized G1 and asynchronous CHO cells were embedded in agarose and irradiated with X-ray doses ranging from 1 to 100 Gy. Following proteolysis by sarcosine and proteinase K, electrophoresis was run for 49 h using graded electric fields with stepwise increasing field strength (0.6, 1.5, 3 and 9 V/cm). Since the molecular size of DNA able to migrate decreased with increasing voltage, each voltage step led to the generation of a distinct DNA band with the largest fragments in band 1, fragments of intermediate size in bands 2 and 3 and the smallest fragments in band 4. Using yeast chromosomal DNA as a reference, the molecular weight of eluted fragments was calculated to range from 1 to 10 Mbp. It could be shown that the fragment size was not the only criterion that discriminates migrating from non-migrating DNA. DNA fragments were found to be retained in the well by an unknown factor presumably associated with DNA conformation. This retention factor increased with increasing fragment size. Graded-field gel electrophoresis also allowed the determination of the absolute number of double-stranded breaks (dsb) induced, which amounted to 11.5 x 10(-12) dsbs Gy-1 Da-1 corresponding to 37 dsbs/G1 cell.

Animals↗
PubMed2002

Fragment generation and support vector machines for inducing SARs.

We present a new approach to the induction of SARs based on the generation of structural fragments and support vector machines (SVMs). It is tailored for bio-chemical databases, where the examples are two-dimensional descriptions of chemical compounds. The fragment generator finds all fragments (i.e. linearly connected atoms) that satisfy user-specified constraints regarding their frequency and generality. In this paper, we are querying for fragments within a minimum and a maximum frequency in the dataset. After fragment generation, we propose to apply SVMs to the problem of inducing SARs from these fragments. We conjecture that the SVMs are particularly useful in this context, as they can deal with a large number of features. Experiments in the domains of carcinogenicity and mutagenicity prediction show that the minimum and the maximum frequency queries for fragments can be answered within a reasonable time, and that the predictive accuracy obtained using these fragments is satisfactory. However, further experiments will have to confirm that this is a viable approach to inducing SARs.

Artificial Intelligence↗
PubMed1969

Isolation of a fragment (C3a) of the third component of human complement containing anaphylatoxin and chemotactic activity and description of an anaphylatoxin inactivator of human serum.

A small fragment of C3, called C3a, which has smooth muscle contracting activity, was isolated by three different methods. At pH 8.6, C3a behaved as cation, and using the Archibald method, its mol wt was determined to be 7000. A specific antiserum to C3a showed the fragment to be antigenically distinct from the rest of the C3 molecule, i.e., the C3b portion. The same antiserum and an anti-whole C3 were able to inhibit the biologic activity of C3a. In addition to anaphylatoxin activity, leukocyte chemotactic activity was shown to reside in C3a. Treatment with trypsin caused the cationic fragment to become anionic and abolished the anaphylatoxin but not the chemotactic activity. C3a fragments with identical biologic activity and comparable cationic properties, as determined by acid disc electrophoresis, were obtained by treatment of C3 with C3 convertase, C3 inactivator complex, trypsin, and plasmin. Thrombin produced a similar C3 fragment which was inactive. It was concluded that C3a corresponds to an unusually basic portion of C3 which may be liberated by attack of a variety of enzymes on a highly susceptible region of the native C3 molecule. C3b was cleaved by trypsin and less efficiently by thrombin or plasmin into two antigenically distinct pieces: the larger C3c fragment corresponding to beta(1A) and the smaller C3d fragment to alpha(2D) of aged serum. The c- and the d-fragments were separated and characterized. Isolated C3a rapidly lost its anaphylatoxin activity when treated with small amounts of a partially purified, thermolabile 10S alpha-pseudoglobulin of human serum. The conditions of inactivation suggested an enzymatic reaction. The anaphylatoxin inactivator also destroyed the activity of C5-derived anaphylatoxin and of lysyl bradykinin.

Anaphylaxis↗
PubMed2003

Are fragments islands? Landscape context and density-area relationships in boreal forest birds.

We investigated the role of matrix type as a determinant of change in bird densities with forest patch area (patch area effect) in two different Fennoscandian landscape types: mature forest fragments surrounded by cut-over or regenerating forest and true forested islands surrounded by water. Since the matrix of forested archipelagoes offers no resources to and impedes movement of forest birds, we predict that patch area effects on bird densities should be stronger on forested islands than in forest patches fragmented by forestry. We compiled correlation estimates of the bird density-patch area relationship from the literature and analyzed the data using meta-analysis. Combined correlation coefficients were significantly positive on islands but were not significantly different from 0 in fragments. Within-species comparisons also showed that correlations were consistently more positive on islands than in fragments. On islands but not in fragments, the densities of forest specialist species were more sensitive to area than were the densities of forest generalists, suggesting that specialists are more sensitive to changes in matrix quality. Migration status was only weakly associated with bird responses to island or fragment area. Thus, forest fragments do not function as true islands. We interpret this as the result of compensatory effects of the surrounding matrix in terms of availability of resources and enhanced connectivity (matrix quality hypothesis). A purely patch-centered approach seems an unrealistic framework to analyze population processes occurring in complex landscapes. The characteristics of the habitat matrix should therefore be explicitly incorporated into the assessment of species' responses to habitat fragmentation.

Animals↗
PubMed1979

Comparison of nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of rat and chicken.

Nucleotide sequences of large T1 ribonuclease fragments of 18S ribosomal RNA of Novikoff rat ascites hepatoma cells and chicken lymphoblastoid cells were determined and compared. Among the 19 large T1 ribonuclease fragments examined of rat 18S ribosomal RNA, 12 fragments were found to be the same in chicken 18S ribosomal RNA. Three fragments of rat 18S ribosomal RNA were not found among large T1 ribonuclease fragments of chicken 18S ribosomal RNA. Four fragments of rat 18S ribosomal RNA were found to be changed in chicken 18S ribosomal RNA. All the changes were point mutations except the change in the largest T1 ribonuclease fragment 1 which is 21 nucleotides long. 2'-0-methylation at the center of the fragment was lost in chicken 18S ribosomal RNA; all the other nucleotides were the same.

Animals↗
PubMed1995

DNA fragmentation induced in high-cell-density culture of primary rat hepatocytes is an active process dependent on energy availability, gene expression, and calmodulin.

We previously reported that internucleosomal DNA fragmentation, a biochemical feature of apoptosis, was induced spontaneously in high-cell-density culture of adult rat hepatocytes. To understand better the intracellular mechanism of the DNA fragmentation in this system, we have examined the effects of several inhibitors of specific intracellular functions on the DNA fragmentation. We found that the DNA fragmentation could be suppressed by treatment of the cells with inhibitors of mitochondrial respiration, KCN and CCCP, or a protein synthesis inhibitor, cycloheximide. We also demonstrated that calmodulin inhibitors, chlorpromazine and W-7, could suppress the DNA fragmentation. Together, these results lead us to conclude that the DNA fragmentation in hepatocytes cultured at high cell density is an active process dependent on energy availability, gene expression, and calmodulin, rather than a passive event resulting from necrosis. However, by analyzing the incidence of apoptotic morphology during culture, we found that only 2-3% of cells exhibited apoptotic morphology, while the incidence of DNA fragmentation was estimated to be much higher. Based on these results, we estimated that the DNA fragmentation may result from the incomplete progression of apoptosis or from the occurrence of another type of active cell death.

Animals↗
PubMed2002

Maintenance of the inner cell mass in human blastocysts from fragmented embryos.

The degree of fragmentation during early cleavage is universally used as an indicator of embryo quality during human in vitro fertilization treatment. Extensive fragmentation has been associated with reduced blastocyst formation and implantation. We examined the relationship between early fragmentation and subsequent allocation of cells to the trophectoderm and inner cell mass in the human blastocyst. We retrospectively analyzed data from 363 monospermic human embryos that exhibited varying degrees of fragmentation on Day 2. Embryos were cultured from Day 2 to Day 6 in Earle balanced salt solution with 1 mM glucose and human serum albumin. Rates of development and blastocyst formation were measured. The number of cells in the trophectoderm and inner cell mass and the incidence of apoptosis were assessed following differential labeling with polynucleotide-specific fluorochromes. Increasing fragmentation resulted in reduced blastocyst formation and lower blastocyst cell numbers. For minimal and moderate levels of fragmentation, the reduction in cell numbers was confined largely to the trophectoderm and a steady number of inner cell mass cells was maintained. However, with extensive fragmentation of more than 25%, cell numbers in both lineages were reduced in the few embryos that formed blastocysts. Apoptotic nuclei were present in both the trophectoderm and inner cell mass, with the lowest incidence in blastocysts that had developed from embryos with minor (5-10%) fragmentation. Paradoxically, higher levels of apoptosis were seen in embryos of excellent morphology, suggesting a possible role in regulation of cell number.

Apoptosis↗
PubMed1996

Occurrence of deoxyribonucleic acid fragmentation during prolactin-induced structural luteolysis in cycling rats.

We determined whether fragmentation of genomic DNA, one of the hallmarks of apoptosis, occurs during structural luteolysis in cycling rats. Corpora lutea (CL) were collected from rats at each estrous cycle stage (1800 h), and fragmented DNA was extracted. Only CL from rats at the proestrous stage showed distinct DNA fragmentation. To determine the period of occurrence of DNA fragmentation, CL were collected at several points between 1200 h on the day of proestrus and 0600 h on the day of estrus. Distinct DNA fragmentation was observed from 1800 h (proestrus) to 2400 h (proestrus), and the extent was significantly lower at 0600 h (estrus). It is known that prolactin (PRL) induces structural luteolysis in rats. To examine the role of PRL in luteal DNA fragmentation, 2-bromo-alpha-ergocryptine (BE) was used to suppress the PRL surge on the day of proestrus. CL collected at 1800 h from BE-treated rats did not show distinct DNA fragmentation, and PRL injection offset the effect of BE. Histochemical analysis with a 3'-end labeling technique confirmed the occurrence of DNA fragmentation in luteal tissue. These results suggest that apoptotic cell death occurs during PRL-induced structural luteolysis.

Animals↗
PubMed2001

Genetic and epigenetic factors affecting blastomere fragmentation in two-cell stage mouse embryos.

We report here that mouse embryos can exhibit a significant incidence of blastomere fragmentation at the two-cell stage. The incidence of this is influenced by both the maternal and paternal genotype. Embryos from C57BL/6 mothers exhibit a very low incidence of fragmentation at the two-cell stage in crosses involving males of C57BL/6, DBA/2, AKR/J, or SJL strains but exhibit a significantly increased incidence of fragmentation in crosses involving C3H/HeJ males. Increased fragmentation is seen in embryos from C3H/HeJ females crossed with C57BL/6 males but not with C3H/HeJ males. Embryos obtained from reciprocal (C57BL/6 x C3H/HeJ) F1 hybrid females also exhibit an increased incidence of fragmentation at the two-cell stage when the hybrid females are mated to either C57BL/6 or C3H/HeJ males. Interestingly, the results differ significantly between reciprocal F1 hybrid females, indicating a parental origin effect, possibly a result of either genomic imprinting or differences in mitochondrial origin. We conclude that the incidence of blastomere fragmentation at the two-cell stage in the mouse is under the control of more than one genetic locus. We also conclude that blastomere fragmentation is affected by both parental genotypes. These results are relevant to understanding the genetic control blastomere fragmentation, which may contribute to evolutionary processes, affect the success of procedures such as cloning, and affect the outcome of assisted reproduction techniques.

Amanitins↗
PubMed2006

Fragmentation of care for frequently hospitalized urban residents.

BACKGROUND: Fragmentation across sites of care may impede efficient healthcare delivery. OBJECTIVES: The objectives of this study were to evaluate fragmentation of hospital care for chronically ill New York City (NYC) residents and its association with enrollment in the New York State (NYS) Medicaid program. RESEARCH DESIGN: We conducted a cross-sectional study using the NYS Department of Health's Statewide Planning and Research Cooperative System discharge database. We identified 53,031 NYC residents admitted 3 or more times to acute care hospitals between 2000 and 2002 with the same principal diagnosis of a specific chronic illness (diabetes, sickle cell anemia, psychosis, substance abuse, cancer, gastrointestinal disease, chronic obstructive pulmonary disease/asthma, coronary artery disease, or congestive heart failure). We also evaluated a larger cohort of 225,421 patients with >or=3 admissions for a specific chronic illness coded as either the principal or a secondary diagnosis. A generalized logit model was used to examine the relationship between fragmentation and each patient's primary insurance adjusted for diagnosis and demographic characteristics. MEASURES: Fragmentation was characterized as high, moderate, or low based on the number of distinct hospitals a patient visited relative to the patient's total number of hospitalizations over the 3-year interval. RESULTS: Among frequently hospitalized NYC residents with select chronic conditions, 17.1% experienced highly fragmented care. This rate was 9.9% for patients with commercial insurance, 24.4% for those with Medicaid, and 9.7% for those with Medicare. The unadjusted odds ratio describing high fragmentation of Medicaid enrollees compared with commercially insured patients was 3.82 (95% confidence interval [CI], 3.50-4.18) and, although attenuated, remained significant after adjustment for demographic characteristics (odds ratio, 1.33; 95% CI, 1.20-1.47). The strongest predictor of fragmentation was a diagnosis of psychosis (OR, 2.81; 95% CI, 2.43-3.25) or substance abuse (OR, 7.58; 95% CI, 6.55-8.77). CONCLUSIONS: In NYC, Medicaid enrollment is associated with greater fragmentation of hospital care, but this is largely attributable to the preponderance of Medicaid enrollees with diagnoses of psychosis and substance abuse. Strategies to improve the efficiency of healthcare delivery should focus on patients with mental illness who are frequently admitted to general hospitals.

Adolescent↗