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PubMed1993

Evaluation of NR-LU-10 with the Neoprobe gamma detector in a mouse model.

The Neoprobe Model 1000 hand-held gamma detector, in combination with the murine monoclonal antibody (MAb) B72.3 can successfully intraoperatively target both primary and recurrent colorectal cancer. Because of the shortcomings of this system (length of time needed to clear unbound MAb and heterogeneous staining of cancer cells), new MAbs are under investigation. NR-LU-10 IgG and its FAB fragment were evaluated with the Neoprobe gamma detector in a nude mouse model. NR-LU-10 is a pancarcinoma IgG2b antibody that recognizes an oncofetal glycoprotein antigen that is expressed by most carcinomas. Animals were injected intraperitoneally with 125I-labeled IgG, 125I, or technetium 99m (99mTc)-labeled Fab fragment. The Neoprobe gamma detector and standard gamma well counts provided biodistribution and pharmacokinetic data. The Fab fragment achieved a 5:1 tumor to blood-pool background (BPB) ratio in only 66 hr, whereas the whole MAb required 14 days. Technetium did not appear to be an adequate isotope for this system because of its short half-life. Autoradiographs performed for both the 125I-labeled NR-LU-10 IgG and its 125I-labeled Fab fragment (but not 99mTc-labeled Fab) localized well in this model and gave adequate tumor to BPB and tissue ratios. Use of the 125I-labeled Fab significantly decreased the time required for clearance of the MAb radionuclide complex from the blood and tissue background, thus providing earlier tumor localization in the Radioimmunoguided Surgery (RIGS) system.

Animals↗

PubMed2003

Dual wavelength parametric test of two-state models for circular dichroism spectra of helical polypeptides: anomalous dichroic properties of alanine-rich peptides.

A two-state helix-coil model underlies all calculations of fractional helicities FH from CD spectra of helical polypeptides. The presence of an isodichroic point near 203 nm is widely assumed to validate this model, but is shown here to provide inadequate validation for alanine-rich peptides. A parametric correlation with constant slope B between CD ellipticities at a pair of wavelengths is introduced as a more rigorous two-state test. Correlations of temperature-dependent [theta](222) vs [theta](208) values are reported for a variety of peptides. Constant slopes B are observed for literature CD data obtained from fragments of helical proteins and dimeric helical coiled coils, but parametric correlations of CD data for alanine-rich peptides consistently exhibit anomalous concave upward curvature, characterized by local slopes that are linearly temperature dependent. Low-temperature CD studies together with parametric correlations at a series of wavelengths demonstrate that the curvature anomaly is maximal at 222 nm and localized in the 215-230 nm wavelength region. Precedented structural variation of the phi, psi dihedral angles of the alpha-helix is suggested as a possible explanation. For the important case of alanine-rich peptides, experiments are proposed that may yield temperature corrections for [theta](222) and permit reliable calculations of FH from [theta](222) values.

Alanine↗

PubMed1984

Recombinant DNA data management at the restriction and functional site level.

We have created a system to manage in a unified manner the restriction and functional site information required for design and analysis of recombinant DNA experiments. Primary source DNA data and recombinant clone specifications are used to generate recombinant restriction maps and restriction fragment lists. Sequence data, restriction-site data, and functional-site data may be combined in the data base. Interaction and output is user-friendly and versatile.

Base Sequence↗

PubMed2005

Genetic variation and phylogeographic analyses of two species of Carpobrotus and their hybrids in California.

Despite the commonality and study of hybridization in plants, there are few studies between invasive and noninvasive species that examine the genetic variability and gene flow of cytoplasmic DNA. We describe the phylogeographical structure of chloroplast DNA (cpDNA) variation within and among several interspecific populations of the putative native, Carpobrotus chilensis and the introduced, Carpobrotus edulis (Aizoaceae). These species co-occur throughout much of coastal California and form several 'geographical hybrid populations'. Two hundred and thirty-seven individuals were analysed for variation in an approximate 7.0 kb region of the chloroplast genome using PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) data. Phylogenetic analyses and cpDNA population differentiation were conducted for all morphotypes. Historic geographical dispersion and the coefficient of ancestry of the haplotypes were determined using nested clade analyses. Two haplotypic groupings (I and II) were represented in C. chilensis and C. edulis, respectively. The variation in cpDNA data is in agreement with the previously reported allozyme and morphological data; this supports relatively limited variation and high population differentiation among C. chilensis and hybrids and more wide-ranging variation in C. edulis and C. edulis populations backcrossed with C. chilensis. C. chilensis disproportionately contributes to the creation of hybrids with the direction of gene flow from C. chilensis into C. edulis. The cpDNA data support C. chilensis as the maternal contributor to the hybrid populations.

Aizoaceae↗

PubMed2006

Obtaining data on patient race, ethnicity, and primary language in health care organizations: current challenges and proposed solutions.

OBJECTIVES: To provide an overview of why health care organizations (HCOs) should collect race, ethnicity, and language data, review current practices, discuss the rationale for collecting this information directly from patients, and describe barriers and solutions. PRINCIPAL FINDINGS: Hospitals and HCOs with data from their own institutions may be more likely to look at disparities in care, design targeted programs to improve quality of care, and provide patient-centered care. Yet data collection is fragmented and incomplete within and across organizations. A major factor affecting the quality of data is the lack of understanding about how best to collect this information from patients. CONCLUSIONS: If HCOs make a commitment to systematically collect race/ethnicity and language data from patients, it would be a major step in enhancing the ability of HCOs to monitor health care processes and outcomes for different population groups, target quality initiatives more efficiently and effectively, and provide patient-centered care.

Data Collection↗

PubMed2000

Increase in fragmented phosphatidylcholine in blood plasma by oxidative stress.

Oxidatively modified phospholipids with fragmented acyl chains have attracted much interest because of their proinflammatory activity and their potential involvement in atherosclerosis. They can be formed in vitro by free radical treatment of unsaturated phospholipids but it is not known under which conditions they accumulate in vivo. We assayed one species of fragmented phosphatidylcholine (PC) in human blood plasma by high performance liquid chromatography after precolumn derivatization with chloromethylanthracene. Structural analysis suggested that fragmented PC was a diacyl species with a palmitoyl group and a short oxidized residue, which most likely had four carbons. The concentration of fragmented PC was higher in elderly individuals with coronary heart disease than in young healthy controls. Smoking one cigarette acutely increased the concentration of fragmented PC in healthy adults. Fragmented PC also increased in the reperfusion period after treatment with cardiopulmonary bypass. The increase coincided with a surge of circulating neutrophils. In rats, the plasma concentration of fragmented PC was elevated by vitamin E deficiency and exposure to high oxygen. The data demonstrate that fragmented PC increases in blood plasma in response to various forms of oxidative stress.

Adult↗

PubMed2006

Fibrinogen fragment D is necessary and sufficient to anchor a surface plasminogen-activating complex in Streptococcus pyogenes.

In this study, the importance of different domains of the fibrinogen molecule in the binding and assembly of a surface plasminogen (plgn) activator has been analyzed. This was achieved using SELDI technology that enabled dissociation of bound fragments from intact bacteria and accurate distinction between fibrinogen fragments based on their molecular mass. These studies indicate that Streptococcus pyogenes binds directly to human fibrinogen fragment D but not fragment E. The predominant surface proteins binding to fragment D were associated with the mrp gene product. Surface-associated fibrinogen fragment D was capable of anchoring a functional surface plgn activator complex. Taken together, these data indicated that fragment D of fibrinogen is necessary and sufficient to anchor a plgn activator complex on the surface of Streptococcus pyogenes.

Fibrin Fibrinogen Degradation Products↗

PubMed2003

Short peptide amyloid organization: stabilities and conformations of the islet amyloid peptide NFGAIL.

Experimentally, short peptides have been shown to form amyloids similar to those of their parent proteins. Consequently, they present useful systems for studies of amyloid conformation. Here we simulate extensively the NFGAIL peptide, derived from the human islet amyloid polypeptide (residues 22-27). We simulate different possible strand/sheet organizations, from dimers to nonamers. Our simulations indicate that the most stable conformation is an antiparallel strand orientation within the sheets and parallel between sheets. Consistent with the alanine mutagenesis, we find that the driving force is the hydrophobic effect. Whereas the NFGAIL forms stable oligomers, the NAGAIL oligomer is unstable, and disintegrates very quickly after the beginning of the simulation. The simulations further identify a minimal seed size. Combined with our previous simulations of the prion-derived AGAAAAGA peptide, AAAAAAAA, and the Alzheimer Abeta fragments 16-22, 24-36, 16-35, and 10-35, and the solid-state NMR data for Abeta fragments 16-22, 10-35, and 1-40, some insight into the length and the sequence matching effects may be obtained.

Amino Acid Sequence↗

PubMedDate not supplied

DNA loop organization and DNA fragmentation during radiation-induced apoptosis in human lymphocytes.

Apoptotic DNA fragmentation induced by gamma-rays has been compared with the DNA loop sizes in G0-human lymphocytes using pulsed field gel electrophoresis (PFGE). Genomic DNA was cleaved into the DNA loops at the topoisomerase II mediated attachment points using short treatment of cells with etoposide. The apoptotic fragmentation, with a distinct cut-off around 50 kb for a maximum length of fragments, appeared 5 h after irradiation when the most part of radiation-induced DNA double strand breaks (DSBs) have been repaired. The data indicate that apoptotic fragmentation of DNA in the G0-human lymphocytes begins when repair of radiation-induced DSBs has been completed. Similar apoptotic DNA fragmentation was also observed following the treatment of cells with etoposide. All genomic DNA was fragmented into 50-kb fragments during the final stages of apoptosis. Most of the DNA in resting lymphocytes is organized into Mb-size loops but loops of sizes down to 50 kb were also observed. A sharp border between the size distributions of DNA loops and apoptotic fragments was found. The data suggest that 50 kb apoptotic fragmentation is not based on excision of the DNA loops. No apoptotic fragments with the sizes more than 5.7 Mb were seen during the whole course of apoptosis. This observation indicates that despite intensive apoptotic fragmentation into the 50-kb fragments the chromosomes maintain integrity during radiation-induced apoptosis in human lymphocytes. We propose a model for radiation-induced apoptotic fragmentation in human lymphocytes that involves four stages: induction of DNA breaks and relaxation of DNA loops; DNA repair followed by reorganization of the DNA loops into the 50-kb units of condensed chromatin; co-operative fragmentation of the reorganized DNA loops into the distinct 50-kb fragments and resealing of the chromosome ends at the sites of this fragmentation; cleavage of the 50-kb fragments at the internucleosomal spacers.

Apoptosis↗

PubMed1994

Acid and base resistance in Escherichia coli and Shigella flexneri: role of rpoS and growth pH.

Escherichia coli K-12 strains and Shigella flexneri grown to stationary phase can survive several hours at pH 2 to 3, which is considerably lower than the acid limit for growth (about pH 4.5). A 1.3-kb fragment cloned from S. flexneri conferred acid resistance on acid-sensitive E. coli HB101; sequence data identified the fragment as a homolog of rpoS, the growth phase-dependent sigma factor sigma 38. The clone also conferred acid resistance on S. flexneri rpoS::Tn10 but not on Salmonella typhimurium. E. coli and S. flexneri strains containing wild-type rpoS maintained greater internal pH in the face of a low external pH than strains lacking functional rpoS, but the ability to survive at low pH did not require maintenance of a high transmembrane pH difference. Aerobic stationary-phase cultures of E. coli MC4100 and S. flexneri 3136, grown initially at an external pH range of 5 to 8, were 100% acid resistant (surviving 2 h at pH 2.5). Aerobic log-phase cultures grown at pH 5.0 were acid resistant; survival decreased 10- to 100-fold as the pH of growth was increased to pH 8.0. Extended growth in log phase also decreased acid resistance substantially. Strains containing rpoS::Tn10 showed partial acid resistance when grown at pH 5 to stationary phase; log-phase cultures showed < 0.01% acid resistance. When grown anaerobically at low pH, however, the rpoS::Tn10 strains were acid resistant. E. coli MC4100 also showed resistance at alkaline pH outside the growth range (base resistance). Significant base resistance was observed up to pH 10.2. Base resistance was diminished by rpoS::Tn10 and by the presence of Na+. Base resistance was increased by an order of magnitude for stationary-phase cultures grown in moderate base (pH 8) compared with those grown in moderate acid (pH 5). Anaerobic growth partly restored base resistance in cultures grown at pH 5 but not in those grown at pH 8. Thus, both acid resistance and base resistance show dependence on growth pH and are regulated by rpoS under certain conditions. For acid resistance, and in part for base resistance, the rpoS requirement can be overcome by anaerobic growth in moderate acid.

Acids↗

PubMed1989

A high-resolution, fluorescence-based, semiautomated method for DNA fingerprinting.

We describe a fluorescence-based method for the automated analysis of DNA fragments on polyacrylamide gels. A single-stranded oligonucleotide primer (18-mer) with a fluorochrome covalently bound to its 5'-end is annealed to a synthetic oligonucleotide to create a double-stranded oligonucleotide linker with a 5'-overhang complementary to a restriction enzyme site. Cosmid or plasmid DNA is digested with the appropriate restriction enzyme and then ligated to the fluorochrome-labeled linker. The labeled restriction fragments are loaded on a denaturing polyacrylamide gel in a commercially available DNA sequencer. As the restriction fragments migrate through the gel, they intersect a laser beam which excites the fluorochrome-labeled fragment. Fluorescence emission data are captured on a computer in real time and analyzed after the completion of electrophoresis. Fragment length is nearly linearly related to migration time. This method offers very near single-base resolution up to 400 bases and the ability to quantitate fragment size up to 2000 bases. The fluorochrome-labeling chemistry relies on straightforward enzymatic reactions and can be performed in a single reaction tube. Because four different fluorochromes can be used, each of 16 lanes on the gel can be used to analyze four different digest reactions, one in each color. One of the fluorochromes can be used to label size standards in each lane, eliminating interlane variability and allowing more precise estimates of fragment size. We apply the method to the analysis of overlapping cosmids.

Animals↗

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↗

PubMed1983

Stimulation of fibrinogen biosynthesis by fibrinogen fragments D and E.

Infusions of either fibrinogen fragment D or fibrinogen fragment E into rabbits were followed by increases in fibrinogen synthesis determined by the rate of incorporation of 75Se-selenomethionine into circulating fibrinogen. The degree of stimulation was proportional to the amount of protein infused. When 4.5 mg of each fibrinogen fragment was administered separately to different groups of animals, fibrinogen fragment D was associated with a fourfold increase in fibrinogen synthesis above that in the control animals compared with 1.5-fold increase induced by fragment E. Fragments D and E were assayed for bound sialic acid, the absence of which facilitates binding, transport and catabolism of many circulating glycoproteins by the liver. Fibrinogen fragment D contained 1.3% sialic acid compared to 1.4% in fragment E. These data indicate conservation of sialic acid during plasmic digestion of fibrinogen. The capacity of these glycopolypeptide fragments to stimulate fibrinogen synthesis appears unrelated to the nearly identical quantities of N-acetyl neuraminic acid found in each fragment.

Animals↗

PubMed2002

Public domain computer-aided surgery (CAS) in orthodontic and maxillofacial surgery.

BACKGROUND: Computer-aided virtual three-dimensional (3D) surgical simulation assists the necessary visual understanding of complex pathological situations but has so far been dependent on expensive hardware and software. METHOD: For the first time a non-commercial, user-orientated application for orthognathic and craniofacial surgical simulation has been introduced, based on freeware NIH Image 1.62 provided by the National Institute of Mental Health (NIMH). Use and limitations are demonstrated here by the example of hypertelorism surgery. The osteotomy lines and the amount of resection for outward positioning of the orbits were determined by the surgeon on a workstation using CT data. Possible movement patterns of the osteotomy fragments were rotations, sagittal and transversal movements or combinations of these. The program then allows the calculation of anatomical distances on the screen in a 1:1 relation. Normative values according to age and pathological anatomy determine the degree of displacement. The program calculates the new position of the osteotomy fragments and transfers the data of the segment movement to the original CT data. RESULTS: 3D visualization of bone and soft tissue contours have been produced with an acceptable quality on a workstation for the demonstration and visual understanding of the surgical plan. Evaluation of a postoperative CT showed that the amount of movement and size of bone resection in the simulation was slightly overestimated rather than underestimated. This was also dependent on the complexity of the pathological anatomy and on how experienced the surgeon was. CONCLUSION: The use of 3D simulation programs allows a better understanding of the pathological anatomy in all dimensions, and in many cases enables limitations to be recognized in advance. The public domain program makes a major contribution to the quality of orthognathic and craniofacial surgical planning through cost-free usability, and supports continuous development and exchange of experience.

Cephalometry↗

PubMed2006

Transition metal complexes of terminally protected peptides containing histidyl residues.

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>>Cu(II)>>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.

Animals↗

PubMed1991

Structural dynamics and functional domains of the fur protein.

Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.

Amino Acid Sequence↗

PubMed2006

Can neutral theory predict the responses of Amazonian tree communities to forest fragmentation?

We use Hubbell's neutral theory to predict the impact of habitat fragmentation on Amazonian tree communities. For forest fragments isolated for about two decades, we generate neutral predictions for local species extinction, changes in species composition within fragments, and increases in the probability that any two trees within a fragment are conspecific. We tested these predictions using fragment and intact forest data from the Biological Dynamics of Forest Fragments Project in central Amazonia. To simulate complete demographic isolation, we excluded immigrants--species absent from a fragment or intact forest plot in the initial census but present in its last census--from our tests. The neutral theory correctly predicted the rate of species extinction from different plots as a function of the diversity and mortality rate of trees in each plot. However, the rate of change in species composition was much faster than predicted in fragments, indicating that different tree species respond differently to environmental changes. This violates the key assumption of neutral theory. When immigrants were included in our calculations, they increased the disparity between predicted and observed changes in fragments. Overall, neutral theory accurately predicted the pace of local extinctions in fragments but consistently underestimated changes in species composition.

Conservation of Natural Resources↗

PubMed1996

The root of the phylogenetic tree of human populations.

Although African populations have been shown to be most divergent from any other human populations, it has been difficult to establish the root of the phylogenetic tree of human populations since the rate of evolutionary change may vary from population to population owing to the fluctuation of population size and other factors. However, the root can be determined by using the chimpanzee as an outgroup and by employing proper statistical methods. Using this strategy, we constructed phylogenetic trees of human populations for five different sets of gene frequency data. The data sets used were two sets of microsatellite loci data (25 and 8 loci, respectively), restriction fragment length polymorphism (RFLP) data (79 loci), protein polymorphism data (15 loci), and Alu insertion frequency data (4 loci). All these data sets showed that the root is located in the branch connecting African and non-African populations, and in the four data sets the root was established at a significant level. These results indicate that Africans are the first group of people that split from the rest of the human populations.

DNA, Satellite↗