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Biomedical subjects

E Johnson

Publications and source records attributed to E Johnson.

At least 343 records · Page 19Linked to original sources
PubMed1983

Comparison of two separation techniques for the determination of blood mononuclear cell magnesium content.

The determination of blood mononuclear cell magnesium content may be a predictor of total body magnesium status. Separation techniques employing Ficoll-Hypaque have been used for this assay; however, Hypaque contains a significant concentration of iodine that may be toxic to the cell membrane and affect the results. We developed a different separation technique using arabinogalactan (Stractan) at concentrations of 13.0 and 17.7%. The blood from 16 normal volunteers was divided and assayed for mononuclear cell magnesium content using both methods. The results (mean +/- standard deviation) with the Ficoll-Hypaque method were 83.6 +/- 30.8 fg/cell, and with the arabinogalactan method they were 78.4 +/- 25.5 fg/cell. A paired sample t test comparing the results between the two methods gave a t value of 1.05 (P greater than .3). The purest population of blood mononuclear cells was obtained with the Ficoll-Hypaque method. Thus, these two separation materials do not have a significant effect on the blood mononuclear cell magnesium content, and the better separation of cells was obtained with Ficoll-Hypaque.

Cell Separation↗
PubMed1983

Complement (C3) receptor-mediated attachment of agarose beads to mouse peritoneal macrophages and human monocytes.

We have determined the receptors on human monocytes and mouse peritoneal macrophages producing agarose binding. By using isolated human complement factors C3, B and D, agarose beads were coated with C3b. In some experiments C3b was converted to C3bi by using human serum diluted 1:20. Agarose beads coated with C3b or C3bi bound strongly to monocytes. Only agarose beads coated with C3bi were attached to mouse macrophages. Trypsinization of agarose beads coated with C3bi abolished the attachment of the beads to macrophages and monocytes, probably because of conversion of C3bi to C3d. Endocytosis by macrophages of agarose preincubated in human serum or in C5-deficient AKR mouse serum reached the same levels, indicating that the amount of C5 present in serum during preincubation is not important for the degree of endocytosis. It is concluded that internalization of agarose by macrophages is mediated via the C3bi receptor.

Animals↗
PubMed1983

Complement (C3)-receptor-mediated phagocytosis of agarose beads by mouse macrophages. II. Extracellular activation of macrophage-derived complement on agarose via the alternative pathway.

Phagocytosis of agarose beads by macrophages cultured under serum-free conditions was studied. 48 h was needed before a plateau in the uptake was reached. The ingested agarose beads were coated extracellularly with macrophage-derived protein before attachment and ingestion of the beads. Intracellularly, the agarose-linked protein was removed from the agarose. If the ingested agarose beads were extracted from the macrophages within 24 h after the plateau in the uptake was reached, a fraction of the beads could attach to new macrophages, demonstrating modification of the agarose beads by opsonin(s). Because of binding of anti-human C3c antibodies to beads extracted from the macrophages after 24 h of phagocytosis and the trypsin sensitivity of the protein on the agarose, we conclude that the main opsonin on the agarose beads is C3bi. Requirements for the stimulatory effect of agarose on macrophages are summarized.

Animals↗
PubMed1983

Complement (C3)-receptor-mediated phagocytosis of agarose beads by mouse macrophages. I. Intracellular degradation of agarose-bound C3bi and C3b by lysosomal enzymes.

The phagocytosis by macrophages of C3bi-coated agarose beads reached a plateau after 15 min, compared with 30 min for C3b-coated beads. By using 125I-labelled C3bi or C3b coupled to the agarose beads, we found that 70% and 95% of total radioactivity were removed from the beads after 12 h and 36 h of intracellular digestion, respectively. Intracellular degradation of C3bi linked to agarose beads was also demonstrated by testing binding of monoclonal antibodies against human C3c, C3g and C3d to beads extracted from the cells after phagocytosis. Such extracted beads also showed reduced attachment to new macrophages compared with non-ingested beads. Treatment of the cells with leupeptin, an inhibitor of the lysosomal enzyme cathepsin B, or with dextran sulphate to inhibit phagosome-lysosome fusion greatly reduced the release of labelled protein from the agarose during the first 12 h. These findings show that C3bi and C3b on agarose is destroyed intracellularly by lysosomal enzymes.

Animals↗
PubMed1983

Monoclonal antibodies against vasoactive intestinal polypeptide: studies of structure and related antigens.

Hybridomas secreting monoclonal anti-vasoactive intestinal polypeptide (VIP) antibodies were constructed from spleen cells sensitized to VIP in vitro. The secreted antibodies were characterized by binding to VIP in indirect radioimmunoassays and enzyme-linked immunosorbent assays. Two monoclonal antibodies, characterized for their binding activities with synthetic fragments of VIP, were found to bind different sites on the VIP molecule. These monoclonal antibodies may recognize tertiary structures of the VIP. A search was conducted for antigens recognized by the monoclonal antibodies in brain: brain proteins separated on polyacrylamide gels were electroblotted onto nitrocellulose filters and were reacted first with the mouse antibody and then with goat anti-mouse immunoglobulin coupled to horseradish peroxidase as a means of detection. The monoclonal antibodies were found to react with a protein of molecular weight 60,000, which was also recognized by polyclonal antibodies, although the latter reacted with a number of additional proteins. The relationship of the protein of molecular weight 60,000 to VIP is discussed.

Adrenal Gland Neoplasms↗
PubMed1983

Circulating antibody to prostate antigen in patients with prostatic cancer.

A reverse enzyme-linked immunosorbent assay (ELISA) modified from a prostate antigen (PA) assay previously reported has been developed to measure circulating PA-binding globulin (PABG). Serum specimens taken from normal males, normal females, male patients with nonprostatic cancers, patients with benign prostatic hypertrophy, and patients with various stages of prostate cancer were analyzed for PABG. Results revealed that only patients with an advanced stage of prostatic cancer exhibited an elevated level of PABG. PABG was then isolated from prostatic cancer patients' serum by PA affinity chromatography. Upon immunodiffusion and immunoelectrophoresis, this PABG preparation reacted with purified PA, anti-PA xenoantibodies and anti-human IgG. By the immunoperoxidase technique, PABG stained positively in prostatic ductal epithelial cells and negatively in all other tissues examined. An additional PABG preparation, which reacted with anti-PA and anti-human IgG and not with purified PA, was also isolated by an anti-PA affinity chromatography. These PABG preparations were separately subjected to high-performance liquid chromatography for further purification. Three major protein peaks at Mr of greater than 240K, 150K, and 34K were obtained. These results demonstrated that circulating IgG antibody reactive with PA was present in patients with metastatic cancer of the prostate, and this in part was in complexed form with PA and was specifically reactive with ductal epithelial elements of the prostate.

Animals↗
PubMed1983

Noninvasive echo-Doppler duplex measurements of common femoral artery blood flow variables during supine exercise and post-occlusive reactive hyperemia.

Noninvasive dynamic measurements of common femoral artery blood flow (CFBF) and stroke volume (CFSV) during supine exercise (SE) and staged post-occlusive reactive hyperemia (PORH) in normal subjects using an echo-Doppler duplex scanner (DS) would provide baseline hemodynamic data in the study of the peripheral circulation in resting, stressed, and diseased conditions. Heart rate (HR), CFBF, and CFSV were determined at rest (R), after 5 minutes of SE on an ergometer (30 rpm) at 30, 60, 90-watt loads and after a 3-minute recovery. The same variables were also calculated after periods of 2-, 5-, and 10-minute thigh occlusions (greater than 240 mm Hg cuff pressure). Measurements of spatial average blood velocities and lumen diameters were used to calculate CFBF. Ankle pressure (AP) and brachial pressure (BP) were determined, and the ratio of systolic AP to systolic BP, the ankle index (AI), was calculated. As in vitro validation of the DS method was accomplished using a hydraulic model of CFBF (r = 0.98). Mean values for maximal CFBF following SE and PORH were 0.81 and 1.53 l/min, respectively. With SE, increases in CFBF and CFSV and decreases in AI were significantly (p less than .05) only at 60- and 90-watt loads. Increases in CFBF and CFSV and decreases in AP and AI were significantly different (p less than .05) from rest for the three occlusion durations and show a linear trend. This study suggests that the DS method can noninvasively and quantitatively measure CFBF and CFSV at rest following SE and during PORH.

Adult↗
PubMed1982

Behavioral effects of early deprivation of nerve growth factor: some similarities with familial dysautonomia.

Female rats immunized with mouse nerve growth factor develop an antibody (anti-NGF) which reaches offspring through the placenta and via the milk. Pups exposed to maternal anti-NGF have fewer dorsal root and sympathetic neurons. When the offspring are examined on a wide variety of behavioral tests, they exhibit severe deficits in response to stress (ulceration, corticosterone levels), and mild deficits on some sensory and cognitive tasks. Exploratory and motor functions, however, are relatively normal. The pathologic and behavioral profiles of the animals closely mimic the sensory and sympathetic aspects of familial dysautonomia.

Animals↗
PubMed1982

Endocytosis of agarose in mouse peritoneal macrophages in vitro.

We have studied the endocytosis of tritium-labelled and of non-radioactive agarose in mouse macrophages in vitro. The endocytosis was greatest and most rapid in syngeneic mouse serum and in human serum, reaching a plateau after 12 h of incubation. Ten per cent serum was the minimum concentration giving optimal ingestion. The endocytosis appeared to be regulated by mechanisms involving complement factors C3 and B. Different pretreatments of sera, inactivating or depleting C3 and B, resulted in 70-80% reduction of endocytosis. Preincubation of agarose in untreated serum increased the endocytosis of agarose in heat-inactivated serum three-fold indicating that the essential factors were bound to agarose. Antibodies against C3 and B reduced endocytosis moderately but significantly.

Animals↗
PubMed1982

The cytotoxic effect of mouse macrophages stimulated in vitro by a beta-1,3-D-glucan from yeast cell walls.

Macrophages stimulated by an insoluble beta-1,3-D-glucan from yeast cell walls were able to destroy tumour cells as measured by the release of radioactive label from prelabelled 14C-thymidine cells. Target cells were B-16 melanoma, P-815 mastocytoma, and the L-929 cell line. A significant target cell killing by macrophages stimulated by glucan was observed after 72-96 h. The cytolysis of L-929 cells was investigated in some detail. No stable soluble cytolytic factor appeared to be released into the medium during the stimulation of macrophages by glucan, since cell-free spent medium had no cytotoxic effect on L-929 cells. The densities of the macrophage monolayers were critical for an effective target cell killing; dense cultures showed more cytotoxicity than less dense cultures. The kinetics of the development of macrophage-mediated cytotoxicity suggests a minimum stimulation period of 4 days for maximal cytolysis.

Animals↗
PubMed1982

Evidence that agarose must be internalized to stimulate mouse macrophages in vitro.

Agarose stimulation of macrophages in vitro was studied. Under conditions where agarose was ingested, stimulation was detected during 24-48 h of incubation at a time when the agarose increasingly was concentrated in the perinuclear region. Removal of extracellular agarose after 24 h when endocytosis had reached a plateau did not reduce the stimulatory effect. Preincubation for 4 days with dextran sulphate in concentrations reported to inhibit phagosome-lysosome fusion potentiated strongly the stimulatory effect. In all situations in which agarose was not internalized--in teflon tubes where the cells remain in suspension, on glass cover slips with inhibitors (2-deoxy-D-glucose, cytochalasin B), or with large, noningestible Sepharose beads--no stimulation was recorded. The possibility is discussed that stimulation of macrophages by agarose may be related to complement activation in phagosomes.

Animals↗
PubMed1982

Experimental nasal infection of normal and leukopenic mice with Pseudomonas aeruginosa.

Histological and ultrastructural changes in the nasal mucosa of normal and leukopenic mice exposed to Pseudomonas aeruginosa were compared and correlated with changes in the distribution of pseudomonads by use of immunoperoxidase labeling. Pseudomonas was limited to the surface of the nasal mucosa of normal mice and was cleared rapidly. Concurrently, granulocytes were recruited across unaltered nasal epithelium and contained phagocytosed bacilli within two hours. Pseudomonas was limited to the surface of the nasal mucosa of most leukopenic mice at two hours. By four hours, pseudomonads had penetrated interepithelial junctions of all leukopenic mice. Granulocytes were not recruited and nasal epithelium underwent necrosis at points of invasion. These results show that neutrophils participate in the clearance of P. aeruginosa from the surface of the nasal mucosa and that the failure to recruit granulocytes may be important in the breakdown of epithelial barriers. Possible mechanisms of mucosal invasion are discussed.

Animals↗
PubMed1982

Spermatogenesis in the grey squirrel (Sciurus carolinensis) and changes during sexual regression.

In the testes of sexually active grey squirrels 8 stages of spermatogenesis were recognized each with a characteristic association of germ cells. The low numbers of spermatozoa in squirrel testes are the result of fewer spermatogonial divisions compared with those of most other mammals as well as a low efficiency of spermatogenesis, with only 42% of the germ cells becoming spermatozoa. A period of testicular regression which may be prolonged follows the breeding season in June/July. In fully regressed testes no stages of spermatogenesis could be recognized, the lumen of the seminiferous tubules was occluded and no stages later than primary spermatocytes were seen.

Animals↗
PubMed1982

Effect of ovariectomy on the course of gestation in the grey squirrel (Sciurus carolinensis).

In the grey squirrel pregnancy continues in the absence of the ovaries from day 16 of gestation. Since the investigation was performed on squirrels caught in the wild the stage of gestation was estimated from plasma progesterone levels, volume of corpora lutea at ovariectomy and date of parturition for those squirrels which did give birth. Three squirrels ovariectomized in early pregnancy did not give birth. Plasma progesterone levels after ovariectomy were lower than those found in normal pregnancy squirrels suggesting that the corpora lutea are a continuing source of progesterone even at a time when pregnancy can continue in their absence.

Animals↗
PubMed1982

Four methods for determining uric acid compared with a candidate reference method.

Uric acid as measured in serum by three different uricase (EC 1.7.3.3) methods (aca, Ektachem, and SMAC) and by the SMAC method with phosphotungstic acid was compared with a candidate Reference Method for uric acid. Serum specimens from 83 patients (uric acid concentrations, 19 to 141 mg/L) were analyzed by all five methods. Results were compared by using linear regression analysis, and the mean difference between results by the candidate Reference Method and the four other methods was calculated. Compared with the candidate Reference Method, the aca method gave the smallest deviation from zero for the intercept and the smallest mean difference, and the SMAC phosphotungstic acid method showed a slope closest to unity. The SMAC uricase method had the largest intercept and greatest deviation of the slope from unity.

Autoanalysis↗
PubMed1981

Progesterone concentrations in peripheral plasma of non-pregnant and pregnant grey squirrels (Sciurus carolinensis).

Plasma concentrations of progesterone in non-pregnant female grey squirrels were never greater than 3.2 nmol/l and no significant differences were found between levels in anoestrous, pro-oestrous and oestrous animals. During pregnancy, plasma concentrations of progesterone increased significantly and reached a maximum level of 318 nmol/l at around day 35 of the 44 day period of gestation. After parturition, plasma concentrations of progesterone fell sharply. The corpora lutea of pregnancy began to regress in size at about day 30 of gestation, before the maximum levels of progesterone in the plasma were reached, which suggests that there is an extra-ovarian source of progesterone. Chromatography of pregnancy plasma extracts showed that no significant amount of 5 alpha- or 5 beta-pregnane-3,20-dione was present and that progesterone accounted for 90% of the assay-positive material in pregnancy plasma from grey squirrels.

Animals↗
PubMed1981

Low results for inorganic phosphorus with the SMAC continuous-flow analyzer.

Serum inorganic phosphorus concentrations as measured with the SMAC are lower than those found with other methods. To resolve this problem we analyzed patients' specimens and performed analytical recovery studies with four different systems (SMAC, AutoAnalyzer II, aca, and the Fiske--SubbaRow method). With the SMAC, results for patients' specimens are significantly lower (p less than 0.0001) than with any of the other three methods. The SMAC recovered only about 87% of the added inorganic phosphorus. The value assigned to SMAC Reference I for inorganic phosphorus was 0.876 of the value obtained when the material was analyzed by the reference method. Thus there is a significant systematic error in the SMAC method for inorganic phosphorus determination, attributable to an erroneous inorganic phosphorus concentration assigned the SMAC calibration material by the supplier (Technicon).

Autoanalysis↗