Mitochondrial tyrosine nitration precedes chronic allograft nephropathy

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2–52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.     

Effects of prepubertal castration on the spinal motor nucleus of the ischiocavernosus muscle of the rat

Summary The location, number and size of the motoneurons innervating the ischiocavernosus muscle, identified by means of horseradish-peroxidase (HRP) retrograde transport, were studied (1) in adult untreated male rats, (2) in adult male rats castrated before puberty, and (3) in adult male rats castrated before puberty and injected with testosterone from the day of castration. After injection of HRP into the ischiocavernosus muscle, labeled motoneurons were found in the dorsolateral and dorsomedial columns of the lamina IX, at the level of L6 and S1 segments of the spinal cord. Morphometric analysis demonstrated that prepubertal castration induces a statistically significant reduction in the somatic and nuclear areas (40% and 35%, respectively, if compared to those of the control rats) of both the dorsolateral and dorsomedial motoneurons, but does not affect their number. The effects of castration are prevented by exogenous testosterone.Preliminary results were presented at the International Conference on Hormones, Brain and Behaviour, Liège, Belgium, August, 1989     

Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat

Abstract: The dopamine (DA) D₃ receptor antagonist PD 58491 {3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]-benzoimidazol-1-yl-butyl]-1 H -benzoimidazol-2-yl]-phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D₃ versus D_2L and D_4.2 receptors transfected into Chinese hamster ovary cells: K _i values of 19.5 n M versus 2,362 and >3,000 n M , respectively. In contrast, the putative DA D₃ receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D₃ versus D_2L and D_4.2 receptors (91 n M vs. 253 and 193 n M , respectively). In vitro, PD 58491 (1 n M −1µ M ) exhibited D₃ receptor antagonist activity, reversing the quinpirole (10 n M )-induced stimulation of [³H]thymidine uptake in D₃ CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ-butyrolactone-induced increase in DA synthesis ( l -3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D₂/D₃ receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D₃-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D₃ autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.     

Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na⁺ + K⁺)-ATPase (ouabain-sensitive), Mg²⁺-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F₁-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.     

Developmental Changes in Polypeptide Composition of, and Precursor Incorporation into, Cellular and Subcellular Fractions of Rat Cerebral Cortex

Abstract: Neuronal-enriched and glial-enriched fractions from rat cerebral cortex at 2. 5, 9, 14 and 23 days postnatally, and subcellular fractions from 2, 14 and 46 day old rat were prepared. The polypeptide composition of all fractions was analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electro-phoresis and quantified by densitometry. Fifty-nine polypeptides (mol. wts., 13,200–251,000) were resolved in the cell fractions of which the majority remained unchanged throughout postnatal development. Three polypeptides (mol. wts., 102,000, 56,000, 53,700) were found to increase in amount devel-opmentally in both cellular fractions, the latter two showing a peak in relative amount on day 14 and a subsequent decline. Three polypeptides (mol. wts., 47,000, 28,200, 17,400) were found to be common to the glial-enriched fraction as well as the myelin fraction, and all showed a developmental increase. The neuronal-enriched fraction was found to be enriched in five polypeptides of which one (mol. wt., 51,900) showed a developmental increase after ten days postnatally, the others (mol. wts., 178,700, 142,000, 109,000, 24,000) showing a decrease. In vitro incorporation of [³⁵S]-methionine into the glial-enriched fraction was carried out, and a developmental decline was observed in the labelling of a polypeptide of 42,000 mol. wt.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Slow Delivery of the Selective Cholecystokinin Agonist pBC 264 into the Rat Nucleus Accumbens Using Microspheres

Abstract: Neuropeptides have been shown to play a critical role in adaptational processes, probably by long-term modulation of neuronal pathways. It could therefore be interesting to study behavioral changes induced by chronic local stimulation of neuropeptide receptors. With this aim poly(lactide-co-glycolide) microspheres loaded with a highly potent, peptidase-resistant, cholecystokinin (CCK)-B-selective CCK peptidomimetic agonist (pBC 264) were prepared by a water in oil in water emulsion solvent evaporation method and stereotaxically implanted into the anterior part of the rat nucleus accumbens. Two different kinds of loaded polymeric microspheres differing only by the stabilizing agent [ovalbumin (OVA) or Pluronic F 68] added to the inner emulsion were used. The histological and behavioral studies done 24 h and 8 days after implantation of nonloaded microspheres in the nucleus accumbens indicated that the microspheres were well tolerated. The in vivo release of the selective CCK-B agonist pBC 264 (associated with a tracer dose of [³H]pBC 264) from microspheres prepared with OVA was very fast (92% after 6 h), whereas only 26% (88 pmol) of pBC 264 was released from the formulation with Pluronic F 68 after 24 h. Eight days after implantation 36% of pBC 264 had diffused from the microspheres, and 8% (∼30 pmol) was still present in the brain concentrated around the site of administration. In all cases the released material was found to correspond to intact pBC 264, thus demonstrating the possibility of obtaining a slow controlled release of peptide in vivo. This method opens up interesting perspectives to study the long-term effects of neuropeptides.     

An iterative learning strategy for the auto-tuning of the feedforward and feedback controller in type-1 diabetes

This paper proposes a scheme for the control of the blood glucose in subjects with type-1 diabetes mellitus based on the subcutaneous (s.c.) glucose measurement and s.c. insulin administration. The tuning of the controller is based on an iterative learning strategy that exploits the repetitiveness of the daily feeding habit of a patient. The control consists of a mixed feedback and feedforward contribution whose parameters are tuned through an iterative learning process that is based on the day-by-day automated analysis of the glucose response to the infusion of exogenous insulin. The scheme does not require any a priori information on the patient insulin/glucose response, on the meal times and on the amount of ingested carbohydrates (CHOs). Thanks to the learning mechanism the scheme is able to improve its performance over time. A specific logic is also introduced for the detection and prevention of possible hypoglycaemia events. The effectiveness of the methodology has been validated using long-term simulation studies applied to a set of nine in silico patients considering realistic uncertainties on the meal times and on the quantities of ingested CHOs.     

In vivo biosynthesis and turnover of 35S-labeled glomerular basement membrane

Renal glomerular basement membrane was labeled in vivo by the injection of tracer amounts of radioactive sulfate into normal adult rats. The biosynthesis and turnover of [³⁵S]glycosaminoglycans in purified basement membrane was determined from the specific activity of ³⁵S in pronase digests of basement membranes isolated 1–7 days after injection. Peak radioactive labeling occurred 24 h after injection following which the specific activity of basement membrane sulfate, expressed as cpm/μg uronic acid, progressively declined over the ensuing period of study. The biologic half-life of radioactive sulfate in basement membrane was estimated at about 7 days, which is within the range previously reported for [³⁵S]glycosaminoglycans in whole renal cortex. The findings indicate that ³⁵S-labeled components of glomerular basement membrane have a relatively rapid turnover.     

Über intraepitheliale nervöse Endformationen in mehrschichtigem Plattenepithel

Zusammenfassung Im Anschluß an frühere lichtmikroskopisch erhobene Befunde (Silberimprägnation; 1924, 1928) weist der Verfasser durch elektronenmikroskopische Untersuchungen nach, daß durch die Interzellularspalten des Epithels (Schnauze der weißen Ratte) Nervenfasern hindurchziehen. Diese Nervenfäserchen, ihre Varikositäten und ihre Endknöpfchen liegen in den Spalten zwischen den Epithelzellen. Die Ultrastruktur der nackten, d.h. von Schwannschen Zellen nicht umhüllten terminalen Nervenfasern und ihrer Endigungen im Epithel wird beschrieben.

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On intraepithelial nerve endings in a stratified squamous epithelium

Summary In addition to former light microscopical observations the author demonstrates electronmicroscopically that nerve fibers pass through the intercellular spaces of the epithelium. The nerve fibers, their varicosities and terminal bulbs lie within the spaces between the epithelial cells. The ultrastructure of the terminal nerve fibers in the epithelium is described.