Quantitative evaluation by computer-based video densitometry of film response to different isotopes and fluorographic treatments

Previous investigations of fluorographic methods have generally reported the minimum level of radioactivity that can be detected for a given length of exposure or the amount of radioactivity needed to reach a given optical density value. Even when the full characteristic curve is given, comparisons with other published values are difficult or, if different lengths of exposures are used, impossible. Computer-based video densitometry was used to evaluate different fluorographic methods. Sections of polyacrylamide gels with uniform distributions of either 35S or 125I radioactivity were embedded with different fluors and used to expose X-ray film for various lengths of time. Due to its compatibility with algorithms used in densitometry, the effectiveness of various treatments was evaluated by fitting relevant parameters to a modified Hill-type equation. The two parameters that were affected by treatments were the amount of radioactivity needed to half-saturate the film (ED50, or the midpoint) and the slope at the midpoint. The relationship between length of exposure and film density was investigated. Since the ED50 values for both 35S and 125I were found to vary in a linear fashion with respect to 1/time, one can determine the ED50 for lengths of exposure not reported. A unique feature of this method of evaluating effectiveness of different treatments, therefore, is that it is possible to compare films that were exposed for different lengths of time. The method of presenting data used here overcomes many of the difficulties in comparing different fluorographic and film treatments, is compatible with the needs of computer-based densitometry, and is suggested as a useful way in which to present the relationship among isotopes, fluors, and films.     

Correction for the inherent error in optical density readings.

Except at very low levels, uncorrected photometric determination of bacterial cell densities showed a decreasing proportionally to actual cell density or dry weight. A standard curve was prepared to convert photometric readings to truly proportional optical density values. With one dry weight determination, optical density values may be converted to absolute dry weight values.     

Correction for the inherent error in optical density readings.

Except at very low levels, uncorrected photometric determination of bacterial cell densities showed a decreasing proportionally to actual cell density or dry weight. A standard curve was prepared to convert photometric readings to truly proportional optical density values. With one dry weight determination, optical density values may be converted to absolute dry weight values.     

Metabolism of lipoproteins in nonhuman primates. Studies on the origin of low density lipoprotein apoprotein in the plasma of the squirrel monkey.

The plasma of squirrel monkeys contains extremely low levels of very low density lipoproteins. The delipidated apoproteins from the different lipoprotein density classes of this species show a heterogeneity similar to that of man and the rat. The biosynthesis of the apoproteins of squirrel monkey lipoproteins was studied in fasted normal and Triton WR1339-treated animals. After intravenous injection of [3-H] leucine, maximal labeling of very low density lipoproteins occurred after 1 h, intermediate density lipoproteins (d 1.006--1.019) in 2 h, and low density lipoproteins after 3 h. At all times, however, low density lipoproteins had the greatest percentage of radioactivity. Polyacrylamide gel electrophoresis revealed that the apoprotein B moiety of very low density and intermediate density lipoproteins contained 62% and 81% of the total radioactivity in these lipoproteins whereas the fast-migrating peptides were minimally labeled. In monkeys injected with Triton WR1339, 70--80% of the radioactivity incorporated into d smaller than 1.063 lipoproteins was in very low density lipoproteins with only 10--15% in intermediate and low density lipoproteins. After injection of 3-H-labeled very low density lipoproteins and [14-C] leucine into Triton-treated monkeys, catabolism of 3-H-labeled very low density lipoprotein to intermediate and low density lipoproteins was small and was significantly less than corresponding values for the incorporation of [14-C] leucine. Thus, breakdown of very low density lipoproteins could not account for all the labeled apoprotein B present in the intermediate and low density lipoprotein fractions. The results indicate that most, but not all, of the newly synthesized apoprotein B enters plasma in very low density lipoproteins and that the low concentrations of this lipoprotein in squirrel monkey plasma are a consequence of its rapid turnover.     

RETENTION OF DISSOLVED COMPOUNDS BY MEMBRANE FILTERS AS AN ERROR IN THE 14C METHOD OF PRIMARY PRODUCTION MEASUREMENT1

Membrane filters retain ¹⁴C bicarbonate, ¹⁴C glycollate, and other ¹⁴C labeled substances from filtrates of algal cultures and lake water. By refiltering different volumes of the filtrate from algal cultures and from lake water after incubation with ¹⁴C bicarbonate, it was shown that the labeled material retained was not proportional to volume but showed a saturation effect with increasing volume filtered. When the radioactivity retained by a filter is divided by the volume filtered, decreasing values are obtained with increased volume filtered. This radioactivity may represent a significant addition to the radioactivity in particulate material on the filters, resulting in a similar type of curve when different volumes of lake water or cultures are filtered. Values of radioactivity per milliliter were constant using Chlorella in Chu 10 medium. However, the curve could, be obtained by increasing pH and bicarbonate concentrations in the medium and on resuspending the algae in Lake Ontario (winter) filtrate. The values of cpm/ml retained from filtrates were low in Cryptomonas cultures and the curve was not obtained unless population density was reduced, thus increasing the relative contribution to the radioactivity on the filters. The curve was not always obtained in lake water. It was significant in 10 out of 14 experiments in Lake Ontario and in 2 out of 5 experiments in Grenadier Pond. Changes in lake water rather than in experimental techniques were probably responsible. On 2 occasions when values of cpm/ml were constant in Lake Ontario, addition of sodium bicarbonate without a pH change resulted, in a significant curve. Our experiments do not disprove the possibility of cell damage during Millipore filtration, however, it has been shown that ¹⁴C labeled substances retained from solutions can account for the entire range of decreasing values as a function of volume filtered.     

Selective determination of mRNA specific radioactivity in HeLa cells without the use of inhibitors

Polysomes from (³H)-uridine pulse-labeled HeLa cells were isolated and the specific radioactivity of polysome-associated mRNA was determined by selective enzymic hydrolysis at 0°C of the $\text{inter}$ribosomal mRNA sections. $\text{Intra}$ribosomal mRNA protected from hydrolysis during ribonuclease treatment and subsequently isolated by the proteinase K method (1) exhibited the same specific radioactivity as the interribosomal mRNA split products.When labeled polysomes were subjected to ribonuclease treatment at 25°C instead of 0°C a higher specific radioactivity of the interribosomal split products resulted, while intraribosomal sections still exhibited the same values as after 0°C treatment. The labeled polysomes used as substrate exhibited one single A₂₆₀ and radioactivity peak in CsCl density gradients. No RNP material banding at ? = 1.35 ? 1.45 could be detected. However, the radioactivity maximum banded at slightly lower densities than the A₂₆₀ peak (? = 1.55 versus 1.57). The shift appears to be caused by a contaminant RNA. These findings as well as the radioactivity pattern of pulse-labeled polysomes in sucrose gradients may indicate the presence of newly synthesized mRNA associated with monosomes (and oligosomes) protected from ribonuclease action at 0°C by (transport?) proteins.     

Measurement of cell proliferation by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine

An ELISA was developed and optimized to measure cell proliferation using a monoclonal antibody to bromodeoxyuridine (BrdUrd). Incorporation of BrdUrd into myoblast monolayers, measured as the optical density at 492 nm, increased in response to fetal calf serum, IGF-I and EGF, the ELISA data correlated closely with data obtained by BrdUrd immunocytochemistry (r = 0.984), cell counting (r = 0.972) and tritiated thymidine uptake by liquid scintillation counting (r = 0.990). The BrdUrd ELISA is a useful alternative to measurement of tritiated thymidine uptake by scintillation counting, and has the added advantages of dispensing with the use of radioactivity and of being less labour intensive.     

MICROSCOPIC DETERMINATION OF BONE PHOSPHORUS BY QUANTITATIVE AUTORADIOGRAPHY OF NEUTRON-ACTIVATED SECTIONS

Longitudinal sections of human cortical bone were submitted to thermal neutrons. γ-ray spectra were recorded repeatedly during 15 days following irradiation. They showed that Na²⁴ is predominant as early as 3 hours after activation and that all the γ-emitters have decayed on the 15th day. When the γ-rays have disappeared, β-rays are still produced by the sections. It was proved by the absorption curve in aluminium that all these β-rays are issued from the P³² induced in the sections by activation of P³¹. Therefore autoradiograms registered 15 days after activation reveal the distribution of P³² in the sections. γ-ray spectra and β-ray absorption curves of neutron activated sections of ivory demonstrated a mineral composition similar to that of bone. Autoradiograms of ivory sections activated for various times were used to establish the relation between the optical density of the autoradiograms and the radioactivity in P³². When the bone autoradiograms are compared with the ivory standards of known radioactivity, the optical densities of single osteons (Haversian systems), can be related to their phosphorus contents. Autoradiograms and microradiograms of the same sections were examined side by side. The least calcified osteons, that contain 80 per cent of the calcium of the fully calcified osteons, also contain about 80 per cent of the phosphorus of the fully mineralized osteons. It is concluded that the Ca:P ratio remains constant while mineralization of bone tissue is being completed.     

Gibberellic acid—Binding proteins from pea stems

Summary The formation of complexes of gibberellic acid (GA₃) and proteins under in vitro conditions was studied. It was shown that labelled GA₃ binds to soluble cytoplasmic proteins, although a considerable amount of radioactivity remains in the pellet containing nuclei and cell debris. GA₃-protein complexes are excluded from Sephadex G-10 column with the void volume. They sediment in linear sucrose density gradients as three distinct peaks, having higher S values than bovine serum albumin, used as a marker. Soluble GA₃-protein complexes can be separated into four zones of radioactivity upon ion exchange chromatography on DEAE-Sephadex A-50 column, each of them eluting with a different KCl concentration. Agarose gel electrophoresis of GA₃-protein complexes reveals two zones of radioactivity at the anodic part of the electrophoretogram. After extraction of the complex with ethanol, more than 90% of radioactivity is found in the ethanolic phase, which indicates that the binding is not covalent. GA₄₊₇ and GA₁₃ decrease the binding of GA₃ to cytoplasmic proteins for 30%, suggesting that some common binding sites exist at the same binding proteins.Abbreviations BAP benzylaldenine - GA gibberellic acid - IAA indolyl-3-acetic acid - TSS tris-sucrose-salts     

Incorporation of [3H]glucosamine into glycosaminoglycans in different cell culture conditions by rabbit aortic smooth muscle cells

This study sought to elucidate the optimal cell culture conditions for studies concerned with the incorporation of [³H]glucosamine into glycosaminoglycans by rabbit aortic smooth muscle cells. The incorporation of radioactivity into extracellular sulphated glycosaminoglycans was linear for at least 72 h and that into pericellular sulphated glycosaminoglycans for up to 24 h. The incorporation of radiolabel into hyaluronic acid was linear only up to 12 h. In the exponential growth phase the incorporation of [³H]glucosamine into sulphated glycosaminoglycans and hyaluronic acid proved to be less marked than in the stationary growth phase, but the highest values were nevertheless obtained immediately after trypsinisation. When studied in the stationary growth phase, cell density and incorporation of [³H]glucosamine were positively correlated in the case of hyaluronic acid, but in the case of sulphated glycosaminoglycans there was a negative correlation. The serum concentration of the incubation medium and the incorporation of radioactivity into hyaluronic acid were positively related. With sulphated glycosaminoglycans this was the case only after a 7-day preincubation in the different serum concentrations. when incorporation was studied without preincubation, the incorporation of radioactivity into sulphated glycosaminoglycans proved to be negatively associated with the serum concentration of the medium. The environmental pH of the cells was associated with the incorporation of radioactivity into hyaluronic acid and sulphated glycosaminoglycans in that between pH values 6.8 and 7.9 the incorporation of radioactivity increased when the pH of the medium was raised.     

Characterization of the Rapidly Labeled Hybridizable RNA Synthesized in L5178Y Mouse Leukemic Cells: Hybridization Lifetime Studies

An attempt is made to characterize the rapidly labeled hybridizable RNA of L5178Y mouse leukemic cells which has been shown to have similar base sequences when synthesized in two different stages of the cell cycle. The size of rapidly labeled RNA molecules was heterogeneous. For labeling times of 20 min or less, the per cent of hybridization was maximal. With longer labeling times, the per cent of hybridization decreased as radioactivity appeared in long-lived species of low hybridization efficiency; the radioactivity profile resembled the optical density profile in sucrose gradients. The lifetime of newly synthesized hybridizable RNA was studied by pulse labeling exponentially growing cells and then “chasing” with nonradioactive uridine. The per cent of hybridization was studied as a function of chase time. Three RNA groups, which comprised different proportions of rapidly labeled hybridizable RNA, were distinguished. The short-lived group had a half-life of 10 min, much less than the values reported in the literature for messenger RNA of mammalian cells. The half-life of 1-1½ hr observed for a medium-lived group more closely corresponds to that of messenger RNA. A long-lived group had a half-life of approximately 20 hr. Specific activity measurements during chase indicate the presence of a “pool” of labeled uridine derivatives. The uridine of this pool appears to be nonexchangeable with but dilutable by exogenous uridine. A nontoxic concentration of actinomycin D was added to the chase media in an attempt to block the “pool effect”. A rapidly degradable RNA was demonstrable both by specific activity and per cent of hybridization measurements.     

Intrahepatocellular site of the catabolism of heme and globin moiety of hemoglobin-haptoglobin after intravenous administration to rats

The intracellular site of incorporation and degradation of heme and globin moiety of hemoglobin-haptoglobin in rat liver cells was investigated in vivo. Hemoglobin-haptoglobin, administered intravenously to rats, is cleared from the circulation and incorporated exclusively into liver parenchymal cells through the receptor specific for the molecule (Kino, K., Tsunoo, H., Higa, Y., Takami, M., Hamaguchi, H., and Nakajima, H. (1980) J. Biol. Chem. 255, 9616-9620). Intracellular distribution of radioactivity was determined after intravenous administration of [3H-Heme,14C-Globin]hemoglobin-haptoglobin to rats. The doubly labeled hemoglobin-haptoglobin was incorporated first in organelles of lower anodic mobility in carrier-free electrophoresis and of low density (density range, 1.05-1.07 g/ml) in Percoll density gradient centrifugation recovered in Golgi subfractions of the liver cells in a substantially intact form. In the subsequent stages, these organelles progressively acquired a higher anodic mobility as well as higher density, presumably through fusion with other organelles. In the resulting organelles of higher anodic mobility in electrophoresis and high density (density range, 1.07-1.15 g/ml) in Percoll, the hemoglobin-haptoglobin first dissociated symmetrically into two 82,000-dalton subunits having intact heme, and then the organelles containing only 3H radioactivity but no 14C radioactivity were separated by electrophoresis. Most of the 3H radioactive materials in these organelles are identified as intact [3H]heme. These investigations suggest that the heme moiety of hemoglobin-haptoglobin in the organelles is detached from globin-haptoglobin and binds to another carrier protein prior to conversion of heme to bilirubin.     

Bioluminescence Assay for Measuring the Number of Bacteria Adhering to the Hydrocarbon Phase in the BATH Test

A thorough validation of the bacterial adherence to hydrocarbons (BATH) test was performed by means of a bioluminescence assay. Ten different gram-negative strains were subjected to the BATH test. For the calculation of the adhesion index, several factors had to be taken into account: ATP leakage, the action of ATP-hydrolyzing enzymes, the change in the extraction efficiency of Nucleotide-Releasing Reagent for Microbial Cells (NRB; Lumac bv) after vortexing and the difference in light production after the addition of NRB. When the adhesion index values obtained by bioluminescence measurement were used as reference, the total plate count technique appeared to be unreliable in estimating the number of bacteria adhering to the hydrocarbon phase. A highly significant correlation was established, however, between those reference values and the adhesion index values obtained by the optical density reading for octane and especially for hexadecane. With xylene, no correlation was found between the optical density reading values and the total plate count or bioluminescence values.     

Synthesis of glycoproteins in the Golgi complex of the mouse gallbladder epithelium during fasting,refeeding, and gallstone formation

Summary The mouse gallbladder epithelium was studied with light microscopic autoradiography and quantitative electron microscopy during fasting, refeeding and experimental gallstone formation. To determine the intracellular pathway of glycoproteins, H³-galactose was injected at different time intervals into the mice. At 10, 25 and 40 min after an intraperitoneal injection the gallbladders were fixed and prepared for light microscopy. As early as 10 min after injection, label was observed in supranuclear cytoplasmic regions and at 25 min, an increased radioactivity was present throughout the apical cytoplasm. At 40 min, silver grains were mainly present at the cell surface. Autoradiographs processed 25 min after an intraperitoneal H³-galactose injection after fasting for 48 h showed decreased supranuclear and apical radioactivity. After refeeding (12 h) there was an enhanced activity in both these regions. Animals fed a lithogenic diet for one month showed a marked increase of radioactive label mainly in cells of crypts and invaginations of the mouse gallbladder mucosa.Morphometric measurements of the Golgi apparatus revealed that deprivation of food significantly diminished the volume density of the Golgi apparatus. Refeeding the animals restored the volume density values to normal levels. In the course of gallstone formation there was a further significant increase in the volume density of the Golgi complexes as compared to controls.     

Secretion of apolipoproteins in very low density and high density lipoproteins by perfused rat liver

The incorporation of labeled amino acids into the peptides of very low density lipoproteins (VLDL) and high density lipoproteins (HDL) secreted by perfused rat liver was studied using a Ringer-albumin solution in the perfusate in place of serum to diminish exchange of peptides between VLDL and HDL. Among the lipoproteins, the greatest release of protein, greatest incorporation of amino acid, and highest specific activity were found in VLDL. After separation of the delipidated peptides by electrophoresis on polyacrylamide gel, the incorporation into VLDL peptides was found to be 5-10 times as great as into HDL peptides. There was virtually no incorporation into the peptides of low density lipoproteins (LDL). Approximately 25% of the radioactivity incorporated into perfusate VLDL failed to enter the 13% polyacrylamide gel. The remaining radioactivity was distributed primarily among three peptide bands; one, found in the upper portion of the gel, contained 45% of the total, most of the remainder being found in two rapidly migrating bands. These three peptides appear to approximate those of human apo-C in relative electrophoretic mobility. Most of the HDL peptide radioactivity entering the running gel was found in a band that migrates slightly faster than the main VLDL band. A portion of the radioactivity of this major HDL band did not enter the running gel unless beta-mercaptoethanol was present. Greater separation of these two bands by polyacrylamide gel electrophoresis for 24 hr confirmed that the major bands in VLDL and in HDL were different. The rapidly moving peptides of HDL were found to contain very little radioactivity. Determination of the intensity of staining of carrier-free perfusate VLDL and HDL peptides produced a pattern similar to the incorporation of labeled amino acids. It is concluded that the rapidly moving peptides, which may contain activators of lipoprotein lipase, are only secreted as part of the VLDL.     

Development of mathematical models (Logistic, Gompertz and Richards models) describing the growth pattern of Pseudomonas putida (NICM 2174)

Bacterial growth curve, which is asymptotic after a certain period, is described using three different mathematical models, namely, Logistic model, Gompertz model and Richards model. The equations for these three models are fitted by evaluating the mathematical parameters involved in these models. This is done by applying the method of partial sums to the data in Table 1 containing the optical density values for different cell mass at different time intervals. The sum of square of residuals between the expected optical density values and the experimental values is calculated for each of these models. In the cases tested, the Logistic model was found to be the best fit for the growth curve of Pseudomonas putida (NICM 2174) and was found to be easy to use. These results fit the data very well at 5% level for more than 70% of the readings.     

Human chylomicron apolipoprotein metabolism.

Chylomicron apolipoprotein metabolism was studied utilizing chylomicrons isolated from the pleural fluid of a patient with a recurrent chylous pleural effusion. Chylomicrons contained apolipoproteins A-I, A-II, B, C-I, C-II, C-III, D, E, and albumin. Following intravenous injection of [¹²⁵I] chylomicrons, almost all of the A apolipoprotein radioactivity was recovered in high density lipoproteins, while only a small amount of the B apolipoprotein radioactivity was recovered in low density lipoproteins. These observations indicate that intestinal chylomicron A apolipoproteins serve as precursors for plasma high density lipoprotein A apolipoproteins and only a small fraction of chylomicron apolipoprotein B is metabolized to form low density lipoprotein apolipoprotein B.     

Effects of CDPcholine and CDPethanolamine on the Alterations in Rat Brain Lipid Metabolism Induced by Global Ischemia

Abstract: The fast turnover pool of rat brain lipids was labeled by intracerebral injection of [³H]acetate. Cerebral ischemia for a duration of 5 min after decapitation caused a 2.2-fold increase in radioactivity in the free fatty acids and loss of more than 20% of the radioactivity from choline and ethanolamine glycerophospholipids. An intracerebral injection of 0.6 μmol each of cytidine diphosphocholine (CDPcholine) and cytidine diphosphoethanolamine (CDPethanolamine) prevented the loss of radioactivity from the glycerophospholipids and decreased the amount of radioactivity in the free fatty acids by 59% as compared with control values and 82% as compared with ischemia values. By GLC assays of the mass of the free fatty acids, there was a threefold increase of free fatty acids in ischemic brains. Pretreatment of ischemic brains with CDPcholine and CDPethanolamine reduced the levels of unesterified fatty acids to 60% of the control values. Thus, a prior injection of cytidine nucleotides prevented the release of free fatty acids observed in ischemic brains.