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PubMed1978

Small intestinal epithelial renewal in the Syrian hamster exposed to cholera enterotoxin.

Epithelial renewal of the small intestine was measured in the Syrian hamster utilizing tritiated thymidine by standard autoradiographic and a scintillation counting technique. Scintillation counting of intestinal replicates proved to be as accurate as standard autoradiography. Average mucosal cell turnover was 71 +/- 3.0 hr in jejunum and 79 +/- 4.2 hr in ileum. Scintillation counting was utilized to study the effect of a maximum cholera enterotoxic secretory stimulus on small intestinal mucosal cell turnover. No significant change in epithelial cell migration occurred during cholera enterotoxin (CT)-induced fluid and electrolyte secretion. The rate of decline in radioactivity as a measure of cell turnover in CT-exposed animals was no different from controls. Epithelial cell proliferation 1 to 42 hr after CT exposure showed no difference from controls. Intestinal fluid and electrolyte secretion persisted for 24 hr after CT exposure. It is concluded that (1) the small intestinal epithelial cell migration was unaltered by this metabolic secretory stimulus, and (2) the data are consistent with the concept that epithelial migration after CT exposure was one factor, although not necessarily the major determinant of the progressive decline in intestinal secretory activity.

Animals↗
PubMed2001

Characterization of the binding of delmopinol to salivary precipitates.

The aim of this study was to quantify the amount of delmopinol recovered in different molecular weight ranges of salivary proteins in the supernatant and pellet of delmopinol-saliva mixes. Unstimulated whole saliva was collected from 5 subjects and mixed with radiolabeled delmopinol to obtain a final drug concentration of 9.7 mM. The salivary-delmopinol solutions were incubated and then centrifuged. The resulting pellets and supernatants were studied in an electrophoresis assay. Each individual sample was run in three different electrophoretic gel lanes. The first lane was stained with silver dyes; the second lane was used for electrophoretic blotting and autoradiography; the third lane was cut in standard slices that were dissolved and analyzed with scintillation counting. The scintillation results demonstrated that higher radioactivity levels were detected at high molecular weight ranges (700-600 kDa). Furthermore, pellet samples were found to contain the highest amounts of delmopinol. The autoradiography results confirmed that delmopinol was bound to proteins of high molecular weight (700-600 kDa).

Adult↗
PubMed1975

Axoplasmic transport in the toad Bufo marinus.

The rate and course of axoplasmic transport from the eighth dorsal root ganglion cell bodies into the sciatic nerve of the toad Bufo marinus were studied. Concentrated tritiated proline was hydraulically injected into a surgically exposed dorsal root ganglion of animals maintained at 19 +/- 0.5 degrees C. At postinjection intervals of 1, 6, and 10 h, the animals were sacrificed and the dorsal root, ganglion, and sciatic nerve were removed bilaterally. The dorsal roots and peripheral nerves were cut into 3 mm segments measured from the ganglion. In some experiments all tissues were prepared for liquid scintillation counting techniques. In others the ganglion and every fifth 3 mm nerve segment were fixed in Bouin's fixative for radioautography, and the remaining tissue segments were prepared for liquid scintillation counting methods. Scintillation counts (counts/min) of consecutive segments along the labeled nerve were plotted against distance (mm) for each animal. Examination of these profiles showed a peak of radioactivity in the injected ganglion for each animal that was followed distally by an abrupt drop in the adjacent segments of the nerve. Radioactivity remained relatively stable in subsequent segments forming a plateau and then dropped to baseline levels forming a wavefront in the distal portion of the peripheral nerves of the 6 and 10 h toads. Movement of this wavefront during the 6 to 10 h time interval provided evidence for an axoplasmic flow rate of about 120 mm/day. Radioautographs of the ganglion and representative segments along the sciatic nerve were examined with both bright- and dark-field microscopy. Accumulations of silver grains were observed overlying the injected ganglion cell bodies and labeled axons of the nerve. Values for the number of silver grains/unit area were obtained from dark-field radioautographs of the nerve segments through the use of a computer-microscope system. The relative amount of radioactivity present in the axons was thereby demonstrated. Radioautographic data confirmed the axoplasmic flow rate of about 120 mm/day determined by liquid scintillation counting methods for the bulk of radioactive materials transported at this fast rate. In addition, the evaluation of radioautographic data suggests that an even faster flow rate of 185-215 mm/day may exist for a small portion of the labeled materials transported in the axon.

Animals↗
PubMedDate not supplied

The absolute standardization of 32P and 204Tl at LNMRI.

32P and 204Tl solutions were standardized within the frame of the international key comparisons organized by bureau international des poids et mesures, in 2002. The activity concentration of 32P was measured by counting solid sources in a 4pibeta proportional gas flow counter and by liquid scintillation counting. The self-absorption in solid sources for 4pibeta counting and the presence of 33P as an impurity were evaluated. The combined standard uncertainty for 32P was 0.59% in the 4pibeta counting and 0.38% in the liquid scintillation counting. Liquid scintillation counting was used to measure the activity concentration of 204Tl with combined standard uncertainty of 0.35%.

Brazil↗
PubMed1991

Dot-blot hybridization: quantitative analysis with direct beta counting.

The suitability of using direct beta counting (DBC) for quantitating radioactivity of the probe:target complex in dot-blot hybridization was evaluated using a Packard Matrix 96. A comparison of blots analyzed using autoradiography followed by densitometry scanning (film/densitometry) with those analyzed using direct beta counting revealed similar data trends with the two methods. However, direct beta counting quantitated the amount of radioactivity in the dot blots directly (without film exposure or additional sample preparation), which significantly reduced the time required to obtain results. Blots analyzed first with direct beta counting and then liquid scintillation counting exhibited similar data trends with both methods. Despite a decreased counting efficiency, analysis with direct beta counting has the following advantages compared with liquid scintillation counting: 1) no additional sample preparation is required (no vials or cocktail are used), 2) no sample destruction occurs due to analysis and 3) quantitative results are obtained more rapidly (since the radioactivity for all 96 samples in a dot blot is simultaneously determined in real time). Analysis with direct beta counting was also shown not to interfere with the successful reprobing of stripped dot blots with either unique sequence or total genomic probes. Overall, direct beta counting provides quick, quantitative results for dot blots while saving considerable time and effort.

Animals↗
PubMed1999

Degradation of basement membrane by prostate tumor heparanase.

BACKGROUND: The degradation of basement membrane (BM) by cancer is an important event that characterizes invasive biological behavior. A component of BM is heparan sulfate proteoglycan (HSPG). The glycanase(s) that degrade HSPG in BM are not yet isolated. We recently identified HSPG-degrading activity (PC-3M heparanase) in the conditioned media (CM) of malignant prostate carcinoma cells (PC-3M and LNCaP C4-2). Antibodies (Abs) to a recently isolated heparanase from human platelets (CTAP-III), cross-react with PC-3M heparanase although they differ in size; under reduced conditions PC-3M heparanase is 60 kDa whereas CTAP-III is 10 kDa by polyacrylamide gel electrophoresis. PC-3M heparanase therefore shares homology with CTAP-III. The purpose of this study was to test the inhibition of PC-3M heparanase by Abs specific to the N- and C-terminals of CTAP-III. MATERIALS AND METHODS: CM from PC-3M and LNCaP C4-2 cells were tested for heparanase activity. Each reaction contained substrate as [3H]glucosamine-labeled HSPG (>50 kDa) from the BM of the EHS tumor, CM from PC-3M or LNCaP C4-2 cells, and inhibitor or buffer (negative control). Protease inhibitors were present throughout. After incubation for 3-20 h at 37 degreesC and pH 5.8, the reaction was stopped with 0.2% SDS. Each reaction mixture was centrifuged in an Ultrafree-MC 30,000 NMWL filter unit (Millipore) and radioactivity in the filtrate counted by scintillation counting. Results. For both cell lines, there was a linear relationship between the amount (microgram) of CM and degradation of HSPG. Degradation was inhibited by 54.1% (mean) using carrageenan lambda (10 microgram/ml), a nonspecific glycanase inhibitor (P < 0.05 by ANOVA). Ab to the N-terminus of CTAP-III (anti-Hep A) reduced degradation by 10-50% (mean 31.1%) and to the C-terminus (anti-Hep C) by 38.8-64.3% (mean 51.1%) (P < 0.003 by ANOVA). CONCLUSIONS: The degradation of HSPG by malignant prostate cancer cell lines is inhibited by both a nonspecific glycanase inhibitor, and specific Abs to a homologous platelet heparanase. Based upon molecular weight, PC-3M heparanase is different from platelet heparanase and degrades BM.

Amino Acid Sequence↗