Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).     

A laser-temperature-jump method for the study of the rate of transfer of hydrophobic ions and carriers across the interface of thin lipid membranes

The first application of a laser-temperature-jump apparatus for the study of ion transport through planar (artificial) lipid membranes is described. The relaxation of the electric current is detected, either continuously at a constant applied voltage or discontinuously by a series of short voltage pulses. The second technique, a combined voltage- and temperature-jump method, is especially appropriate to investigate the kinetics of the adsorption/desorption process of hydrophobic ions and neutral carriers of cations at the membrane interface and to separate this phenomenon from the diffusion process through the unstirred aqueous layers adjacent to the membrane. The aim is to determine the rate-limiting step of transport. The permeation rate of the hydrophobic anion 2,4,6-trinitrophenolate is limited by the inner membrane barrier. For tetraphenylberate the rate constant of translocation across the inner barrier and that of desorption from the membrane into water are found to be of comparable magnitude. The membrane permeability of the neutral macrocyclic ion carrier enniatin B is strongly interface limited by its comparatively small rate of desorption into water. These results show that the frequently used a priori assumption of partition equilibrium at the membrane interfaces during transport is not justified.     

Pain modulation by adrenergic agents and morphine as measured by three pain tests

The effects of several adrenergic agents on pain and morphine analgesia were assessed using three pain tests in rats. These tests--Tail-Flick, Hot-Plate, and Formalin--allow comparison of the effects of different noxious stimuli and different motor responses. Each pain test yielded a unique constellation of adrenergic influences, suggesting that variation of stimulus and response parameters can change the functional expression of adrenergic systems related to pain processing. The salient drug effects include: 1) a pronounced, relatively selective analgesic effect of yohimbine in the Hot-Plate test; 2) a selective analgesic effect of clonidine in the Formalin test; 3) a strikiny, but variable, antagonism of morphine analgesia by a combination of yohimbine and propranolol in the Formalin test; 4) a nonlinear dose-response curve for antagonism of morphine analgesia by propranolol in the Hot-Plate test; and 5) a generalized interference with pain responding and enhancement of morphine analgesia by most drrgs in the Formalin test. The data suggest that the type of pain test is crucial in determining the pattern of drug influences that is revealed.     

Stimulation of polypeptide synthesis by spermidine at the level of initiation in rabbit reticulocyte and wheat germ cell-free systems

It is shown that the stimulation of eukaryotic polypeptide synthesis by spermidine is due to the stimulation at the level of initiation by following reasons. The incorporation of formylmethionine into polypeptides was stimulated by spermidine at the same degree to the incorporation of leucine into polypeptides. Fluorography of the polypeptides formed showed that the number of chains of individual protein synthesized was larger when spermidine was added. The formation of the complex of Met-tRNA_f, globin mRNA and 40-S ribosomal subunits was stimulated by spermidine.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

Kinetics of microtubule formation after cold disaggregation of the mitotic apparatus

The rate of spindle-fiber reformation following cold treatment of the giant amoeba, Chaos carolinensis, has been determined and used to test a single growth point, subunit incorporation model of microtubule assembly. Mitotic apparatuses isolated at one-minute intervals after rewarming contain progressively longer spindle fibers; re-assembly begins at the kinetochore region, proceeds at a rate of 1·5 μm per minute, then slows as the normal length of 5 μm is approached. From information on microtubule ultrastructure, the total number of 40-Å subunits in mitotic apparatuses per amoeba, and hence the concentration released during disassembly, was calculated to be 1·0 × 10¹⁵ molecules per cm³. Calculation of diffusion and assembly kinetics indicates that this concentration of microtubule subunits is equal to the concentration required to produce a growth rate of 1·5 μm per minute by diffusion of single subunits to one assembly point per microtubule.     

Clinical Validation of Automatable Gaussian Normalized CBV in Brain Tumor Analysis: Superior Reproducibility and Slightly Better Association with Survival than Current Standard Manual Normal Appearing White Matter Normalization

PURPOSE: To validate Gaussian normalized cerebral blood volume (GN-nCBV) by association with overall survival (OS) in newly diagnosed glioblastoma patients and compare this association with current standard white matter normalized cerebral blood volume (WN-nCBV). METHODS: We retrieved spin-echo echo-planar dynamic susceptibility contrast MRI acquired after maximal resection and prior to radiation therapy between 2006 and 2011 in 51 adult patients (28 male, 23 female; age 23-87 years) with newly diagnosed glioblastoma. Software code was developed in house to perform Gaussian normalization of CBV to the standard deviation of the whole brain CBV. Three expert readers manually selected regions of interest in tumor and normal-appearing white matter on CBV maps. Receiver operating characteristics (ROC) curves associating nCBV with 15-month OS were calculated for both GN-nCBV and WN-nCBV. Reproducibility and interoperator variability were compared using within-subject coefficient of variation (wCV) and intraclass correlation coefficients (ICCs). RESULTS: GN-nCBV ICC (≥0.82) and wCV (≤21%) were superior to WN-nCBV ICC (0.54-0.55) and wCV (≥46%). The area under the ROC curve analysis demonstrated both GN-nCBV and WN-nCBV to be good predictors of OS, but GN-nCBV was consistently superior, although the difference was not statistically significant. CONCLUSION: GN-nCBV has a slightly better association with clinical gold standard OS than conventional WM-nCBV in our glioblastoma patient cohort. This equivalent or superior validity, combined with the advantages of higher reproducibility, lower interoperator variability, and easier automation, makes GN-nCBV superior to WM-nCBV for clinical and research use in glioma patients. We recommend widespread adoption and incorporation of GN-nCBV into commercial dynamic susceptibility contrast processing software.     

Salt-induced hypertension in chronic renal failure: Evidence for a neurogenic mechanism

We investigated the possibility that blood pressure elevation induced by salt excess may be secondary to a neurogenic mechanism. The compound SK&F 64139 (50 mg/kg) known to inhibit central and peripheral phenylethanolamine N-methytransferase (PNMT) the enzyme necessary for the conversion of norepinephrine to epinephrine, was given by oral gavage to two groups of subtotally nephrectomized rats maintained for five days on either a high salt (HS) or low salt (LS) diet respectively. Blood urea nitrogen (BUN) and hematocrit were not different between the two groups, while body weight and serum Na were significantly higher in the HS animals. Baseline mean blood pressure (BP) was higher in the HS animals (HS 154 ± 4.7 vs LS 121 ± 3.7 mmHg, p<0.001) and decreased by 39 ± 6.9 mmHg one and one half hour post SK&F 64139 to normotensive levels in the HS as opposed to a decrease of 10 ± 1.8 mmHg in the LS group. Baseline heart rate (HR) was higher in the LS group (474 ± 17 beats/min) vs the HS group (408 ± 17, p<0.05), and decreased significantly after SK&F 64139 in both groups to the same extent (by 17.6% in the HS vs 13.3% in the LS). A third group of subtotally nephrectomized rats maintained for five days on a HS diet were given by oral gavage the compount SK&F 29661 (100 mg/kg), a PNMT inhibitor which does not cross the blood-brain barrier. Following SK&F 29661, there was no significant decrease in mean BP (153 ± 5 to 149 ± 4 mmHg) and a less than 2% decrease in HR. Baseline plasma norepinephrine (NE) was higher in the HS as compared to the LS group (1.50 ± 0.16 vs 0.904±0.15 ng/ml, respectively, p<0.05) and a significant correlation was found between plasma NE level and decrease in BP following SK&F 64139 (r=0.65, p<0.01). Not withstanding possible effects of some ancillary properties of SK&F 64139, these data support the hypothesis that a neurogenic component, possibly mediated via central epinephrine containing neurons, contributes to the BP elevation induced by salt excess.     

Occurrence of sym-homospermidine as the major polyamine in nitrogen-fixing cyanobacteria

We analyzed the amount of polyamines in a variety of cyanobacteria including nitrogen-fixing and nonfixing species. All the cyanobacteria capable of fixing nitrogen, contained sym-homospermidine as the major polyamine. The concentration of putrescine, spermidine and spermine was extremely low in these cyanobacteria. The cyanobacteria which normally fail to fix nitrogen contained spermidine as the major polyamine, while the sym-homospermidine content was very low or under the limits of detection. Apparently there is a close relationship between the sym-homospermidine content and the ability to fix nitrogen in cyanobacteria.     

Effects of early embryonic exposure to genistein on male copulatory behavior and vasotocin system of Japanese quail

Genistein is a phytoestrogen, particularly abundant in soybeans that can bind estrogen receptors and sex hormone binding proteins, exerting both estrogenic and antiestrogenic activity. In this study we used the Japanese quail embryo as a test end-point to investigate the effects of early embryonic exposure to genistein on male copulatory behavior and on vasotocin parvocellular system. Both differentiate by the organizational effects of estradiol during development and may therefore represent an optimal model to study the effects of xenoestrogens. We injected two doses of genistein (100 and 1000 microg) into the yolk of 3-day-old Japanese quail eggs. Other eggs were treated with either 25 microg of estradiol benzoate or sesame oil as positive and negative controls. At the age of 6 weeks, behavioral tests revealed a significant decrease of all aspects of copulatory behavior (in comparison to the control group) in estradiol-treated birds. In contrast, genistein-treated animals demonstrated various degrees of decrease in the mean frequencies of some aspects of the sexual behavior. The computerized analysis of vasotocin innervation in medial preoptic, stria terminalis and lateral septum nuclei revealed a statistically significant decreased immunoreactivity in treated animals compared to control ones. These results demonstrate that genistein, similarly to estradiol, has an organizational effect on quail parvocellular vasotocin system and on copulatory behavior. In conclusion, present results confirm, in this avian model, that embryonic exposure to phytoestrogens may have life-long effects on sexual differentiation of brain structures and behaviors.