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D L Taylor

Publications and source records attributed to D L Taylor.

At least 217 records · Page 12Linked to original sources
PubMed1979

Distribution of fluorescently labeled actin in living sea urchin eggs during early development.

Rabbit skeletal muscle actin was labeled with 5-iodoacetamidofluorescein (5-IAF) and purified by gel filtration, ion-exchange chromatography, and polymerization-depolymerization. The resultant fluorescent conjugates retained full biochemical activities. The labeled actin was incorporated into unfertilized eggs of Lytechinus pictus by direct microinjection and the distribution of fluorescence was investigated after fertilization through the first division cycle. The results were interpreted by comparing the images with those of control eggs injected with fluorescein isothiocyanate (FITC)-labeled ovalbumin. After fertilization of eggs containing IAF actin, the membrane-cortical regions showed dramatic increases in fluorescence intensity which were not observed in FITC ovalbumin controls. During the first division, spindle regions of both IAF-actin-injected eggs and control eggs became distinctly fluorescent. However, no distinctly fluorescent contractile ring was detected in the cleavage furrow. After cytokinesis, the surface between blastomeres containing IAF actin exhibited an increase in fluorescence intensity. These observations have been compared with those of previous studies using different methods, and the possible implications have been discussed in relation to cellular functions.

Actins↗
PubMed1979

The contractile basis of ameboid movement. VI. The solation-contraction coupling hypothesis.

The contracted pellets derived from a high-speed supernate of Dictyostelium discoideum (S3) were investigated to determine the functional activity associated with this specific subset of the cellular motile apparatus. A partially purified model system of gelation and contraction (S6) was prepared from the contracted pellets, and the presence of calcium- and pH-sensitive gelation and contraction in this model demonstrated that a functional cytoskeletal-contratile complex remained at least partially associated with the actin and myosin during contraction. Semi-quantitative assays of gelation and solation in the myosin-free preparation S6 included measurements of turbidity, relative viscosity, and strain birefringence. The extent of gelation was optimal at pH 6.8 and a free calcium ion concentration of approximately 3.0 x 10(-8) M. Solation was favored when the free calcium ion concentration was greater than 7.6 x 10(-7) M or when the pH was increased or decreased from pH 6.8. Gelation was reversibly inhibited by increasing the free calcium ion concentration to approxomately 4.6 x 10(-6) M at pH 6.8. The solation-gelation process of this model has been interpreted to involve the reversible cross-linking of actin filaments. The addition of purified D. discoideum myosin to S6 served to reconstitute calcium- and pH-regulated contraction. The results from this study indicate that contraction is coupled functionally to the local breakdown (solation) of the gel. Therefore, solation has been identified as a structural requirement for extensive shortening during contraction. We have called this concept the solation-contraction coupling hypothesis. Fractionation of a preparation derived from the contracted pellets yielded a fraction consisting of actin and a 95,000-dalton polypeptide that exhibited calcium-sensitive gelation at 28 degrees C and a fraction composed of actin and 30,000- and 18,000-dalton polypeptides that demonstrated calcium-sensitive genlation at 0 degrees C.

Calcium↗
PubMed1978

Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells.

Actin labeled with 5-iodoacetamidofluorescein has been incorporated into the functional pool of actin in Chaos carolinensis and Physarum polycephalum by direct microinjection. The functional activity of the labeled actin has been analyzed at three levels of organization as: (a) with the purified actin, (b) in motile extracts of cells, and (c) in living motile cells. The labeled actin exhibited normal polymerization and activated myosin ATPase to a similar extent as unlabeled controls. Labeled actin and endogenous actin were incorporated into contracted pellets to approximately the same extent in motile cell extracts. After labeled actin had been microinjected into single C. carolinensis cells, the fluorescent actin spread into both the endoplasm and etoplasm without forming distinct fibrils. In contrast, fluorescent bundles developed in the ectoplasm of P. polycephalum following microinjection of labeled actin. This experimental method in conjunction with fluorescence spectroscopic techniques could become a powerful tool for studying the intracellular distribution and structural changes of components in living cells.

Actins↗
PubMed1978

In vivo evaluation of the radiosensitizing and cytotoxic properties of newly synthesized electron-affinic drugs.

We are synthesizing and testing various 2-nitroimidazole analogs with different octanol/water partition coefficients for their radiosensitizing and cytototoxic properties. Our hypothesis is that, unlike the situation in vitro, lipophilicity will play a major role in determining the extent of radiosensitization of hypoxic tumour cells in vivo and also the degree of animal toxicity. The radiosensitivity testing involves determination of the surviving fraction of EMT6 tumour cells irradiated in vivo under different conditions following drug injection, and subsequent assay in vitro. Data obtained to date on eight 2-nitroimidazoles do not indicate any compound clearly superior to misonidazole (Ro-07-0582), although one or two are close and may prove better at the completion of testing. Cytotoxicity of the compounds is assayed by cell survival and histologically. Data obtained on the toxicity of misonidazole to the cells of EMT6 tumours suggest that a large proportion of the anoxic cells in these tumours are not near necrotic areas, but rather result from the temporary closure of blood vessels.

Animals↗
PubMed1977

The contractile basis of amoeboid movement. V. The control of gelation, solation, and contraction in extracts from Dictyostelium discoideum.

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.

Actins↗
PubMed1977

Comparative study of three methods of identification of Enterobacteriaceae.

Three separate hospital clinical microbiology laboratories using three different identification systems participated in the identification of Enterobactericeae from a central pool of 'unknown" clinical isolates. With conventional tubed media, API-20E (Anlytab Products Inc.) and R/B tube (Corning Diagnostics) systems, there was a 91.1% agreement in the species designation. No significant differences at the 95% confidence level were found among the systems. Evaluation of individual tests within the systems used revealed lysine decarboxylase of the conventional and citrate of the API-20E system to be significantly different from the same test within the other two systems. The lysine decarboxylase of the conventional system had species relatedness, whereas the differences in citrate of the API-20E system were not related to a particular species. These individual test variations did not affect final organism identification. Reproducibility, evaluated as the system's ability to designate the same identification on two separate occasions, was 92 to 94% for each system. Exact duplication of selected sets of reactions was 60% for conventional, 45% for API-20E, and 61% for R/B. The variations in sets of reactions differed with the system and with the organism involved. The findings suggest equivalency among the three systems in ability to identify common clinical isolates of Enterobacteriaceae and point out the limited usefulness of these systems for biochemical biotyping.

Bacteriological Techniques↗
PubMed1977

The contractile basis of amoeboid movement III. Structure and dynamics of motile extracts and membrane fragments from Dictyostelium discoideum and Amoeba proteus.

Motile extracts from D, discoideum and A. proteus have been characterized in order to compare the structural dynamics and chemical regulation of movement in 2 different types of amoeboid cells. The structural dynamics of both extracts involve the formation of a nonmotile cytoskeleton followed by the contraction of actin and myosin to generate both direct contractile force and cytoplasmic streaming. The contractions are regulated by calcium ions and a threshold of ca. 1.0 X 10(-6) M calcium induces a transformation of actin to the free F-actin state which is capable of interacting with myosin. Furthermore, 3 low molecular weight proteins are concentrated along with actin and myosin during contraction and might play a regulatory role in movement. Several common characteristics of amoeba cytoplasm have been exhibited by these two types of amoeboid cells. The major contractile and "associated" proteins are similar, actin and associated proteins are structurally dynamic, and movement is regulated by calcium. The different modes of movement observed in different types of amoeboid cells could result from the site, rate, and extent of actin transformation followed in some regions by contractions.

Actins↗
PubMed1976

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts.

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.

Actins↗
PubMed1975

Birefringence changes in vertebrate striated muscle.

The changes in birefringence in the rigor to relax transition of single Triton-extracted rabbit psoas muscle fibers have been investigated. The total birefringence of rigor muscle fibers was dependent on sarcomere length and ranged from (1.46 +/- 0.08) X 10(-3) to (1.60 +/- 0.06) X 10(-3) at sarcomere lengths from 2.70 mum to 3.40 mum. An increase in total birefringence was measured dependent on sarcomere length when 55 single fibers were relaxed from the rigor state with Mg-ATP. Pyrophosphate relaxation produced a smaller increase in retardation when compared to Mg-ATP. The expected change in intrinsic birefringence during the rigor to relax transition was calculated assuming a hinge function of the subfragment 2 moiety of myosin. The changes in birefringence during isometric contraction and relaxation have been discussed in relation to possible structural changes.

Adenosine Triphosphate↗