Megalodiscus temperatus: absorption and incorporation of tritiated tyrosine, thymidine, and adenosine

Autoradiographic and liquid scintillation counting studies indicate adults of Megalodiscus temperatus can absorb large amounts of thymidine and adenosine, but use little of the thymidine in in vitro exposures of up to 72 hr. Labeling of worms exposed to ³H-adenosine in periods as short as 3 hr was achieved especially over young eggs, testes, and the ovary. Large amounts of tyrosine are found in the free amino acid pool of this worm but little is incorporated into tissues for exposure periods of up to 24 hr. Ligation experiments and autoradiograms of frozen-dried worms show thymidine and adenosine can enter via the tegument. Adenosine added to the exposure medium did not inhibit absorption of tyrosine but did markedly reduce the uptake of ³H-thymidine in 8-hr in vitro exposures. Bacteria adhering to the glycocalyx of M. temperatus were identified as Escherichia coli, but their role in uptake phenomena is unknown. The association of thymidine with the lymph vessels indicates a circulatory or excretory role for this system in M. temperatus. Adenosine was considered the best compound of the three to use for labeling cells for studies on the reproductive system.     

Brugia pahangi: uptake and incorporation of adenosine and thymidine.

The uptake and incorporation of adenosine and thymidine by infective larvae, 10-day-old juvenile, and adult stages of Brugia pahangi were investigated using scintillation counting and autoradiographic techniques. No evidence of thymidine incorporation by the worm was obtained in this study. Scintillation counting methods demonstrated that ¹⁴C-labeled adenosine was incorporated by all three stages of this filarial worm. Autoradiography, performed on worms incubated in [³H] adenosine from 5 min through 2 hr, revealed that following 5–15 min incubation the greatest degree of adenosine incorporation occurred in the hypodermis and somatic cords. Adenosine incorporation into the deeper body tissues, including the gut, increased significantly with longer periods of incubation. The results obtained further support the concept that nutrient uptake in B. pahangi occurs by a transcuticular route.     

Echinostoma caproni: mating behavior and the timing of development and movement of reproductive cells

The development and movement of reproductive cells were determined in Echinsostoma caproni on autoradiograms by labeling nuclei of stem cells during exposure to [3H]thymidine and then transplanting the worms to mice for various times. The development and movement of sperm, primary oocytes, and vitelline cells were much more rapid in E. caproni than other digenetic trematodes investigated previously. Mating behavior was determined by labeling the sperm of 1 adult by in vitro exposure to [3H]tyrosine and transplanting alone or with unlabeled worms to mice for 4 and 6 days. Echinostoma caproni adults self-inseminated when isolated and self- and cross-inseminated when in groups. This behavior is similar to that found for the frog rectal fluke Megalodiscus temperatus but unlike that determined for eyeflukes in the genus Philophthalmus. Worm size was not a barrier to insemination in E. caproni. Cross-insemination could not be detected in 6-day transplants probably because of dilution or elimination of radioactive sperm due to rapid turnover or frequent sperm transfer. An increased number of structural anomalies was noted in the transplanted worms. The most common anomaly was an accumulation of vitelline cells in the vitelline reservoir and ducts.     

The uptake in vitro of dyes, monosaccharides and amino acids by the filarial worm Brugia pahangi.

The uptake in vitro of various substances by Brugia pahangi was investigated using infective larvae obtained from Aedes aegypti and worms removed from Meriones unguiculatus at 2, 3, 10, 20 and 90 days post-infection. Worms incubated in growth medium 199 containing 1% Trypan blue possessed demonstrable dye in the oral orifice, the anterior oesophageal lumen and the external openings of the vulva and the cloaca or anus but the dye was not found in the gut lumen even after incubation for 24 h. No uptake of ferritin particles into the intestine of the worms was found and no fluorescence could be demonstrated in the gut lumen of worms incubated in medium containing 50% (v/v) fluorescein isothiocyanate-conjugated calf serum for up to 24 h. Trypan blue uptake by the gut of Aspiculuris tetraptera was clearly observed after incubation for several hours. The uptake of D-glucose and L-leucine by B. pahangi was demonstrated using autoradiographic and scintillation counting techniques and incorporation into worm tissues was detected. Glucose was found to be readily incorporated in the apical, glycogen-rich areas of the myocytes of worms of all ages studied and in the uterine epithelium of the adult female. In contrast, a lower incorporation of D-glucose was found in the eggs, embryos and vas deferens and especially in the gut. The incorporation of L-leucine occurred throughout the tissue of the worms during a 30 min incubation. Labelling was also located over the surface of the cuticle of the worms, when incubated for a period of 15 to 60 min in L-[H]leucine. Scintillation counting techniques demonstrated that there was no uptake of 14C-labelled L-glucose or sucrose by B. pahangi. The data presented on the uptake in vitro of nutrients or other compounds by infective larvae and adult stages of B. pahangi did not demonstrate an intestinal route of uptake but indicated that the transcuticular route of uptake may be employed.     

Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasite's behavioral and nutritional status.     

Schistosoma mansoni: cholesterol uptake by paired and unpaired worms

Mature males and females of Schistosoma mansoni were incubated for 24 hr in medium containing [3H]cholesterol. Worms thus labeled were paired for 24 hr with unlabeled partners, in vitro or in vivo by surgical implantation into hamsters. Controls consisted of additional unlabeled worms that did not pair. Scintillation counting of thin layer chromatographic separations of lipid extracts of schistosome tissues and of the culture medium indicated that [3H]cholesterol underwent no major metabolic changes during the course of the experiment. During the period of time allowed for pairing, labeled worms lost up to 65% of their [3H]cholesterol, which was detected in the pairing medium. In both unlabeled males and females which had paired with labeled partners, levels of [3H]cholesterol were higher than in unpaired controls. This suggests that normal cholesterol transfer in worm pairs is bidirectional and that it is facilitated by physical contact between juxtaposed membranes. Cholesterol exchange in schistosome worm pairs may be partly or wholly a consequence of normal tegumental turnover of the molecule.     

Studies on the reproductive system of Hymenolepis diminuta using autoradiography and transplantation.

Adult Hymenolepis diminuta exposed in vitro for 3 hr to 3H-thymidine showed incorporation of the isotope on autoradiograms over nuclei of actively dividing cells in the testes, ovary, vitellaria, and developing embryos in the eggs. Timing studies utilizing labeled worms transplanted to uninfected hosts showed that it took 18 hr for spermatogonia to develop to primary spermatocytes, 24 hr to secondary spermatocytes, 36 hr to spermatids, and 48 hr to sperm bundles. Self-insemination was confirmed in single worm transplants of 3 days by the presence of labeled sperm in the seminal receptacles. In multiple worm transplants labeled worms inseminated themselves in each case and cross-inseminated with 92% of the unlabeled worms present.     

Rate of incorporation of [3H]thymidine in various tissues of the mouse. A basis for the evaluation of genotoxic effects of chemicals

[3H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible. The present paper deals with some practical aspects on the use of [3H]thymidine in vivo. [6-3H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and DNA-associated radioactivities. A rapid increase of the [3H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities associated with DNA closely correlated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [3H]thymidine incorporation into DNA.     

Diurnal migration of Echinostoma caproni (Digenea: Echinostomatidae) in ICR mice

Twenty-four female ICR mice, 12 acclimated to a 12 ∶ 12 light-dark cycle and 12 to a 12 ∶ 12 dark-light cycle for 7 days, were each infected with 10 metacercariae of Echinostoma caproni. Infected mice were maintained on their respective lighting regimes for 28 days. Six mice (3 from each group) were necropsied at 4-hr intervals beginning at 0700 hr. The small intestine was removed, opened, and the position of individual worms and worm clusters was measured to the nearest 0.1 cm. Each intestine was subsequently divided into 20 equal segments and individual worms and worm clusters were assigned to the appropriate segment based on the original measurements. All worms were found in the posterior 55% of the intestine (ileum). All posterior segments (10-20), with the exception of segment 18, harbored at least 1 worm at some time. A Monte Carlo simulation of worm abundance in segments 10-17 over all time periods indicated a random distribution, while the same analysis of segments 10-20 indicated a non-random distribution due to large numbers of worms in segment 20 and to the absence of worms in segment 18. To analyze temporal changes in worm distribution, mice were grouped by time of necropsy as follows: night (1900 and 2300 hr), morning (0300 and 0700 hr), and day (1100 and 1500 hr). During the night and morning, E. caproni was heavily concentrated in segments 10-17 and, during the day, worms were located more posteriorly, with a heavy concentration in the last segment (20).     

Macrophage-lymphocyte clusters in the immune response to soluble protein antigen in vitro: III. DNA synthesis of lymphocytes in clusters

The lymphocytes which engage in DNA synthesis during the in vitro immune response to PPD (purified protein derivative of tuberculin) were studied by scintillation counting and in autoradiographs prepared from cultures of macrophages and immune T-lymphocyte-enriched lymphocytes. The lymphocytes in these cultures were located in three compartments: lymphocytes in macrophage-lymphocyte clusters, lymphocytes attached to macrophages but not involved in clusters, and not macrophage-attached lymphocytes. One of the cluster cells, the central lymphocyte, which is attached directly to the macrophage, was identified as the only DNA-synthesizing lymphocyte in the cluster early in the culture period. In cultures extended beyond 20 hr the increase in percentage of DNA-synthesizing lymphocytes in the cluster and macrophage-attached compartments exceeded the increase in the compartment of not macrophage-attached lymphocytes. The total amount of radiolabeled thymidine incorporated into lymphocytes in a blast transformation assay was directly proportional to the number of macrophage-lymphocyte clusters produced by the same lymphocytes in a cluster assay.     

Rate of Incorporation of R3hlthymidine In Various Tissues of the Mouse A basis for the evaluation of genotoxic effects of chemicals

Abstract. [³H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [³H]thymidine in vivo. [6-³H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [³H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [³H]thymidine incorporation into DNA.     

Autoradiographic study of protein and RNA formation during early development of Drosophila eggs

Permeabilized eggs of Drosophila melanogaster were incubated in tritiated uridine, valine, and phenylalanine. The uptake and incorporation into TCA-insoluble material were measured by scintillation counting. There was very little incorporation of uridine before the blastoderm stage. At the blastoderm stage, the egg took up 2.4 pmoles/hr of uridine and incorporated 0.13 pmoles into RNA (assuming no dilution of specific activity of the precursor). The uptake of amino acids varied with the age of the embryo; virgin eggs synthesized about as much protein as fertilized eggs. Autoradiography of eggs incubated in uridine showed a lack of RNA synthesis in nuclei until the start of the blastoderm formation. The small amount of uridine incorporation before this stage was due to mitochondria. Incorporation of amino acids was uniform in the cytoplasm until the blastoderm; there was no incorporation by yolk granules. Regional difference in labeling appeared during gastrulation. The pole cells did not form RNA during the blastoderm stage, formation started during gastrulation. Protein labeling of the pole cells, on the contrary, was very strong in the blastoderm and early gastrula. These results indicate that the expression of zygotic genome before the blastoderm stage is unlikely.     

Trichinella spiralis: mediation of the intestinal component of protective immunity in the rat by multiple, phase-specific, antiparasitic responses.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.     

Detection of Plutonium Contamination in Humans by the Autoradiographic Method

The autoradiographic technique, using 5 μ paraffin sections in contact with 5 μ NTA emulsion for 10 hr. and similar sections of sputum for 3 days, was found to be more sensitive for detecting contamination with Pu²³⁹ than tests with a scintillation counter. Both human and pig skin were tested. The autoradiograms showed characteristic alpha tracks in the emulsion at sites of Pu deposition following its uptake either in solution or in particulate form. Autoradiography is recommended as a routine method to supplement information gained from chemical analyses and counting procedures.     

Schistosoma mansoni: patter of release of secretory products.

Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, N-acetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems.     

The Measurement of Cell Turnover Rates Using Stable Isotope-Labelled Thymidine: Experiments On the Small Intestine and On A Transplantable Lymphatic Tumour of the Mouse

Multilabelled, non-radioactive thymidine and tritiated thymidine were incorporated simultaneously into the DNA of a rapidly-growing, transplantable mouse lymphoma and the DNA of mouse small intestine by a single intraperitoneal injection of both labelled nucleosides. Mice were killed at selected times during the next 7 days, and the specific activities of the tissues and extracted DNA and the 132/126 mass ratio of thymine isolated from the DNA were determined by scintillation counting and by field ionization mass spectrometry respectively. the rates of decrease of the concentrations of tritiated and stable isotope-labelled thymine in the DNA of the tumour or of the intestine were essentially identical. These results indicate the feasibility of using thymidine multilabelled with stable isotopes for measurement of cell turnover rates in conjunction with cancer therapy.     

The measurement of cell turnover rates using stable isotope-labelled thymidine: experiments on the small intestine and on a transplantable lymphatic tumour of the mouse.

Multilabelled, non-radioactive thymidine and tritiated thymidine were incorporated simultaneously into the DNA of a rapidly-growing, transplantable mouse lymphoma and the DNA of mouse small intestine by a single intraperitoneal injection of both labelled nucleosides. Mice were killed at selected times during the next 7 days, and the specific activities of the tissues and extracted DNA and the 132/126 mass ratio of thymine isolated from the DNA were determined by scintillation counting and by field ionization mass spectrometry respectively. The rates of decrease of the concentrations of tritiated and stable isotope-labelled thymine in the DNA of the tumour or of the intestine were essentially identical. These results indicate the feasibility of using thymidine multilabelled with stable isotopes for measurement of cell turnover rates in conjunction with cancer therapy.     

Reproductive development of female Schistosoma mansoni (Digenea: Schistosomatidae) following bisexual pairing of worms and worm segments

Maturation and maintenance of normal reproductive function in female Schistosoma mansoni require a permanent association with the male, but the nature of this relationship is not well understood. The regional localization of a stimulatory factor in the male and its target in the female were investigated. Unisexual female and mature male worms were transected into segments of various lengths. Various combinations of transected male and female segments and intact worms were transferred to the mesenteric veins of recipient hamsters and were also maintained in vitro. In hamsters and in vitro, pairing took place between intact worms of each sex and segments of the other, and between segments of both sexes. The majority of female worms and segments so paired showed some reproductive development, as assessed by vitelline gland differentiation. In intact unisexual females paired with small male segments, vitelline gland development was limited to that portion of the worm that had been held by the male. Worm segments continued to display normal body contractions throughout 24 days of in vitro maintenance and morphological integrity was retained. It is concluded that 1) in the absence of a functioning gut, worm segments can survive for prolonged periods on nutrients absorbed through the tegument; 2) worm pairing, male stimulation, and the female developmental response are independent of central nervous control by the cerebral ganglia; 3) males have no centralized localization for the female-stimulating factor; 4) vitelline gland differentiation in the female requires local stimulation through male contact, and this is not propagated throughout the worm.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.     

Uptake of tritiated thymidine in the root tip meristem of Vicia faba (L)

The use of tritium-labeled thymidine (3H-TdR) in biological research made it necessary to develop a quick and accurate method for determination of tritium activity in tissue. After 3H-TdR incorporation into the root tip meristem of Vicia faba, total 3H activity as well as 3H-DNA activity was measured by liquid scintillation counting. The incorporation rate of 3H-TdR using various parameters was examined-for example, the amount of incorporated 3H-TdR as a function of duration of treatment or as a function of thymidine concentration in the nutrient solution. The experimental results together with other data allow the calculation of the average number of incorporated thymidine molecules per labeled cell nucleus. This is necessary to interpret quantitatively the biological effects of incorporated radionuclides.