The synthesis of radioactive fluorescein isothiocyanate

¹⁴C-labelled fluorescein isothiocyanate was synthesized from uniformly labelled [¹⁴C]benzene. Radioactive measurements of the amount of ¹⁴C-labelled dye in a globulin conjugate gave a ratio of 14.7 μg of dye per mg of globulin. The potential uses of this double tracer are discussed.     

Studies of cerebrospinal fluid flow and penetration into brain following lateral ventricle and cisterna magna injections of the tracer [14C]inulin in rat

Parasynaptic communication, also termed volume transmission, has been suggested as an important means to mediate information transfer within the central nervous system. The purpose of the present study was to visualize by autoradiography the available channels for fluid movement within the extracellular space following injection of the inert extracellular marker [¹⁴C]inulin into the lateral ventricle or cisterna magna. Bolus injections of 5 μl of 1 μCi of [¹⁴C]inulin were made in awake rats via chronically implanted cannulae. After survival times ranging from 5 min to 4 h, brains were processed for in vivo autoradiography. At 5 min the tracer distributed throughout the ventricles, subarachnoid spaces and cisterns “downstream” of the injection sites. Penetration into the brain from these sites was complex with preferential entry along the ventral side of the brain, especially into the hypothalamus and brainstem. By 4 h virtually the entire brain was labeled irrespective of the site of tracer application. Sustained tracer entry from subarachnoid spaces suggests that some areas act as depots to trap circulating material. This mechanism may contribute to the pattern of deep penetration at later time-points. The spatial and temporal characteristics of fluid movement throughout the brain are instructive in the interpretation of many experimental procedures involving injection of molecules into the cerebrospinal fluid.     

Fastigial nucleus projections to the brain stem in beagles: Pathways for autonomic regulation

Efferent connections from a portion of the cerebellar fastigial nucleus were investigated using autoradiography. Bipolar stimulating electrodes were placed in the fastigial nucleus of anesthetized beagles and the area that produced increases in blood pressure and heart rate was localized. A mixture of [³H]leucine and [³H]proline (4:1) was injected into the area and autoradiograms of transported material were prepared. Injections filled the rostral and various parts of the caudal fastigial nucleus. Labeled axons reached the brain stem via two routes, the ipsilateral juxtarestiform body and the contralateral uncinate fasciculus. Ventral portions of the lateral vestibular nucleus were labeled bilaterally, projections to the inferior vestibular and medial vestibular nuclei are contralateral. Nucleus tractus solitarius was heavily labeled on the side opposite the injection. The contralateral medial reticular formation contained many labeled terminals and axons. Label was found in the nucleus reticularis ventralis, lateral reticular nucleus, nucleus gigantocellularis, nucleus pontis caudalis and the paramedian reticular nucleus. No terminal labeling was found in nucleus parvocellularis or nucleus ambiguus.Stimulation of the rostral fastigial nucleus produces increases in blood pressure and heart rate by generalized sympathoexcitation. Many cell groups which facilitate the activity of preganglionic sympathetic neurons do not receive direct fastigial input. It is suggested that sympathoexcitation resulting from stimulation of the fastigial nucleus occurs through multisynaptic connections in the brain stem.     

Measurement of hepatic protein synthesis in unrestrained mice-evaluation of the ‘flooding technique’

Controversy exists regarding the validity of various techniques for estimating rates of protein synthesis in viva. In the present report, we have compared estimates of hepatic protein synthesis in normal mice with a pulse labelling of [1-¹⁴C]leucine and calculated hepatic protein synthetic rates in a conventional two-pool model and in a five-pool compartment analysis. Results obtained with pulse labelling were also compared to those obtained in animals receiving a flooding dose of 1.5 μmol L-phenylalanine and 0.4 μCi [U-¹⁴C]phenylalanine per gram of body weight or 1.0 μmol L-leucine and 0.4, μCi [I-¹⁴C]leucine per gram of body weight. Estimates of protein synthesis were calculated with plasma free amino acid, liver acid-soluble fraction and acylated tRNA specific radioactivities as being representative of the precursor pool for protein synthesis. Rates of hepatic protein synthesis obtained with pulse labelling and either leu-tRNA or acid-soluble fractions of liver leucine as the precursor for protein synthesis gave similar results (37 pL 5 vs 42 pL 5% per day) in a two-pool model, but disagreed in a fivepool model (37 pL 5 us 6 pL 2% per day). Estimates based on plasma enrichment in leucine were only one fifth of values obtained with tRNA in labelling experiments. When the plasma pool with tracer amino acids was used to indicate the precursor labelling of protein synthesis, values obtained with the flooding dose of either phenylalanine or leucine agreed with those obtained with pulse labelling and enrichment in tRNA (30 pL 3 nmol min-¹ us 28 pL 4 nmol min-¹); with however no agreement when the enrichment in the liver mixed tissue pool was used (76 pL 5 nmol min-¹). Complete equilibration of the amino acid pools did not occur despite flooding. Therefore, the flooding technique may only represent an approximate method to measure protein synthesis in vivo, although it gives absolute values that agree well with results from labelling techniques based on tRNA enrichment provided the plasma pool is used as the precursor enrichment.     

In vivo [14C]amino acid profiles in discrete hypothalamic regions during push-pull perfusion in the unrestrained rat.

In the unanesthetized rat, the differential release of [¹⁴C]amino acids into a perfusate from a push-pull cannula was analyzed for 20 circumscribed areas of the hypothalamus. To serve as an exogenous precursor for [¹⁴C]amino acid synthesis. [U-¹⁴C]glucose was injected in a vol of 0.5–1.0 μl into a discrete hypothalamic region 20 min prior to a push-pull perfusion. Then, at 15min intervals, the labeled site was perfused for 5 min with an artificial cerebrospinal fluid usually at a rate of 10μl per min. Each sample of perfusate was extracted and assayed by two-dimensional, thin-layer chromatography for the presence of eight amino acids and residual glucose.The pattern of recovery of ¹⁴C-labeled substances as well as the distribution of ¹⁴C between γ-aminobutyric acid, glutamate, aspartate, alanine, taurine and glycine depended solely upon the neuroanatomical region perfused. Near the lateral edge of the ventromedial nucleus, 67% of the amino acid activity was accounted for by [¹⁴C]GABA. Perfusion sites medial to or within this nucleus, or others near the anterior hypothalamus or paraventricular nucleus, yielded a recovery of labeled GABA that was equal to that of glutamate, together accounting for two-thirds of the total ¹⁴C recovery. Within many perfusion loci, small amounts of [¹⁴C]alanine and [¹⁴C]aspartate were detected, whereas within most sites the respective recovery of [¹⁴C]aspartate, [¹⁴C]alanine, [¹⁴C]taurine and [¹⁴C]glycine was negligible. Finally, the metabolism of [¹⁴C]amino acids in the rat's hypothalamus decreased exponentially over time with little identifiable content obtained 80 min following injection of the glucose precursor.These results demonstrate that putative amino acid transmitters can be recovered from the brain of a freely moving animal, and can be characterized in vivo within specific neuroanatomical regions.     

Digitalis-like compounds and Na+, K+-ATPase activity in bovine lens

 Digitalis-like compounds in bovine lens capsule, cortex and nucleus were determined quantitatively, following extraction, by their ability to inhibit [³H]ouabain binding to red blood cells. These compounds were found to be highly concentrated in the epithelium capsule and were significantly diminished in the cortex and nucleus. Na⁺, K⁺-ATPase density in the different regions was determined by [³H]ouabain binding to membranes and by autoradiography of lens slices. The highest concentration of [³H]ouabain-binding sites was observed to occur in membranes prepared from the epithelial cells of the capsule, and was almost 100- and 200-fold higher than the concentrations observed in membranes prepared from fiber cells of the cortex and nucleus, respectively. In the autoradiography studies, strong labeling of [³H]ouabain appeared in the epithelial cell zone, and only weak specific labeling appeared in the lens cortex and nucleus. Almost all (99%) of the Na⁺,K⁺-ATPase specific activity was found to be in the capsule epithelium and only 0.5% was measured in the cortex and no activity was detected in the nucleus. These results indicate that the digitalis-like compounds and Na⁺, K⁺-ATPase are concentrated in the lens capsule epithelium and are present only at low levels in the cortex and nucleus, thus implying that the lens capsular epithelial layer is the major region of the lens responsible for the homeostasis of ions and water in this tissue. Received: 20 September 1996 / Received after revision and accepted: 17 October 1996     

Para-chloro-2-[18F]fluoroethyl-etomidate: A promising new PET radiotracer for adrenocortical imaging

Introduction: [¹¹C]Metomidate ([¹¹C]MTO), the methyl ester analogue of etomidate, was developed as a positron emission tomography (PET) radiotracer for adrenocortical tumours and has also been suggested for imaging in primary aldosteronism (PA). A disadvantage of [¹¹C]MTO is the rather high non-specific binding in the liver, which impacts both visualization and quantification of the uptake in the right adrenal gland. Furthermore, the short 20-minute half-life of carbon-11 is a logistic challenge in the clinical setting.Objectives: The aim of this study was to further evaluate the previously published fluorine-18 (T_1/2=109.5 min) etomidate analogue, para-chloro-2-[¹⁸F]fluoroethyl etomidate; [¹⁸F]CETO, as an adrenal PET tracer.Methods: In vitro experiments included autoradiography on human and cynomolgus monkey (non-human primate, NHP) tissues and binding studies on adrenal tissue from NHPs. In vivo studies with [¹⁸F]CETO in mice, rats and NHP, using PET and CT/MRI, assessed biodistribution and binding specificity in comparison to [¹¹C]MTO.Results: The binding of [¹⁸F]CETO in the normal adrenal cortex, as well as in human adrenocortical adenomas and adrenocortical carcinomas, was shown to be specific, both in vitro (in humans) and in vivo (in rats and NHP) with an in vitro K_d of 0.66 nM. Non-specific uptake of [¹⁸F]CETO in NHP liver was found to be low compared to that of [¹¹C]MTO.Conclusions: High specificity of [¹⁸F]CETO to the adrenal cortex was demonstrated, with in vivo binding properties qualitatively surpassing those of [¹¹C]MTO. Non-specific binding to the liver was significantly lower than that of [¹¹C]MTO. [¹⁸F]CETO is a promising new PET tracer for imaging of adrenocortical disease and should be evaluated further in humans.     

Central transport and distribution of labelled glutamic and aspartic acids to the cochlear nucleus in cats: an autoradiographic study.

Tritiatedl-glutamic acid orl-aspartic acid were injected unilaterally into the cochleas of adult cats, and 4 h-7 days later the localization of label was studied by light-microscopic autoradiography in sections of the brain stem. All experiments with [³H]glutamate and most with [³H]aspartate produced visibly denser grains and quantitatively higher grain counts in the cochlear nucleus of the injected side than in the cochlear nucleus of the noninjected side. In general, [³H]glutamate produced dense label ipsilaterally in all major subdivisions of the cochlear nucleus except the molecular layer of the dorsal division and the granular cell cap of the ventral division, in patterns like those after injections of [³H]leucine. The most heavily labelled areas were those that received the densest inputs of primary cochlear fibers. By contrast, [³H]aspartate produced dense grains in the ventral, but not in the dorsal, cochlear nucleus. The results with [³H]glutamate closely resembled those after [³H]leucine or [¹⁴C]leucine injections, but the results with [³H]aspartate showed consistent differences from those after [³H]leucine. After [³H]aspartate, label was densely located around cell bodies and basal dendrites, but not around primary dendrites of octopus cells, in contrast to the dense perisomatic and peridendritic label after [³H]glutamate. Also, relatively low grain counts occurred in the nerve root entry zone and in the deep dorsal cochlear nucleus after [³H]aspartate, although high counts occurred in those same zones after either [³H]glutamate or [³H]leucine injections.Consistent differences in labelling after glutamate and after aspartate suggest differences in their uptake, metabolic conversion and/or transport to the cochlear nucleus by cochlear fibers. The morphological differences shown here agree with the distribution of those two amino acids in the cat cochlear nucleus as shown by microchemical analyses.     

Distribution of cocaine recognition sites in rat brain: In vitro and ex vivo autoradiography with [I]RTI-55

The distribution of binding sites of [¹²⁵I]RTI-55 (3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester), a phenyl tropane analog of cocaine, and the selective labelling of the dopamine transporter (DAT) were studied by in vitro and ex vivo autoradiography in the rat whole brain. Recent evidence has shown that RTI-55 binds to not only DAT but also serotonin transporter (5HTT). In the present study, in vitro autoradiography revealed that [¹²⁵I]RTI-55 bound to the olfactory tubercle, the caudate putamen, the accumbens nucleus, the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the substantia nigra compact part, the subthalamic nucleus, the ventral tegmental area, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus. Further, in the presence of clomipramine, a selective ligand for 5HTT, [¹²⁵I]RTI-55 binding was remarkably inhibited in the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus, while [¹²⁵I]RTI-55 binding remained in the olfactory tubercle, the caudate putamen, the accumbens nucleus, the substantia nigra compact part, the subthalamic nucleus, and the ventral tegmental area. These findings suggest that [¹²⁵I]RTI-55 binds to 5HTT in the former areas and to DAT in the latter areas. It is therefore concluded that RTI-55 is a suitable ligand for studying the action of cocaine in whole brain regions, including the thalamus, the hypothalamus and the dorsal raphe nucleus, regions in which cocaine is thought to act evoking several neurological effects, e.g., analgesia and elevation of adrenocorticotropic hormone. DAT was also labelled selectively both in vitro and in vivo using [¹²⁵I]RTI-55 combined with clomipramine. Therefore, radiolabelled RTI-55, combined with unlabelled clomipramine, which displaces its binding to 5HTT, also appears to be suitable for the selective imaging of DAT in vivo.     

Type A and B monoamine oxidase activities in the human and rat kidney

The present work has examined the distribution of the two isoforms of monoamine oxidase (MAO), type MAO-A and MAO-B, in the cortex and medulla of the human and rat kidney. Homogenates of renal cortex and renal medulla were prepared in 67 mmoles 1^-1phosphate buffer (pH = 7.2) and MAO activity was determined with [³H]5-hydroxytryptamine ([³H]5HT) and [¹⁴C]β-phenylethylamine ([¹⁴C]β-PEA) as preferential substrates of type A and type B MAO, respectively. K_m and V_max values for the two substrates were also calculated. Both MAO-A and MAO-B are present in the cortex and the medulla of the human and rat kidneys. In the human kidney, MAO-A activity was found to be similar in the cortex (V_max= 142.70±45.05 nmoles mg^-1protein h^-1) and medulla (V_max= 133.91±35.51 nmoles mg^-1protein h^-1); MAO-B activity was found to be higher in the cortex (V_max= 166.19±19.75 nmoles mg¹protein h^-1) than in the medulla (V_max= 92.91±13.22 nmoles mg^-1protein h^-1). In the rat kidney, MAO-A was also found to be similar in the cortex (V_max= 62.35±1.74 nmoles mg^-1protein h^-1) and the medulla (V_max= 59.42±0.97 nmoles mg^-1protein h^-1) and higher than the activity of MAO-B in the two renal areas (cortex, V_max= 31.06±1.09 nmoles mg^-1protein h^-1; medulla, V_max= 14.93±0.97 nmoles mg^-1protein h^-1). No statistically significant differences were found between the K_m values towards [³H]5HT and [¹⁴C]β-PEA in the cortex and the medulla of the human and rat kidneys. The results show that in both renal areas, activity of the enzyme is higher in the human kidney than in the rat kidney. Furthermore, in the human kidney, in contrast with the rat kidney, MAO-B activity closely follows MAO-A activity.     

Autoradiographic determination of glucose content and estimation of the lumped constant in intracerebral neuronal tissue transplants.

The quasi-steady-state distribution volume of brain glucose was measured using 3-O-[14C]methylglucose quantitative autoradiography in a group of rats (n = 5) which 12-15 weeks previously had undergone unilateral ibotenic acid-induced lesion of the nucleus basalis magnocellularis, followed by implantation into ipsilateral neocortex of primordial basal forebrain cell suspensions. The effects of the lesion and the presence of transplanted tissue in neocortex were visualized by acetylcholinesterase histochemistry. The 3-O-[14C]methylglucose tracer was distributed homogeneously throughout the host brain areas analysed, with no side-to-side differences, despite a marked unilateral depletion of acetylcholinesterase activity ipsilateral to the lesion site. Whilst the transplants were indistinguishable from the homogeneity of surrounding host frontal cortex, there was a 70% increase in the apparent distribution volume of methylglucose localized around the ibotenate injection site and associated needle tract. Brain glucose content is an important experimental variable affecting the lumped constant of the 2-deoxyglucose technique. There was no evidence of any significant difference in the lumped constant between transplant and host tissue which might compromise the validity of the 2-deoxyglucose technique when used together with intracerebral implantation of fetal neuronal cell suspensions.     

Metabolic effects of nicotine on human adipose tissue in organ culture

Summary Fragments of human adipose tissue were maintained in culture for 1 week in a medium containing 1 mU/ml insulin and 100 ng/ml dexamethasone. Under these conditions lipoprotein lipase activity was present in human adipose tissue fragments which converted [¹⁴C]glucose to ¹⁴C0₂ and [¹⁴C]triglyceride. Both metabolic parameters studied were affected by human tumor necrosis factor and brefeldin A. When fragments of human adipose tissue after 1 week in culture were incubated with nicotine tartrate for 20 h, a slight but significant increase in lipoprotein lipase activity was observed, and an increased conversion of [¹⁴C]glucose to ¹⁴CO₂ and [¹⁴C] triglyceride occurred. Nicotin was taken up by human adipose tissue, but no conversion to cotinine was observed. Our data demonstrate a direct effect of nicotine on human adipose tissue metabolism. Furthermore, it is suggested that weight loss in smokers is a multifactorial phenomenon, and one of the important factors to be considered is the direct effect of nicotine within the tissue.Abbreviations LPL lipoprotein lipase - MEM minimal essential medium - rHuTNF recombinant human tumor necrosis factor - BFA brefeldin A - TG triglyceride     

The interrelations between cell groups in the caudal diencephalon of the rat projecting to the striatum and to the medulla oblongata

Summary In order to investigate the topographical relationships between the caudal diencephalic cells of origin of ascending and descending projections in the rat, one fluorescent retrograde tracer was injected into the striatum and another into the medulla oblongata. The medullary injections were mainly centered in the inferior olive. Cells labeled from the striatal injections densely filled the thalamic parafascicular nucleus. Cells labeled from the medullary injections were seen ventrally to the fasciculus retroflexus in the subparafascicular nucleus. The two populations were mixed in a small area at the ventromedial border of the fasciculus retroflexus. No double labeled cells were observed. The present results indicate that caudal diencephalic cells which ascend to the striatum are different from those descending to the medulla oblongata and that they partially overlap.Supported in parts by CNR grants N. 80.00515-04, 81.00283.04     

The effect of cochlear nerve lesion on the release of glutamate,aspartate, and GABA from cat cochlear nucleus,in vitro

Summary Pool studies of glutamate and aspartate have suggested a transmitter role for these amino acids in cochlear nerve endings. As further evidence. the K⁺-evoked release of glutamate, aspartate and GABA was measured in cat cochlear nucleus slices in vitro and compared to the release following a cochlear nerve lesion. Using [³H]glutamine as precursor, the K⁺-evoked release of glutamate and -aminobutyric acid (GABA) was respectively 4.1 and 7.2 times the spontaneous release. Using [¹⁴C]glutamate as a marker, the K⁺-evoked release of glutamate and GABA was respectively 7.1 and 2.8 times the basal release. All K⁺-evoked releases were Ca⁺⁺-dependent. Nine days after unilateral lesion of the cochlear nerve in the cat, the glutamate release decreased by about 75% on the lesioned side compared to the intact one, whereas the GABA release was not decreased. The labelled tissue glutamate, either synthesized from [³H]glutamine or labelled with [¹⁴C]glutamate, was also markedly decreased on the lesioned side. In comparison, the evoked release of aspartate, newly synthesized from [¹⁴C]glutamate, remained low and was only decreased by about 45% after cochlear nerve lesions. Comparing cat with rat cochlear nucleus, the glutamate release was similar in both animals, whereas the GABA release was much higher in the rat.It is concluded that glutamate and to a lesser extent aspartate are likely to be released from cochlear nerve terminals, supporting a transmitter role in these nerve fibres for both amino acids.     

Reduction of local cerebral blood flow to pathological levels by endothelin-1 applied to the middle cerebral artery in the rat

Endothelin-1 (1 nmol) was applied to the exposed left middle cerebral artery (MCA) in anaesthetised adult male Sprague-Dawley rats. Local cerebral blood flow (1CBF), using [14C]iodoantipyrine and quantitative autoradiography, was measured in 27 anatomically defined structures, 10 min after topical application of endothelin-1. In those areas supplied by the MCA, 1CBF was markedly reduced beyond the threshold for ischaemic damage (e.g. dorsolateral caudate nucleus reduced from 131 +/- 3 to 29 +/- 25 ml.100 g-1.min-1, sensorimotor cortex from 109 +/- 5 to 31 +/- 21 ml.100 g-1.min-1). Distant areas were not affected.     

The time course of retrograde transsynaptic transport of tetanus toxin fragment C in the oculomotor system of the rabbit after injection into extraocular eye muscles

Summary The aim of this study was to determine the optimal survival time for labelling those neurons that monosynaptically terminate on extraocular motoneurons, i.e. the premotor neurons, after an injection of tetanus toxin fragment C, a retrograde transsynaptic tracer substance, into the eye muscle of the rabbit. Concentrated fragment C was injected into the inferior rectus or inferior oblique muscle and detected immunocytochemically in the brain after survival times of 8 h, 17 h, 2 d, 3 d, 4 d, 5 d, 6 d, 8 d and 12 d. Immunoreactivity was confined to granules within motoneuronal and premotor neuronal cell bodies, but became associated with punctate profiles outlining the somata with longer survival times. The strongest and most consistent labelling of premotor cell bodies was seen after 4 days survival time. The transsynaptic labelling pattern was shown to vary for individual premotor pathways.Abbreviations III oculomotor nucleus - IV trochlear nucleus - Vmes mesencephalic trigeminal nucleus - Vmt motor trigeminal nucleus - VI abducens nucleus - VIacc accessory abducens nucleus - VII facial nucleus - BC brachium conjunctivum, co cochlear nucleus - CR restiform body - d dentate nucleus - DAB diamino-benzidine-tetrahydrochloride - HRP horseradish peroxidase - iC interstitital nucleus of Cajal, iv inferior vestibular nucleus - lgn_d lateral geniculate nucleus dorsalis - lgn_v lateral geniculate nucleus ventralis - lv lateral vestibular nucleus - mgn medial geniculate nucleus - MLF medial longitudinal fasciculus - mv_p medial vestibular nucleus pars parvocellularis - mv_m medial vestibular nucleus pars magnocellularis (= ventral part of the lv) - NIII oculomotor nerve - NV trigeminal nerve - NVII facial nerve - NVIII vestibular nerve - PC posterior commissure - pg periaquaeductal grey - ppH nucleus praepositus hypoglossi - riMLF rostral interstitial nucleus of the medial longitudinal fasciculus - rn red nucleus - sc superior colliculus - sn substantia nigra - so superior olive - sv superior vestibular nucleus - sv_c superior vestibular nucleus contralateral - sv_i superior vestibular nucleus ipsilateral - TR tractus retroflexus - Y Y-group zi zona incerta     

Calibration of [14C]plastic standards for quantitative autoradiography of [125I]labeled ligands with Amersham Hyperfilm beta-max

The densitometric response of Hyperfilm beta-max (HFBM) to [14C]plastic standards was calibrated to tissue-equivalent concentrations of [125I]. Plastic sections with standard concentrations of [125I] and [14C] were apposed to HFBM for 1, 3, 5, and 9 days, with liver and muscle slices (20 microns thick) labeled with [125I]insulin. The relative optical densities (ROD = log 10 [1/gray level x 256(-1)] produced by [125I]- and [14C]plastic standards were converted to equivalent tissue [125I] concentrations (dpm/mm2). The response of HFBM to the [125I]- and [14C]plastic standards was similar (P less than 0.001). Standard curves of tissue [125I] (dpm/mm2) vs plastic [14C] (microCi/g) concentrations fit second order polynomials (r2 = 0.995-0.999). The results show that [14C]plastic standards are valid for measuring [125I] radioactivity in tissue slices by autoradiography with HFBM.     

Studies on the serum proteins of chimeras: III. Detection of donor-type C′5 in allogeneic and congenic post-irradiation chimeras

Allogeneic and congenic post-irradiation chimeras were produced by bone marrow transfer from C′5 active donor mice into C′5 defective recipients. During the first 4 weeks after transfer many of the chimeras contained haemolytic complement activity in their sera. B6AF₁→A chimeras developed higher levels of activity than did B10D2 (new line)→ B10D2 (old line).

Spleen tissue, but not liver tissue, taken from the chimeric animals during this time period incorporated [¹⁴C]amino acid into MuB1 as demonstrated by autoradiography of immunoelectrophoretic patterns, suggesting localization of the active donor cells in the spleen rather than in the liver. Formation of donor-type IgG remained demonstrable for a more extended period after induction of chimerism than formation of MuB1.

A transplantable hepatoma in C57L/J, a C′5 active mouse strain, also incorporated [¹⁴C]amino acid into MuB1.

     

Distribution and binding of radioactivity in the starling after intravenous administration of [14C]3-chloro-p-toluidine.

Ring labeled avicide [¹⁴C]3-chloro-p-toluidine HCl (CPT) was injected (14.7 μCi) by intravenous route to starlings. The radioactivity was found to be unevenly distributed in different parts of the body. The retention half-life of radioactivity in brain, spleen, heart, and bone marrow was approximately similar to that of the plasma and ranged from 3–6 hr. A longer retention of radioactivity (8–14.6 hr) than that of the plasma was found in the muscle, lung, liver, and kidney. Of the nine tissues examined, a substantial amount of [¹⁴C]CPT radioactivity was found covalently bound only to liver and kidney proteins. The extent of covalent binding to kidney protein always exceeded the binding to liver protein. Microscopic examination of liver and kidney sections prepared from the birds 12 hr after [¹⁴C]CPT injection revealed marked histopathological changes in the liver and somewhat less striking changes in the kidney. The changes in liver were vacuolation and varying degrees of necrosis ranging from focal to diffuse. In kidney, the pathological changes included hyaline granularity of the cytoplasm. Pretreatment of birds with a microsomal enzyme inducer, phenobarbital, or tissue glutathione depletor, diethylmaleate, had no effect on covalent binding of [¹⁴C]CPT radioactivity to liver protein. These pretreatments, however, enhanced the binding significantly to kidney protein without producing any significant effect on kidney pathology. SKF 525A in phenobarbital pretreated birds had a marked inhibitory effect on covalent binding of [¹⁴C]CPT radioactivity to liver protein. Various explanations have been offered to account for the discrepancy regarding the lack of a positive correlation between the extent of covalent binding of [¹⁴C]CPT radioactivity and tissue lesion in starlings. It has been postulated that an hydroxylamine metabolite of the parent compound possibly binds to liver protein and is responsible for hepatic necrosis.