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J M Harter

Publications and source records attributed to J M Harter.

7 recordsLinked to original sources
PubMed1994

H2O2-induced increases in cellular F-actin occur without increases in actin nucleation activity.

Previous work has shown that H2O2 causes an increase in polymerized actin (F-actin) inside cells. To test the hypothesis that increased polymerization resulted from a mechanism involving increased actin nucleation activity, we employed methods utilizing pyrene-labeled actin to quantify the actin nucleation activity of cell lysates and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-phallacidin binding assays to quantify the amount of F-actin in P388D1 cells. H2O2 increased polymerized actin (NBD-phallacidin assay) in a dose-dependent manner with an effective dose giving 50% response (ED50) approximately 1 mM. Five millimolar H2O2 caused a 1.6-fold increase in NBD-phallacidin staining. In contrast, actin nucleation activity decreased in a dose-dependent manner with a similar ED50. Five millimolar H2O2 caused a 30-40% decrease in actin nucleation activity. The effect was rapid, occurring within 5 min of H2O2 addition. The results indicate that H2O2 causes cytoskeletal changes that enhance NBD-phallacidin binding without increasing actin nucleation activity. Fractionation studies showed that the nucleation activity in H2O2-treated cells and controls sedimented with the Triton X-100-insoluble cytoskeleton, and the cytosolic fraction appeared to contain an inhibitor of actin polymerization.

Actins↗
PubMed1992

A threshold level of coupled G-proteins is required to transduce neutrophil responses.

Chemoattractant-induced activation of human polymorphonuclear leukocytes involves receptor coupling to guanine nucleotide binding proteins (G-proteins). Treatment of polymorphonuclear leukocytes with pertussis toxin, which ADP-ribosylates neutrophil G-proteins and uncouples G-proteins from receptors, causes a conversion of cells from responders to nonresponders rather than a gradual decrease in the ability of all cells to respond (Omann, G. M., and J. M. Harter. 1991. Cytometry 12:252; Omann, G. M., and M. M. Porasik-Lowes. 1991. J. Immunol. 146:1303). Flow-cytometric methods were used to measure N-formylpeptide-induced cytosolic Ca2+ elevation and actin polymerization over a wide range of ADP-ribosylation levels and showed that although the percentage of responding cells varied markedly, the responding cells were stimulated equivalent to controls. The conditions of pertussis toxin (PT) treatment did not interfere with non-G-protein-mediated pathways as assessed by measurement of phagocytosis, a complex process involving the cytoskeleton. We tested the explanation that the all-or-none effect may have been due to heterogeneous insertion of the catalytic subunit of PT into the cells such that responders had no ADP-ribosylation and nonresponders were completely ADP-ribosylated. Measurement of the binding of fluorescent N-formylpeptides to permeabilized cells, which allows the distinction between completely ribosylated and normal cells, showed that all cells treated with a submaximal concentration of PT had intermediate levels of receptor-coupled G-proteins. Thus, partial ADP-ribosylation had occurred in all cells and the all-or-none insertion of the catalytic subunit of PT was ruled out. Thus, there is a threshold of coupled G-proteins required to transduce responses. The ability of PT to inhibit N-formylpeptide-induced actin polymerization and cytosolic calcium elevation was compared and showed that both responses have essentially the same threshold of G-proteins required to transduce the responses. Thus, the pathways regulating actin polymerization and calcium elevation appear to be coupled with equal efficiency to the G-proteins.

Actin Cytoskeleton↗
PubMed1991

Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry.

Flow cytometric methods were utilized to determine N-formylpeptide-induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo-3. Fluo-3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo-3 extrusion was minimal (less than 10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N-formyl-met-leu-phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP-induced cytosolic calcium levels were similar in cells loaded over a 20-fold range of Fluo-3-acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A-protomer of PT or a very sharp threshold of coupled G-proteins required to transduce the responses.

Aniline Compounds↗
PubMed1978

Factors affecting methionine toxicity and its alleviation in the chick.

Young male crossbred chicks were fed crystalline amino acid diets containing excess L-methionine or DL-homocysteine to evaluate factors causing methionine toxicity. Chicks were fed diets containing graded levels of excess methionine from 0% to 2.0%. Rate of gain was reduced at all levels of excess methionine, but the magnitude of depression was greater between 1% and 2% than between 0% and 1% excess methionine. Methionine accumulated in plasma of birds fed excess methionine, but plasma levels of homocysteine, cystathionine and cystine remained essentially unchanged. Spleen iron levels increased linearly and blood hemoglobin decreased linearly when chicks were fed diets containing greater than 1% excess methionine, a level equivalent to about 3 times the chicks' requirement. Chicks fed 1.36% homocysteine had reduced gain and gain:feed values, but spleen iron and hemoglobin levels were unchanged. 3-Methylthiopropionate, a possible metabolite in a proposed alternate pathway, caused a precipitous increase in spleen iron levels. Various methyl sources (betaine, choline, methyl acetate) when fed in excess failed to increase spleen iron levels. Methyl mercaptan and methyl mercaptoacetate likewise did not result in an increase in spleen iron deposition. Both the hemosiderosis condition and the reduced food utilization caused by excess methionine were reversed by supplemental glycine plus threonine.

Animals↗
PubMed1978

D-cystine utilization by the chick.

Young chicks were fed graded levels of either L-cystine or DL-cystine in a purified crystalline amino acid diet made adequate in methionine but void in cystine. Gain performance and slope-ratio efficacy comparisons indicated that the D-isomer of cystine could not be utilized by the chick.

Animals↗