Effect of Vinblastine On the Cell Cycle and Migration of Ameloblasts of Mouse Incisors As Shown By Autoradiography Using 3H-Thymidine

Abstract. The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with ³H-thymidine ([³H]TdR) and radioautography. A group of mice received 2 μg/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1μCi/g body weight of [³H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days.
The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. the thymidine labelling index of the treated animals was 50% higher than the controls. the velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 μ/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 μm/hr respectively).
The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.     

Tumour Cell Recruitment of the Jb-1 and L 1210 Ascites Tumour Determined Directly By Double Labelling With [14C]- and [3H]-Thymidine

Abstract. Tumour cell recruitment of the JB-1 and L 1210 ascites tumour has been demonstrated directly by a double-labelling method with [¹⁴C]- and [³H]-thymidine (TdR). After [¹⁴C]-labelling of all proliferating tumour cells by multiple injections of [¹⁴C]TdR, recruitment of resting cells was stimulated by removal of the majority of tumour cells, i.e. by maximum aspiration of ascitic fluid. the number of recruited resting cells in the remaining tumour that re-enter the cell cycle after stimulation was demonstrated directly by a single injection of [³H]TdR given at different times after stimulation. the increase in the percentage of purely [³H]-labelled cells, i.e. recruited cells, with increasing time after stimulation, shows that recruitment is not a synchronous but a continuous process, the maximum of which occurs earlier in the case of the L 1210 than the JB-1 tumour. This suggests that there seems to be a relationship between the time required for maximum recruitment and the corresponding cell cycle parameters of the unperturbed tumour. There is a transitory increase of the growth fraction to about 100% and a considerable shortening of the cycle time at the maximum of recruitment.     

The Effect of Dichlorophenyldimethylurea on Light-Stimulated 22Na+, 42K+ and 86Rb+ Absorption in Different Tissues of Phaseolus vulgaris Leaves

The light-stimulated absorption of ⁸⁶Rb⁺ by Phaseolus vulgaris L. leaf slices was found to be sensitive to dichlorophenyldimethylurea in air as well as in nitrogen, whereas light-stimulated ²²Na⁺ absorption in nitrogen was not sensitive to this inhibitor. The absorption of ²²Na⁺ is not affected by light in air. The absorption of ⁴²K⁺ is enhanced by a dichlorophenyldimethylurea-insensitive light effect under anaerobic conditions and further increased by light in the absence of the inhibitor. Light-enhanced ⁴²K⁺ absorption in air was also inhibited by dichlorophenyldimethylurea. Previous work showed that light-stimulated ⁸⁶Rb⁺ and ⁴²K⁺ absorption by Phaseolus vulgaris leaf slices is restricted to the guard cells. The present results are discussed with reference to the effect of light on stomatal opening.     

On the Specificity of 125I-α-Bungarotoxin Binding to Axonal Membranes

Abstract: ¹²⁵I-α-Bungarotoxin (α-BGT) was used to characterize the binding sites for cholinergic ligands in lobster walking leg nerve membranes. The toxin binding component has been visualized histochemically on the external surfaces of intact axons and isolated axonal membrane fragments. Binding of α-BGT to nerve membrane preparations was demonstrated to be saturable and highly reversible ( K _D^app± 1.7 ± 0.32 × 10^-7 M; B _max± 249 ± 46 pmol/mg protein) at pH 7.8, 10 mM-Tris buffer. Binding showed a marked sensitivity to ionic strength that was attributable to the competitive effects of inorganic cations (particularly Ca²⁺ and Mg²⁺) in the medium. ¹²⁵I-α-BGT binding could be inhibited by cholinergic drugs (atropine ≅ d -tubocurarine > nicotine > carbamylcholine ≅ choline) and local anesthetics (procaine > tetracaine = lidocaine), but was unaffected by other neuroactive compounds tested (e.g., tetrodotoxin, 4-aminopyridine, quinuclidinyl benzilate, octopamine, bicuculline, haloperidol, ouabain). The pharmacological sensitivity of toxin binding resembles that of nicotine binding to axonal membranes, but differs significantly from nicotinic cholinergic receptors described in neuromuscular junctions, fish electric organs, sympathetic ganglia, and the CNS. The possible physiological relevance of the axonal cholinergic binding component and its relationship to α-BGT binding sites in other tissues are discussed.     

Characterization of Binding Sites for the Angiotensin II Antagonist 125I-[Sarc1,Ile8]-Angiotensin II on Murine Neuroblastoma N1E-115 Cells

The murine neuroblastoma N1E-115 cell line contains binding sites for the angiotensin II (Ang II) receptor antagonist 125I-[Sarc1,Ile8]-Ang II (125I-SARILE). Binding of 125I-SARILE to N1E-115 membranes was rapid, reversible, and specific for Ang II-related peptides. The rank order potency of 125I-SARILE binding was the following: [Sarc1]-Ang II = [Sarc1,Ile8]-Ang II greater than Ang II greater than Ang III = [Sarc1,Thr8]-Ang II much greater than Ang I. Scatchard analysis of membranes prepared from confluent monolayers revealed a homogenous population of high affinity (KD = 383 +/- 60 pM) binding sites with a Bmax of 25.4 +/- 1.6 fmol/mg of protein. Moreover, the density, but not the affinity, of the binding sites increased as the cells progressed from logarithmic to stationary growth in culture. Finally, agonist, but not antagonist, binding to N1E-115 cells was regulated by guanine nucleotides. Collectively, these results suggest that the murine neuroblastoma N1E-115 cell line may provide a useful model in which to investigate the signal transduction mechanisms utilized by neuronal Ang II receptors.     

The transmission of 125I labelled globulins to the circulation in young rats

The total and bound concentration quotients (C.Q.s) developed after intraperitoneal administration of globulin preparations are considerably greater than those developed after oral administration in young rats aged 15, 20, 23 and 27 days.
Over this age range the total C.Q.s developed after intraperitoneal administration decline more sharply than those developed after oral administration, suggesting that the mechanism of protein absorption from the peritoneum becomes comparatively less efficient during this period. At 27 days of age, contrasting with earlier stages, considerable degradation of the intraperitoneally administered dose occurs.
The duodenum of unweaned 28 day old rats is capable of transmitting to the circulation large quantities of labelled globulin with apparently little degradation or selection. This ability is virtually lost by 33 days. The jejunum of unweaned 28 day old rats transmits only very small quantities of labelled globulin to the circulation, and there is considerable degradation of the injected dose.
These several factors are discussed in the general context of the postnatal transfer of passive immunity in this species.     

Characterization, Regional Distribution, and Subcellular Distribution of 125I-Tyr1-Somatostatin Binding Sites in Rat Brain

Abstract: ¹²⁵I-Tyr₁-somatostatin binds reversibly, in a saturable manner, and with high affinity to membranes from rat brain. Kinetic and saturation data measured at equilibrium lead to K_Dvalues of 0.4 nM for cortical membranes. The binding is not affected significantly by seven neuropeptides and drugs unrelated structurally to somatostatin (SRIF) while native SRIF, Tyr₁-SRIF, and D-Trp⁸-D-Cys¹⁴-SRIF displace ¹²⁵I-Tyr₁-SRIF in a dose-dependent manner, with K_i of 0.23 nM, 0.90 nM, and 0.11 nM, respectively. Binding sites for ¹²⁵I-Tyr₁-SRIF were found in 9 out of 11 central structures; there was a significant correlation between binding capacity and endogenous SRIF levels measured by radioimmunoassay. In each of the two structures containing the most binding sites, the cortex and the preoptic area, Scatchard analysis suggests a single population of sites with apparent affinities of 0.8 nM and 1.4 nM, respectively. Subcellular fractionation of these two regions reveals that more than 60% of ¹²⁵I-Tyr₁-SRIF specific binding of the homogenate is found in the crude mitochondrial pellet (P₂), which contains synaptosomes. When P₂ is further fractionated on a discontinuous sucrose gradient, most of the initial P₂ binding is recovered from membrane fractions. Each of nine SRIF analogs, with a single alanine substitution, displaces ¹²⁵I-Tyr₁-SRIF binding on cortical membranes in the same order of potency as on adenohypophyseal membranes (r= 0.84). The data demonstrate the presence of SRIF binding sites in the rat brain, with kinetic characteristics comparable to those found in the adenohypophysis, and they provide a biochemical basis for the multiple functions of SRIF in brain.     

Comparison of 125I-SCH 23982 and [3H]SCH 23390 as Ligands for the D-1 Dopamine Receptor

125I-SCH 23982, an antagonist with high affinity and selectivity for the D-1 subtype of dopamine receptors, has recently been synthesized. Densities of D-1 receptors in rat brain obtained from autoradiographic studies using this iodinated ligand are 5- to 10-fold less than densities reported with tritiated analogues such as [3H]SCH 23390. A direct comparison of these two ligands using striatal homogenates confirmed this discrepancy. One explanation for this difference is that 125I-SCH 23982 labels a subset of the sites labeled by [3H]SCH 23390. However, the distributions of sites labeled by the ligands in autoradiograms of horizontal sections of rat brain were virtually identical. Furthermore, 127I-SCH 23982 displaced 100% of the specifically bound [3H]SCH 23390 in striatal homogenates with a Hill coefficient of approximately 1. These results are not consistent with the existence of a subset of receptors recognized by 125I-SCH 23982 and suggest that both ligands label the same population of receptors. An alternative explanation for the discrepancy in Bmax values is that an unlabeled inhibitor is present in commercial preparations of 125I-SCH 23982. When all of the solvent (including any volatile inhibitors) was removed from commercial preparations of 125I-SCH 23982 prior to use in radioligand binding experiments, the discrepancy in Bmax values was eliminated.     

[3H]Neurokinin B and 125I-Bolton Hunter Eledoisin Label Identical Tachykinin Binding Sites in the Rat Brain

[3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD = 4.3 nM; Bmax = 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB greater than eledoisin (E) greater than kassinin greater than physalaemin greater than neurokinin A (NKA) greater than substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I-Bolton Hunter (BH) NKA (127I-BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I-BHNKA as a ligand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I-BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I-BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I-BHE. The similarity of the [3H]NKB and 125I-BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I-BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.     

Cell Kinetics In the Erythroid Compartment of Guinea Pig Bone Marrow: A Model Based On 3H-Tdr Studies

A model of steady-state erythropoiesis in the guinea pig is described. the model incorporates an unidentified progenitor compartment, as well as compartments representing proerythroblasts, basophilic, polychromatic and orthochromatic cells. A computer representation of the model permits a simulation of the labeling curves obtained in pulse and intermittent labeling regimes. It was found that a reasonable fit to the data can be achieved when the parameters for the various compartments are essentially identical. the results of a preliminary sensitivity analysis, carried out by perturbing the duration of S phase from the best fit value, are reported. the fit achieved to the data supports the hypothesis underlying the model that each compartment corresponds to one generation and that the flux within and between compartments is sequential.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Effect of Hypoglycemic Encephalopathy upon Amino Acids, High-Energy Phosphates, and pHi in the Rat Brain In Vivo: Detection by Sequential 1H and 31P NMR Spectroscopy

Metabolic alterations in amino acids, high-energy phosphates, and intracellular pH during and after insulin hypoglycemia in the rat brain was studied in vivo by 1H and 31P nuclear magnetic resonance (NMR) spectroscopy. Sequential accumulations of 1H and 31P spectra were obtained from a double-tuned surface coil positioned over the exposed skull of a rat while the electroencephalogram was recorded continuously. The transition to EEG silence was accompanied by rapid declines in phosphocreatine, nucleoside triphosphate, and an increase in inorganic orthophosphate in 31P spectra. In 1H spectra acquired during the same time interval, the resonances of glutamate and glutamine decreased in intensity while a progressive increase in aspartate was observed. Following glucose administration, glutamate and aspartate returned to control levels (recovery half-time, 8 min); recovery of glutamine was incomplete. An increase in lactate was detected in the 1H spectrum during recovery but it was not associated with any change in the intracellular pH as assessed in the corresponding 31P spectrum. Phosphocreatine returned to control levels following glucose administration, in contrast to nucleoside triphosphate and inorganic orthophosphate which recovered to only 80% and 200% of their control levels, respectively. These results show that the changes in cerebral amino acids and high-energy phosphates detected by alternating the collection of 1H and 31P spectra allow for a detailed assessment of the metabolic response of the hypoglycemic brain in vivo.     

Inability of [125I]Sar1, Ile8-Angiotensin II to Move Between the Blood and Cerebrospinal Fluid Compartments

The angiotensin II competitive antagonist [125I]-Sar1, Ile8-angiotensin II was not transported from the vascular space to the cerebroventricular space in either intact or nephrectomized rats. In addition [125I]Sar1, Ile8-angiotensin II lacked the capacity to move in the opposite direction over a 20-min collection period following cerebroventricular infusion. These data suggest that angiotensins lack the capacity to move freely between the blood and cerebrospinal fluid compartments and are consistent with the notion that blood-borne and cerebroventricular angiotensins access different receptor populations.     

The Activity of Rat Pineal and Brain Tyrosine Hydroxylase During the Daily Cycle of Light and Darkness as Determined by the Modified 14CO2 Assay Method

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a two-step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X-100 were as follows. The apparent Km values of the brain enzyme for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-L-erythro-biopterin (BPH4) as cofactor and for BPH4 with 62 microM-L-tyrosine as substrate were approximately 25 microM and 85 microM, respectively. The Km's for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-6-methylpterin (6MPH4) as cofactor and for 6MPH4 with 210 microM-L-tyrosine as substrate were 68 microM and 270 microM, respectively. The marked substrate inhibition by high concentrations of L-tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver-Burk plot of reciprocal velocity versus the reciprocal concentration of BPH4 with 62 microM-L-tyrosine as substrate deviated downward at a BPH4 concentration of about 100 microM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (6L- and 6D-isomers) of the tetrahydrobiopterin cofactor, with apparent Km for BPH4 of 23 microM and 1025 microM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.     

Cell Kinetic Studies of Chick Brain Cells In Culture: an Autoradiographic Study With [3H]- and [14C]-Thymidine

Labelling index, S-phase duration and cell-cycle time of proliferating brain cells from 6-day-old chick embryos in culture were investigated autoradiographically after labelling with [³H]- and/or [¹⁴C]-thymidine. the dissociated cells were cultured in the absence or in the presence of brain extract from 8-day-old chick embryos. Cultures contained essentially two cell types, which could be easily distinguished by the size of their nuclei: small nuclei identified as belonging to precursor cells of neurons and large nuclei corresponding to astroglial cells. the labelling index of astroglial cells (16.4%) was about 2 times higher than that of the neuronal cells (9.9%). Under the influence of brain extract the labelling index of neuroblasts was nearly doubled while that of the astroglial cells remained nearly unchanged. From double-labelling experiments with [³H]- and [¹⁴C]-thymidine, the same S-phase duration of about 7 hr was found for both cell types cultured with or without brain extract. A cell-cycle duration of 39 hr for neuronal and of 29 hr for astroglial cells was found. the cycle times remained constant under the influence of brain extract. From the measured data mentioned above, a growth fraction of 50% (neuroblasts) and 68% (astroglial cells) was calculated in control cultures without brain extract. After addition of brain extract, the growth fraction increased for both cell types (neuroblasts: 92%; astroglial cells: 80%). the results demonstrate that more cells proliferate in the presence of brain extract, but the durations of the S-phase and the cell cycle remain unchanged.     

Brain Transfer Coefficients for 67Ga: Comparison to 55Fe and Effect of Calcium Deficiency

The transfer coefficients (Kin) for the uptake of gallium-67 (67Ga) into brain and CSF were determined in unanesthetized male Fischer-344 rats fed either a normal or a low-Ca diet. Kin for 67Ga was also compared with transfer coefficients for the uptake of iron-55 (55Fe) and 125I-albumin in control animals. The value of CSF 67Ga Kin was 3 x 10(-7) ml.g-1.s-1 and was 50% larger in low-Ca animals. Brain regional Kin values for 67Ga were 3-9 x 10(-7) ml.g-1.s-1 with no differences in Kin between normal and low-Ca rats. CSF Kin values for 55Fe were 40% and those for albumin were 15% of Kin for 67Ga. For brain, Kin values for 55Fe were 15-40% smaller than for 67Ga, but for albumin the Kin values were 85% less than for 67Ga. 67Ga was found to be 99% bound to plasma proteins, whereas 55Fe was 99.9% bound. The results indicate that metals that are primarily bound to transferrin enter the CSF and brain very slowly. Uptake of both metals was faster than albumin, which may indicate that metal bound to small chelates contributes significantly to brain uptake. In addition, Ca deficiency does not enhance entry of Ga into the brain.     

Sequestration of 125I-Labeled p Nerve Growth Factor by Embryonic Sensory Neurons

Abstract: Nerve growth factor (NGF) binds to two specific receptors on sensory nerve cells. These two receptors are characterized by different equilibrium dissociation constants. The higher affinity (type I) receptors have an equilibrium dissociation constant of 3.3 × 10^-11 M. The lower affinity (type II) receptors have an equilibrium dissociation constant of 1.7 × 10^-9M. These two receptors are not a result of negative cooperatively, but apparently are different receptors. At 22°C the rate of association is 1 × 10⁷ M^-1 S^-1 and the rates of dissociation are 6.5 × 10^-4 s^-1 (type I) and 3.2 × 10^-2 s^-1'(type II). After binding, a time-dependent process occurs that makes the NGF inaccessible to the external milieu (sequestered). The sequestration process is energy-dependent, but apparently temperature-independent. The data suggest that only the type I receptors are involved in the sequestration process. This process is similar to that observed on sympathetic neurons and may be the first step in the internalization of NGF by responsive cells.     

Secretin Receptors on Neuroblastoma Cell Membranes: Characterization of 125I-Labeled Secretin Binding and Association with Adenylate Cyclase

Secretin, a gut-brain peptide, elicited cyclic AMP production in a clone of neuroblastoma cells derived from the C1300 mouse tumor. Adenylate cyclase (EC 4.6.1.1) in plasma membranes from these cells was stimulated by secretin greater than vasoactive intestinal peptide greater than peptide histidine isoleucine amide, but not by the related peptides glucagon, gastric inhibitory polypeptide, or human growth hormone releasing factor. Hill coefficients for stimulation approximated one and the response to submaximal peptide concentrations was additive, as expected for hormones competing for a single receptor associated with the enzyme. Binding of 125I-labeled secretin to the neuroblastoma plasma membranes was saturable, time-dependent, and reversible. The KD determined from kinetic and equilibrium binding studies approximated 1 nM. The binding site displayed marked ligand specificity that paralleled that for stimulation of adenylate cyclase. The secretin receptor was regulated by guanine nucleotides, with guanosine 5'-(beta, gamma-imino)-triphosphate being the most potent to accelerate the rate of dissociation of bound secretin. These findings demonstrate the functional association of the secretin receptor with adenylate cyclase in neuronally derived cells.     

Effect of digestion on the δ13C and δ15N of fish-gut contents

Gut contents of sand goby Pomatoschistus minutus showed higher C and N isotope values than the food before consumption. This enrichment was more pronounced in the hindgut than in the foregut, probably because of preferential assimilation of ¹²C and ¹⁴N along the gastro-intestinal tract. The results indicated that the shift towards higher values in the alimentary canal occurs in the first 2 h after feeding.