Evidence of Brucella infection in Parafilaroides lungworms in a Pacific harbor seal (Phoca vitulina richardsi).
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Animals↗
Biomedical subjects
M M Garner
Publications and source records attributed to M M Garner.
Shell disease in river cooters (Pseudemys concinna) and yellow-bellied turtles (Trachemys scripta) in a Georgia (USA) lake.
A disfiguring shell disease was detected in river cooters (Pseudemys concinna) and yellow-bellied turtles (Trachemys scripta) from Lake Blackshear, Georgia (USA). The turtles used were part of a mark-recapture study conducted from September 1991 to June 1993. Histologic changes on four turtles included acute segmental necrosis of the epidermis, followed by ulceration, necrosis of the underlying dermis and dermal bone, and exaggerated remodeling of bone. Additional findings included visceral inflammatory lesions and bacterial infection, sepsis and marked trematode ova granulomatosis. The cause of the shell lesions was not determined.
Animals↗
Mutagenic DNA base modifications are correlated with lesions in nonneoplastic hepatic tissue of the English sole carcinogenesis model.
Hydroxyl radical-induced mutagenic base modifications have been linked to neoplasia in a number of biological systems, including English sole from chemically contaminated urban environments. However, virtually no information exists on the relationship between the mutagenic base modifications and preneoplastic and other lesions found in tumor-free tissues prone to cancer. We studied six hepatic lesions in immature, neoplasm-free English sole exposed to an urban and reference environment and established correlations between the lesion incidence and concentrations of the mutagenic base modifications 8-hydroxyguanine and 8-hydroxyadenine. The lesions were putatively preneoplastic basophilic foci, hepatocellular karyomegaly, megalocytic hepatosis, hepatocellular vacuolar change, hyalin droplet formation, and apoptosis. With the exception of hepatocellular vacuolar change, significant positive correlations were found between the lesions and the mutagenic base modifications. The hydroxyl radical may be a common etiological factor in the formation of the base modifications and hepatic lesions.
Adenine↗
Fatal cytauxzoonosis in a captive-reared white tiger (Panthera tigris).
Fatal (Panthera tigris) cytauxzoonosis was diagnosed in a 7-year-old female white tiger. The tiger presented with a 2-day history of anorexia and lethargy. She was mildly dehydrated, with a temperature of 105.2 F and a hematocrit of 26%. Over the next day, icterus developed, and her physical condition progressed to recumbency, coma, and death. Hematologic findings obtained shortly before death included icteric plasma, severe thrombocytopenia, mild anemia, hematuria, and parasites consistent with Cytauxzoon felis in circulating erythrocytes. Gross necropsy findings included generalized icterus, generalized petechiae and ecchymoses, splenomegaly, and peribronchial edema. Histologic changes included large numbers of intravascular macrophages containing developmental stages of Cytauxzoon felis that partially or completely occluded blood vessels in the lung, spleen, liver, and bone marrow. Except for an experimental infection of a bobcat, fatal cytauxzoonosis has not previously been diagnosed in felids other than domestic cats. These findings raise questions regarding the pathogenicity of this organism in felids and may impact husbandry and interstate transfer of captive large cats.
Anemia↗
Staining and morphologic features of bone marrow hematopoietic cells in desert tortoises (Gopherus agassizii).
OBJECTIVES: To determine optimal site for collection of bone marrow from desert tortoises, and to characterize cytologic staining and morphologic features of bone marrow hematopoietic cells. ANIMALS: 16 desert tortoises. PROCEDURE: Bone marrow was obtained at necropsy from the pelvis, proximal portion of the humerus, femur, and thickened portions of the cranial to craniolateral and caudal to caudolateral margins of the carapace and plastron for histologic and cytologic examinations. Cytocentrifuged preparations of marrow cells were evaluated for reactivity to cytochemical stains. RESULTS: Histologic sections were adequate for evaluating acidophils, acidophil precursors, and erythrocyte precursors. It was difficult to differentiate among monocytes, lymphocytes, thrombocytes, and blast cells, and eosinophils could not be differentiated from heterophils. Basophils were in rare, small clusters of 3 to 12 cells. A few lymphoid follicles were found in the pelvis and long bones. Use of cytochemical staining accomplished differentiation between agranular heterophil precursors and granulated heterophils, and between granulated eosinophils and basophils. Monocytes, azurophils, and monoblasts had similar staining features. Staining of erythrocyte precursors with Sudan black B differentiated them from lymphocytes. Only a few small cells with periodic acid-Schiff-positive cytoplasm were identified as thrombocytes. Lymphocytes did not stain with any of the cytochemical stains. CONCLUSIONS: For histologic and cytologic evaluation of bone marrow hematopoietic cells, pelvis, proximal portion of the humerus, femur, and thickened portions of the peripheral cranial and caudal regions of the carapace and plastron are suitable sites to collect specimens. There are distinct cytochemical markers for heterophil, monocyte, and erythrocyte precursors, as well as later stage heterophils, eosinophils, basophils, monocytes, and azurophils.
Animals↗
Water release associated with specific binding of gal repressor.
Water release coupled to the association of gal repressor with DNA is measured from the sensitivity of the binding constant to the solution osmotic pressure, using neutral solutes that are typically excluded from polar protein and DNA surfaces. Differences in water release for binding of repressor to different sequences are linked with differences in specificity and binding energies. With sucrose, the specific binding of repressor to operator sequences is accompanied by the release of 130 water molecules. No water release is seen for the weak, non-specific binding of repressor to poly(dI-dC).(dI-dC). A difference in the release of six water molecules is seen even for the binding of gal repressor to two different operator sequences that differ in affinity by only a factor of two.
Binding, Competitive↗
Fluorescent labeling of DNA with ethidium homodimer without measurable decrease in DNA mobility: application to automated gel electrophoresis apparatus.
Mobilities of DNA, fluorescently labeled with ethidium homodimer (EtD), and normalized to the mobility of the 50-bp fragment [Rf(50)], are constant with time of electrophoresis, permitting one to conduct studies on DNA mobility in an automated electrophoresis apparatus with fluorescence detection. DNA mobility decreases with an increasing binding density of ethidium homodimer, as previously reported by others and expected since the dye decreases both the net charge and the conformational flexibility of the DNA. However, it was found that this effect of ethidium binding on electrophoretic mobility becomes undetectable at binding densities less than 1 EtD/40 bp. This implies that for the purposes of electrophoretic analysis in an apparatus with fluorescence detector, DNA with ethidium homodimer intercalated at dye-DNA ratios less than 1/40 bp behaves like unlabeled DNA and may be assumed to be in a conformation similar to that of the unliganded DNA. Necessarily, the low labeling ratio lowers the sensitivity of detection and, in particular, increases the load requirement for a full-scale band height required for the analysis of bandwidth and band shape during electrophoresis.
DNA↗
Commercial automated gel electrophoresis apparatus: application to DNA, band dispersion, nonlinear Ferguson curves, and isolation.
Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 micrograms load for a full-scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching.(ABSTRACT TRUNCATED AT 250 WORDS)
Buffers↗
Capillary electrophoresis of rat liver microsomes in polymer solutions.
Rat liver microsome components were separated by capillary zone electrophoresis in buffers containing substituted agarose, agarose crosslinked polyacrylamide, polyacrylamide, polyethylene glycol and dextran of various molecular weights. The best resolution of the components was obtained with polymers of 10-21 nm geometric mean radius. Both the crude and the purified preparations of microsomes exhibit a single major peak. In a Tris-borate-EDTA (TBE) buffer, containing polyacrylamide of 5 x 10(6) molecular weight, it has a retardation coefficient, KR, of 0.77 +/- 0.02. Translation of the KR value to geometric mean radii, R, on the basis of the standard curve applicable to polymer solutions, KR vs. R, with polystyrene carboxylate size standards in both dextran and polyacrylamide solution allows one to estimate a value of R as 13-16 nm for the major microsome component. The value is smaller than expected from electron microscopic measurements (100-250 nm), possibly due to the chemical and geometric differences between microsomes and the polystyrene particles used as size standards. The crude preparation also contains a minor component which is smaller and less charged than the major component. Another component, apparently very much larger than the major one, is seen in TBE buffer but not in a potassium-N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer and is therefore thought to be an artifact of interaction with borate. After a short incubation under conditions promoting delayed microsome fusion, i.e. in presence of GTP and Mg++ and in the absence of polyethylene glycol, the electrophoretic pattern changes dramatically: it now exhibits five unretarded, highly mobile and, therefore, presumably large components in addition to the two original retarded components of the microsome and a less highly charged species similar in KR to the smaller of the original two components.
Acrylic Resins↗
Recovery of SDS-protein and DNA using commercial automated gel electrophoresis apparatus.
The HPGE-1000 apparatus (LabIntelligence, Menlo Park, CA) is a gel electrophoresis instrument with intermittent fluorescence scanning of the migration path and with preparative capability. An electroelution cup sealed with gel is placed onto the band of interest, identified and located under computer control, and the band is electroeluted into the cup at a right angle to the orientation of the resolving gel. The correct location of the eluted band and the degree of its recovery into the elution cup are then verified on the gel pattern, visualized on the computer screen. Using that procedure, SDS-conalbumin-FLUOS was electrophoresed at 5 V/cm in a discontinuous tricinate-chloride-Tris system at loads of 0.25 to 20 micrograms, using 5% agarose (MetaPhor, FMC), 0.03% SDS gel at 5 degrees C. The horizontal gel was partitioned at the sample loading slit between a gel in Tris-tricinate (prepared at the concentrations of an operative phase ZETA) and in Tris-chloride (prepared as phase BETA). The elution cup was sealed with the latter gel and overlayered with buffer of the composition of the former. This arrangement should provide for electroelution of the band as a highly concentrated stack. At electroelution times of 2, 3.5, 4-5, 12, 15 and 15 min at 15 V/cm yields were 58, 60, 54-76, 99, 99 and 84% for loads of 0.25, 0.5, 1, 4, 10 and 20 micrograms, respectively. At the most sensitive scale of detection (13), a full-scale peak was obtained at a load of 1.7 micrograms when the fluorophore (FLUOS, Boehringer-Mannheim) to protein ratio was 10:1. Similarly, homogeneous nucleosomal DNA (146 bp), electrophoresed in 0.2 x TBE buffer at a load of 5 micrograms, was near-quantitatively recovered into the same buffer by electroelution at 15 V/cm for 2.5 or 6 min.
Autoanalysis↗
Molecular sieving of polystyrene carboxylate of a diameter up to 10 microns in solutions of uncrosslinked polyacrylamide of M(r) 5 x 10(6) using capillary zone electrophoresis.
Capillary zone electrophoresis of polystyrene carboxylate (PSC) up to 10 microns in diameter in 0.1 to 0.9% solutions of uncrosslinked polyacrylamide (PA) of M(r) 5 x 10(6) demonstrates effective molecular sieving in that medium. The reduced mobility, however, is not constant in Tris-borate-EDTA (1x TBE) buffer containing the polymer but increases in proportion to the load of PSC and inversely to the concentration of PA unless the TBE concentration is increased 10-fold or 50 mM 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid is added to the solution or in the absence of PA. Both the Ferguson plots [log(mobility) vs PA concentration] of PSC obtained at various PSC loads and the effects of the dissociating conditions, high ionic strength and detergent, signify that the variable mobilities are those of different PSC aggregation states. Thus, only the fully associated or dissociated states of PSC provide mobilities that are constant independently of PSC load. The study shows that the information inherent in Ferguson plots regarding particle and fiber properties can be exploited for particles up to 10 microns in diameter by capillary zone electrophoresis in polymer solutions even if these particles form various aggregation states. Thus, such an analysis appears feasible in application to biologically relevant and functional complexes such as subcellular-sized particles or DNA-protein complexes, providing that these particles maintain their integrity and functionality under the selected electrophoretic conditions.
Acrylic Resins↗
Macromolecular crowding and confinement in cells exposed to hypertonicity.
The nonideal properties of solutions containing high concentrations of macromolecules can result in enormous increases in the activity of the individual macromolecules. It has been proposed that molecular crowding and confinement occur in cells and are major determinants of the activity of the proteins and other intracellular macromolecules. This concept has important implications for cell volume regulation because, under crowded conditions, relatively small changes in concentration, consequent to alterations of water content, lead to large changes in macromolecular activity. This review considers several aspects of macromolecular crowding and confinement, including: 1) the physical chemical principles involved; 2) in vitro demonstrations of the effects; 3) relation to water activity; 4) estimates of the actual intracellular activity of water and macromolecules; 5) relation to osmotic regulation in various types of cells, including bacteria, red blood cells, and complex nucleated cells; and 6) the relation to inorganic ions and organic osmolytes in cells stressed by hypertonicity. We conclude that, while there is compelling evidence for important effects of molecular crowding in vitro and in red blood cells, the role of macromolecular crowding and confinement in osmotic regulation of more complex cells is an open question that deserves the extensive attention it is currently receiving.
Animals↗
Z-DNA binding and inhibition by GTP of Drosophila topoisomerase II.
A Z-DNA binding protein has been isolated and characterized by biochemical means from Drosophila melanogaster tissue culture cells and embryos. This protein shares the following properties with the known, cloned Drosophila topoisomerase II: (1) expression of an ATP-dependent relaxation activity on supercoiled DNA; (2) a monomer mass of 165 kDa in SDS denaturing gels; (3) a sedimentation coefficient, S20,w, of approximately 10 S for the active enzyme; (4) cross-reactivity for the respective monoclonal and polyclonal antibodies; (5) generation of covalent enzyme-DNA intermediates at preferred cutting sites in the Drosophila HSP70 intergenic spacer region; (6) inhibition of DNA relaxation activity by antitumor drugs, e.g., the etoposide VM26, and by monospecific antibodies raised against the protein; and (7) in vitro phosphorylation by a casein kinase activity. However, we have identified new properties for our topoisomerase II preparation not previously reported for the conventionally isolated enzyme: (1) The enzyme binds to Z-DNA with an affinity 2 orders of magnitude greater than that for B-DNA. (2) The binding to Z-DNA is increased 5-10-fold by GTP or GTP-gamma-S. (3) GTP and GTP-gamma-S inhibit the catalytic activity of topoisomerase II through a proposed allosteric mechanism. (4) Z-DNA inhibits the relaxation of closed circular supercoiled DNA. (5) The preparation consists of a single polypeptide chain of 165 kDa on denaturing SDS gels with no evidence of proteolytic degradation. We postulate that the Z-DNA binding activity of undegraded topoisomerase II may be important in targeting the enzyme both to structural motifs required for chromatin organization and to sites of local supercoiling. Some of these features arise during processes such as replication and gene expression and may be more frequent during embryogenesis and early development.
Animals↗
Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis.
Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well-characterized DNA-protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same length. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an "equivalent sphere" (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3-9%T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.
Animals↗
Resolution of circular, nicked circular and linear DNA, 4.4 kb in length, by electrophoresis in polyacrylamide solutions.
Circular DNA of more than 1,400 bp in size is known not to migrate into polyacrylamide gels. The migration of supercoiled plasmid pBR322 DNA (4,363 by) into uncrosslinked polyacrylamide (Mw 5 x 10(6)) solutions and its separation, on the basis of conformation, from its nicked form is demonstrated in this study. Migration of the supercoiled, nicked circular and linear forms of the plasmid DNA is retarded in proportion to the concentration of uncrosslinked polyacrylamide, the degree of retardation being highest for the nicked circular form. Decreasing the level of supercoiling of the covalently closed circular form by decreasing the concentration of the intercalating dye (ethidium homodimer) shows that the degree of retardation decreases in proportion to the superhelix density.
DNA, Bacterial↗
Computer-aided analysis of DNA curves on transverse gradient gels.
Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.
Buffers↗
Minimal electrophoresis time for DNA sequencing.
This study presents a mathematical approach that allows one to determine the shortest electrophoresis time and migration path length required for DNA sequencing. The calculation was applied to the capillary electrophoresis of a DNA sequencing separation and showed that acceptable resolution could be obtained using a shorter path length than anticipated.
Electrophoresis↗
Electrophoresis with intermittent scanning of the migration path: detection of resolution within shortened time.
Intermittent optical scanning (by detection of optical density or fluorescence) of the electrophoretic migration path was applied to the resolution of two dyes under an arbitrary set of conditions. Scanning at 5-min intervals allows for detection of resolution between the two zones at least 3 times faster than conventional automatic zone detection employing a detector at the end of the migration path. This result promises that replacing stationary by mobile detectors in general would result in a substantial time saving for automated detection of electrophoretic zones.
Densitometry↗